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Properties and organization of the proteins in the outer membrane of Escherichia coli Reithmeier, Reinhart A. F. 1976

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PROPERTIES AND ORGANIZATION OF THE PROTEINS IN THE OUTER MEMBRANE OP ESCHERICHIA COLI By REINHART A.F. REITHMEIER B.Sc, Carleton University, 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Biochemistry Faculty of Medicine We accept t h i s thesis as conforming to the required standard October, 1976 University of B r i t i s h Columbia © Reinhart A.F. Reithmeier, 1976 In presenting this thesis in partial fulfilment of the r e q u i r e m e n t s f o r an advanced degree at the University of B r i t i s h C olumbia, I agree that the Library shall make it freely available for r e f e r e n c e and study . I further agree that permission for extensive copying of t h i s t h e s i s for scholarly purposes may be granted by the Head of my Department o r by his representatives. It is understood that c o p y i n g o r p u b l i c a t i o n o f this thesis for financial gain shall not be allowed without my written permission. Department of V^ >%oOwew%.\vV The University of Brit ish Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date Wove^ev- \ 1 7 G i ABSTRACT Two major proteins of the outer membrane of Escherichia  c o l i , the matrix protein, A (M.W. 3 6 , 5 0 0 ) and the heat-modifiable protein, B were p u r i f i e d and p a r t i a l l y characterized. Both have a low content of cysteine, . an excess of aci d i c amino acids over basic and a moderate content of hydrophobic amino acids. Protein B (M.W. 2 8 , 5 0 0 ) was converted to form B* (M.W. 3 3 j ^ 0 0 ) upon heating i n the presence of sodium dodecyl sulfate at temperatures higher than 50°C. Physical studies showed that protein B unfolds upon heating without a large increase i n binding of sodium dodecyl sulfate. It is'proposed that protein B as extracted from the membrane contains some native structure which i s lost upon heating. The l e v e l of protein A-^ , a major outer membrane protein i n glucose-grown c e l l s , was decreased i n c e l l s grown on other carbon sources with a concomitant increase in the amount of protein A^. Both proteins were t i g h t l y as-sociated with the peptidoglycan and had sim i l a r amino acid composition, suggesting that they play the same role i n the outer membrane. The organization of proteins i n the outer membrane of E. c o l i was studied by proteoly t i c digestion, co-valent l a b e l l i n g and crosslinking. The proteins of the outer membrane were inaccessible to pronase i n intact c e l l s and the c e l l s altered i n the i r lipopolysaccharide component. The protein components of is o l a t e d outer membrane preparations va-ri e d i n t h e i r rates of digestion and l a b e l l i n g with fluoresca- ' mine, suggesting that they are asymmetrically arranged i n the i i membrane. The proteins most rapidly degraded (proteins B,C-,p^ and E) were judged to be exposed at the surface of the membrane, while those resistant to digestion (proteins A-^,A^ and D 2) must be protected by t h e i r arrangement i n the membrane. Digestion of outer membrane preparations with pronase l e f t a fragment derived from protein B (protein Bp) embedded i n the membrane. This fragment was not enriched i n hydrophobic amino acids re-l a t i v e to protein B. Protein B could be reassociated with i t s e l f j without phospholipid or lipopolysacchari.de such that pronase digestion of the reassociated material gave protein Bp. These results suggest that protein B may not be held i n the membrane primarily by hydrophobic interactions. The resistance of proteins A-^  and A^ to protease digestion i s l i k e l y due to protein-protein interactions since oligomers of protein A could be is o l a t e d . Treatment of protein A^- or A2~peptidogly-can complexes with d i t h i o b i s (succinimidyl propionate) or glutaraldehyde produced dimer, trimer and higher oligomers of protein A. No crosslinking of protein A to the peptidoglycan was detected. The proteins of the is o l a t e d outer membrane varied i n t h e i r ease of crosslinking. Protein B, but not the pronase-resistant fragment, protein Bp, was readily crosslinked to give high molecular weight oligomers, while protein A formed dimer s« and • .triiaer sunder the same conditions. No crosslinking o f protein A to B was detected. Crosslinking of c e l l wall preparations showed that protein B and the free form of the lipo p r o t e i n , F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. i i i These results suggest that s p e c i f i c protein-protein i n t e r -actions occur i n the outer membrane. A model for the arrange-ment of the proteins i n the outer membrane of E. c o l i , summarizing the re s u l t s of p r o t e o l y t i c digestion, covalent l a b e l l i n g and cross l i n k i n g , i s presented. i v TABLE OP CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS xv TABLE OF CONTENTS i v LIST OF TABLES i x LIST OF FIGURES x ABBREVIATIONS x i v INTRODUCTION 1 C e l l envelope o f gram-negative b a c t e r i a 1 Cytoplasmic membrane 1 Pe p t i d o g l y c a n 3 Outer membrane. 3 Components of the outer membrane 5 L i p o p o l y s a c c h a r i d e 5 Ph o s p h o l i p i d s 7 Outer membrane p r o t e i n s 8 P r o p e r t i e s o f the outer membrane p r o t e i n s 12 Bound l i p o p r o t e i n 12 Free l i p o p r o t e i n 14 Matr i x p r o t e i n 15 Heat-modifiable p r o t e i n 19 Other major outer membrane p r o t e i n s 21 Functions of the outer membrane p r o t e i n s 22 Role of p r o t e i n s i n the b a r r i e r f u n c t i o n of the outer membrane. 22 V Page Role of outer membrane p r o t e i n s i n maintaining the shape of the organism 25 Role of the outer membrane p r o t e i n s i n DNA r e p l i c a t i o n 26 Involvement of outer membrane p r o t e i n s i n high a f f i n i t y t r ansport systems 28 Organization of Pr o t e i n s i n the outer membrane of E. c o l i 29 B i o s y n t h e s i s and assembly of outer membrane pr o t e i n s 42 Bi o s y n t h e s i s 42 Assembly of the p r o t e i n s of the outer membrane. 46 R e c o n s t i t u t i o n 47 METHODS AND MATERIALS 50 Reagents 50 B a c t e r i a l s t r a i n s 50 Growth con d i t i o n s 51 C e l l f r a c t i o n a t i o n P r e p a r a t i o n of T r i t o n - e x t r a c t e d c e l l envelopes. 52 Sequential e x t r a c t i o n of c e l l w a l l 53 Preparation of p r o t e i n A-peptidoglycan'com-plexes 53 Preparation and l y s i s of spheroplasts 53 Release of outer membrane from spheroplasts.... 54 Separation of inner and outer membrane by sucrose d e n s i t y gradient c e n t r i f ugation 55 P u r i f i c a t i o n of outer membrane p r o t e i n s 55 P u r i f i c a t i o n of p r o t e i n B 55 P u r i f i c a t i o n of p r o t e i n B f o r r e c o n s t i t u t i o n . . . 56 P u r i f i c a t i o n of p r o t e i n A 57 v i Page P u r i f i c a t i o n of protein Bp, the pronase-resistant fragment derived from protein B .• 58 Preparation of lipopolysacchardide 59 Small-scale preparation of t o t a l outer membrane proteins 60 Digestion of membrane preparations with p r o t e o l y t i c enzymes 60 Crosslinking of outer membrane proteins 6 l Removal of SDS from protein samples 62 Cyanogen bromide cleavage of proteins 63 A n a l y t i c a l techniques 63 SDS-polyacrylamide tube gel electrophoresis.... 63 SDS-polyacrylamide tube gel electrophoresis at an alkaline pH 64 Preparation of samples for SDS-polyacrylamide tube gel electrophoresis 64 Staining polyacrylamide gels for protein 65 Staining polyacrylamide gels for carbohydrate.. 65 Discontinuous SDS-polyacrylamide gel electrophoresis 66 Staining of polyacrylamide slab gels 69 Analysis of DSP-crosslinked proteins by two-dimensional SDS-gel electrophoresis 69 Polyacrylamide gel electrophoresis i n the presence of urea 70 Determination of molecular weight by gel f i l t r a t i o n 71 Sucrose density gradient centrifugation of proteins B and B* 71 Measurement of binding of SDS to proteins 72 Measurement of i n t r i n s i c v i s c o s i t y 72 v i i S p e c t r a l measurements Amino a c i d a n a l y s i s . . , RESULTS Use of various c e l l f r a c t i o n s P u r i f i c a t i o n and p r o p e r t i e s of the outer membrane pro t e i n s Page ?2 73 74 74 Determination of c y s t e i n e . 73 Amino-terminal a n a l y s i s P r e p a r a t i o n of dansyl-amino acids Determination of p r o t e i n 75 Determination of carbohydrate 75 Determination of l i p o p o l y s a c c h a r i d e 75 Determination of phosphate 76 77 77 79 79 83 87 Major p r o t e i n s of the outer membrane H e a t - m o d i f i a b i l i t y of p r o t e i n B P u r i f i c a t i o n of p r o t e i n B* Amino-acid composition of p r o t e i n B..... 91 Cyanogen bromide cleavage of p r o t e i n B 91 Amino t e r m i n a l a n a l y s i s of p r o t e i n B 95 P h y s i c a l p r o p e r t i e s of p r o t e i n s B and B* 95 E f f e c t of growth conditions on the outer membrane p r o t e i n s 118 P u r i f i c a t i o n of p r o t e i n A 119 Amino a c i d composition of pro t e i n s . Aj_ and A 2 . . . 126 Cyanogen bromide cleavage of p r o t e i n A 126 Organization of p r o t e i n s i n the outer membrane 127 E f f e c t of p r o t e o l y t i c enzymes on the p r o t e i n s of the outer membrane i n i n t a c t c e l l s 127 v i i i Rage Effe c t of p r o t e o l y t i c enzyme on the proteins of the outer membrane i n isola t e d membrane preparations 130 Labelling of outer membrane proteins with f luorescamine 136 Association of oligomers of protein A with the peptidoglycan 139 Reassociation of protein B and the formation of protein Bp 146 Crosslinking of protein A-peptidoglycan complexes 153 Crosslinking of outer membrane 157 Interaction of proteins B and P with peptidoglycan . 159 DISCUSSION 169 Properties of the outer membrane proteins 169 Protein B 169 Protein A 172 Organization of proteins i n the outer membrane 174 BIBLIOGRAPHY 182 APPENDIX 198 i x LIST OP TABLES Table Page I. Nomenclature for the outer membrane proteins of E. c o l i 13 I I . Amino acid composition of SDS extracts 92 I I I . Amino acid composition of p u r i f i e d proteins. 93 IV. P a r t i a l s p e c i f i c volumes and sedimentation c o e f f i c i e n t s of proteins B and B* 110 V. Conditions for reassociation of protein B... 152 X LIST OF FIGURES Figure ^ Page 1. Model of the c e l l envelope of E. c o l i 2 2. Structure of the peptidoglycan-lipoprotein complex of E. c o l i 4 3 . Structure of S_. typhimurium lipopolysaccharide 6 4 . Electrophoresis on polyacrylamide gels of proteins of the E. c o l i outer membrane 9 5 . Models proposed for the organization of the components of the c e l l envelope of E. c o l i 30 a. Martin (1963) 30 b. De Petris (1967) 30 c. Schnaitman (1971) 31 d. Nikaido (1973) 31 e. Leive (1974) 32 f. Costerton, Ingram and Cheng ( 1 9 7 4 ) . . . . 33 g. Inouye (1975) 34 h. Costerton and Cheng (1975) 35 i . Inouye (1975) 36 6 . Schematic representation of procedures used to prepare c e l l fractions 78 7. Resolution of outer membrane proteins by SDS-polyacrylamide gel electrophoresis 80 8. E f f e c t of heating on the migration of outer membrane proteins i n SDS-polyacrylamide gels.. 82 9 . E f f e c t of heating on the migration of protein B i n SDS-polyacrylamide gels 84 LO.. Arrhenius plot for the conversion of protein B to B* 85 L l . E ffect of heating on. the. elution position of protein B from a column of Sephadex G-100 86 x i Figure Page 1 2 . U l t r a v i o l e t spectrum of T r i t o n X-100 , 88 1 3 . Standard curve for the determination of T r i t o n X-100 89 14. Separation of proteins of extract 1 by gel f i l t r a t i o n 90 15 . Cyanogen bromide cleavage of protein B 94 16 . Amino-terminal analysis of proteins B and Bp. 96 17. SDS-polyacrylamide gel electrophoresis of p u r i f i e d protein B 97 18 . Determination of the molecular weights of proteins B and B* 98 19. Reduced v i s c o s i t i e s of proteins B and B* 100 2 0 . Equilibrium d i a l y s i s of protein B 102 2 1 . Binding of SDS to proteins B and B* 103 2 2 . Chromatography of proteins B and B* on Sepharose 6B i n 1% SDS 105 2 3 . Centrifugation of proteins B and B* i n 5 to 20% sucrose gradients prepared i n 0.1% SDS 107 24. Centrifugation of proteins B and B* i n 5 to 20% sucrose gradients prepared i n 0.2% SDS 108 2 5 . Centrifugation of proteins B and B* i n 5 to 20% sucrose gradients prepared i n 0.3% SDS 109 2 6 . Digestion of proteins B and B* by pronase in 1% SDS 112 2 7 . U l t r a v i o l e t absorption of proteins B and B*. . 114 2 8 . Optical rotatory dispersion and c i r c u l a r dichroism spectra of proteins B and B* 115 2 9 . E f f e c t of heating protein B i n the absence of SDS 117 3 0 . Growth of E.: c o l i NRC 482 on d i f f e r e n t carbon sources..... 120 x i i F igure Page 3 1 . E f f e c t of growth c o n d i t i o n s on outer membrane p r o t e i n s 123 3 2 . Cyanogen bromide cleavage of p r o t e i n s A x and A 2 124 3 3 . Gel f i l t r a t i o n chromatography of p r o t e i n s A1 and A 2 125 3 4 . E f f e c t of p r o t e o l y t i c d i g e s t i o n on the outer membrane . p r o t e i n i n c e l l s 128 3 5 . Release of l i p o p o l y s a c c h a r i d e from c e l l s by EDTA 129 36. E f f e c t of a l t e r a t i o n i n l i p o p o l y s a c c h a r i d e s t r u c t u r e on the d i g e s t i o n of outer membrane pr o t e i n s by pronase 131 37. K i n e t i c s of d i g e s t i o n of outer membrane pr o t e i n s by pronase 133 3 8 . K i n e t i c s of d i g e s t i o n of outer membrane pr o t e i n s by t r y p s i n 134 3 9 . K i n e t i c s of d i g e s t i o n of outer membrane pr o t e i n s by chymotrypsin 135 40. Slab g e l e l e c t r o p h o r e s i s of fluorescamine-l a b e l l e d c e l l s of E. c o l i JE 1011 and N S - 1 . . . 137 41. Slab g e l e l e c t r o p h o r e s i s of fluorescamine-l a b e l l e d outer membrane p r o t e i n s 138 42. Resistance of p r o t e i n s A]_ and A 2 to pronase d i g e s t i o n 140 4 3 . SDS-polyacrylamide g e l scans of p r o t e i n A ext r a c t e d from P r o t e i n A-peptidoglycan complexes 142 4 4 . E x t r a c t i o n of p r o t e i n A-peptidoglycan complexes wi t h urea 143 4 5 . Gel f i l t r a t i o n of p r o t e i n A oligomers 144 46. Gel. f i l t r a t i o n of p r o t e i n s s o l u b i l i z e d from i s o l a t e d outer membrane by SDS 145 x i i i Figure Rage 47. P u r i f i c a t i o n of protein Bp by gel f i l t r a t i o n i n the presence of' SDS... 148 48. SDS-polyacrylamide gel scan of p u r i f i e d protein Bp 149 49. Production of protein Bp by pronase digestion of reassociated protein B .151 5 0 . Crosslinking of protein A-peptidoglycan complexes with DSP 154 5 1 . Crosslinking of protein A-peptidoglycan complexes with glutaraldehyde 155 5 2 . Lack of crosslinking of protein A to the peptidoglycan layer by DSP 156 5 3 . Crosslinking of c e l l wall and outer membrane with DSP 158 5 4 . Crosslinking of pronase-treated outer membrane with DSP 160 5 5 . Two-dimensional gel of products from crosslinked outer membrane proteins l 6 l 5 6 . Two-dimensional gel of products from crosslinked c e l l wall 163 57. Crosslinking of proteins B and F to the peptidoglycan layer 164 5 8 . Two dimensional gel of products from crosslinked c e l l wall, at low l e v e l of DSP.. 166 5 9 . Two dimensional gel of products from crosslinked c e l l wall at low l e v e l of DSP... 168 6 0 . Model for the organization of the proteins i n the outer membrane of E ... :aoli l 8 l x i v ABBREVIATIONS cAMP: 3 ' 3 5 ' c y c l i c adenosine monophosphate CD: C i r c u l a r dichroism DNA: Deoxyribonucleic a c i d DNase: Deoxyribonuclease DSP: D i t h i o b i s ( s u c c i n i m i d y l propionate) EDTA: et h y l e n e d i a m i n e t e t r a a c e t i c a c i d Hepes: N-2-hydroxyethylpiperazine N'-2-ethane-s u l f o n i c a c i d mRNA: Messenger r i b o n u c l e i c a c i d ORD: o p t i c a l r o t a t o r y d i s p e r s i o n RNase: Ribonuclease SDS: Sodium dodecyl s u l f a t e TEMED: N,N3N',N* tetramethylethylenediamine T r i s : Tris-(hydroxymethyl)-aminomethane uv: u l t r a v i o l e t Other a b b r e v i a t i o n s used i n f i g u r e s are defined i n the legends. XV ACKNOWLEDGEMENTS There 'are a number of people to whom I am Indebted f o r help throughout these s t u d i e s : Mr. J . Durgo and Mr. L. Bryson f o r s k i l f u l t e c h n i c a l a s s i s t a n c e ; Dr. S.H. Zbarsky and Dr. G.M. Tener f o r use of equipment; Mrs. C. Hou, Dr. A.P. 'Sin and Dr. I.G. G i l l a m f o r u s e f u l d i s c u s s i o n s and the members of my Ph.D. committee, Dr. D.E. Vance and Dr. E.P.M. Candido f o r t h e i r advice and i n t e r e s t i n t h i s r e s e a r c h p r o j e c t . I e s p e c i a l l y wish t o thank my s u p e r v i s o r , Dr. P.D. Bragg f o r h i s guidance and encouragement throughout my graduate work and the Medical Reasearch C o u n c i l of Canada f o r f i n a n c i a l support. Protein "He was supposed to be our commencement speaker," said Sandra. "Who was?" I asked. "Dr. Hoenikker - the old man." "What did he say?" "He didn't show up." "So you didn't get a commencement address?" "Oh, we got one. Dr. Breed, the one you're gonna see tomorrow, he showed up, a l l out of breath, and he gave some kind of t a l k . " "What did he say?" "He said he hoped a l o t of us would have careers i n science," she said. She didn't see anything funny i n that. She was remembering a lesson that had impressed her. She was repeat-ing i t gropingly, d u t i f u l l y . "He said, the trouble with the world was . . . " She had to stop and think. "The trouble with the world was," she con-tinued h e s i t a t i n g l y , "that people were s t i l l superstitious instead of s c i e n t i f i c . He said i f everybody would study science more, there wouldn't be a l l the trouble there was." "He said science was-going to discover the basic secret of l i f e someday," the bartender put i n . He scratched his head and frowned. "Didn't I read i n the paper the other day where they'd f i n a l l y found out what i t was?" "I missed that," I murmured. "I saw that," said Sandra. "About two days ago." "That's r i g h t , " said the bartender. "What i s the secret of l i f e ? " I asked. "I forget," said Sandra. "Protein," the bartender declared. "They found out something about protein." "Yeah," said Sandra, "that's i t . " Prom Cat's. Cradle by Kurt Vonnegut. 1 INTRODUCTION C e l l Envelope of Gram-negative B a c t e r i a The c e l l envelope of gram-negative b a c t e r i a such as E s c h e r i c h i a c o l i i s composed of three p h y s i c a l l y and c h e m i c a l l y d i s t i n c t l a y e r s ( F i g . 1 ) , the i n n e r or cytoplasmic membrane, the murein or p e p t i d o g l y c a n , and the outer membrane ( 1 - 8 ) . The p e p t i d o g l y c a n and the outer membrane comprise the c e l l w a l l l a y e r . The p e p t i d o g l y c a n , l o c a t e d between the two membranes, v a r i e s i n t h i c k n e s s i n d i f f e r e n t gram-negative b a c t e r i a (5) and can be removed by d i g e s t i o n with lysozyme ( 1 0 ) . The i n n e r and outer membranes show a t y p i c a l u n i t membrane (20) appearance, 75 A t h i c k , under the e l e c t r o n microscope ( 9 , 1 0 ) . The i n n e r and outer membranes can be separated by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of s p h e r o p l a s t s , prepared by l y s i n g c e l l s t r e a t e d with lysozyme In the presence of EDTA ( 1 4 , 3 5 , 7 1 , 7 3 - 7 6 ) . C e l l s can a l s o be d i s r u p t e d i n a French P r e s -sure C e l l and the c e l l w a l l , c o n s i s t i n g of the outer membrane and p e p t i d o g l y c a n , can be r e a d i l y separated from the i n n e r membrane by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (13) or e l e c t r o p h o r e s i s ( 7 0 ) . Cytoplasmic membrane The i n n e r membrane of gram-negative b a c t e r i a i s composed of p h o s p h o l i p i d (40$) and p r o t e i n s (60%) and has a s t r u c t u r e s i m i l a r to that of most b i o l o g i c a l membranes ( 1 1 - 1 5 ) . The p h o s p h o l i p i d s , c o n s i s t i n g mainly of p h o s p h a t i d y l ethanolamine F i g . 1. Model of the c e l l envelope of E. c o l i . OM, outer membrane; P, peptidoglycan*, CM, cytoplasmic membrane; fU phospholipid: V , lipopolysaccharide; , protein; ?, l i p o p r o t e i n . • 3 (78$) and phosphatidyl g l y c e r o l (11$), are arranged as a b i l a y e r , p r o v i d i n g a hydrophobic zone which i s cleaved during f r e e z e -etching (16,17). The p r o t e i n components of the membrane are seen as globules i n these preparations (18,19). The p r o t e i n s of the cytoplasmic membrane Include those r e s p o n s i b l e f o r e l e c -t r o n t r a n s p o r t and o x i d a t i v e phosphorylation (21,22), a c t i v e t r a n s p o r t of solutes (23-26), and the b i o s y n t h e s i s of phospho-l i p i d s (27-30), peptidoglycan (2,31-33) and l i p o p o l y s a c c h a r i d e (2,6,14,34-38). Peptidoglycan The peptidoglycan i s composed of a l t e r n a t i n g residues of N-acetylmuramic a c i d and N-acetylglucosamine j o i n e d b y j g ( l , 4 ) linkages (33). Peptide c r o s s b r i d g e s , l i n k e d to the carboxyl groups of the chains, r e s u l t i n the formation of a huge macro-molecular net (40). F i g . 2 presents the s t r u c t u r e of the peptidoglycan from E. c o l i . The peptidoglycan may be involved i n m aintaining the shape of the organism since the c e l l s bounded only by t h e i r peptidoglycan ( 4 l ) and i s o l a t e d s a c c u l i (42,43) r e t a i n the o r i g i n a l shape of the bacterium. The peptidoglycan l a y e r may al s o play a r o l e i n the assembly of the outer membrane components since i t i s attached to the outer membrane by a l i p o p r o t e i n (44). Outer membrane The outer membrane of gram-negative b a c t e r i a Is a unique membrane i n a number of re s p e c t s . As already s t a t e d , the outer membrane i s l i n k e d to the peptidoglycan by l i p o p r o t e i n (44). 4 CH2-0-Fotty acid I CH-O-Fatty acid I CH 2 I S I CHj Cys Ser 1 Asn Alo Lys Me Asp Glu Leu Ser Ser Asp Val Gin Thr Leu 1 I Asn Ala Lys Val Asp Glu Leu Ser Asn Asp Vol Asn Ala Met Arg 1 I Ser Asp Vol Gin Ala Ala Lys 1 I Asp Asp — — Ala Ala Arg 1 I - A* a Asn Gin — Arg 1 58 Leu — Asp Asn Met — Ala Thr Lys Tyr Arg Lys H \MurNAc-GlcNAc V -\-L-Ala-D-Glu-r-L-Alo-D-Glu-Opm-D-Ala-Mur NAc-GlcNAc — MurNAc-GlcNAc l-L-Ala-D-Glu-Dpm-D-Ala Hi H = Dpm-D-Ala r-L-Ala-D-Glu-Dpm--MurNAc-GlcNAc-a: _| Repeating disaccharide units MurNAc-GlcNAc LL-Ala-D-Glu-meso-Dpm-D-Ala > Cross-linkage between two disaccharide units joining two neighbouring polysaccharide chains F i g . 2. Structure of the peptidoglycan l i p o p r o t e i n complex of E. c o l i . The c y l i n d r i c a l section indicates a part of the. murein with the shape of the rod-like E. c o l i c e l l . It i s not known i n which d i r e c t i o n the glycan chains span the c e l l , r e l a t i v e to the long axis of the cylinder. In t h i s model they are a r b i t r a r i l y drawn p a r a l l e l . The murein i s composed of roughly 10 s repeating units, to which approximately 10 6 l i p o -protein molecules are covalently bound. The lipoprotein replaces d-alanine on the diaminopimelate residue. The amino.acid sequence i s represented i n a way that demonstrates the possible evolution of t h i s molecule from a.gene that coded o r i g i n a l l y for 15 amino acids, which was duplicated, and then only the C-terminal half was added four times. The dashes represent hypothetical deletions of amino acids that may have occurred during evolution. The stars indicate the hydrophobic amino acids at every 3.5th position. Taken from Braun ( 4 4 ) . 5 A second unique feature i s that the outer membrane contains l i p o p o l y s a c c h a r i d e (20%) i n a d d i t i o n to phospholipid (20%) and p r o t e i n (60%) (14). T h i r d l y , the outer membrane i n the pre-sence of magnesium ions i s r e s i s t a n t to d i s r u p t i o n by detergents such as T r i t o n X-100 (66,67,72) and sodium l a u r y l s a r c o s i n a t e (69). The cytoplasmic membrane i s completely s o l u b i l i z e d under these c o n d i t i o n s . F i n a l l y , the p r o t e i n composition of the outer membrane i s much simpler than that of the inner membrane, con-t a i n i n g only a few p r o t e i n s , present i n major amounts (12,13). Components of the Outer Membrane Lipopolysaccharide The s t r u c t u r e , b i o s y n t h e s i s and assembly of t h i s unique c e l l w a l l component has been e x t e n s i v e l y studied (2,6,14,34-38,52-58). The l i p o p o l y s a c c h a r i d e molecule i s composed of three regions ( F i g . 3), l i p i d A, inner and outer core, and 0-antigen chains c o n s i s t of o l i g o s a c c h a r i d e r e p e a t i n g u n i t s which determine the s e r o l o g i c a l grouping of the b a c t e r i a l s t r a i n . The 0-antigen re g i o n i s synthesized separately from the r e s t of the l i p o p o l y s a c c h a r i d e molecule and i s then t r a n s f e r r e d to a completed core. The core region i s made by s e q u e n t i a l a d d i t i o n of sugars. A s e r i e s of mutants of E. c o l i and Salmonella have been i s o l a t e d i n which the l i p o p o l y s a c c h a r i d e i s p r o g r e s s i v e l y more d e f e c t i v e i n the core region ( F i g . 3)- The s e r i e s progresses from smooth s t r a i n s which contain 0-antigen side chains to deep-rough mutants which contain only the l i p i d A p o r t i o n of the l i p o p o l y s a c c h a r i d e . The l i p i d A p o r t i o n of 6 Rho •Gal—/-•Man 0 side chain R core 1 Core oligosaccharide Lipid A : ; II — I PorP-EIN KDO—P-EtN (Fatly acids)6 "n, I. r ® •Hep.—'•Hep,-h» KDO "I ' j I I. . • KDO 0GlcN GlcN — ® Re LPS Rd2 LPS Rbj LPS Rb2 LPS Rb| LPS Ro LPS F i g . 3 . Structure of S_. typhimurium LPS. This structure shows a basic "monomer" unit, which i s presumably cross-linked with other units. Abbreviations: Abe, abequose; Man, D-mannose; Rha, L-rhamnose; Gal, D-galactose; GlcNAc, N-acetyl-D-glucosamine; Glc, D-glucose; Hep, L-glycero-D-manno-heptose; KDO, 3-deoxy-D-manno-octulosonic acid; EtN, ethanolamine; Ac, acetyl. To some sugar residues i n the core oligosaccharide region roman numeral subscripts have been added i n order to d i f f e r e n t i a t e one from the other. A l l sugar residues are <x.-anomers except where JS-conformation i s s p e c i f i e d . The biosynthesis of LPS starts at the l i p i d A portion and the core oligosaccharide i s elongated toward the " l e f t " ( i . e . , non-reducing end) i n th i s scheme. Thus, mutants defective at various stages of core oligosaccharide produce incomplete LPS; the structure of these LPS molecules and t h e i r "chemotype" are indicated by dotted l i n e s . Taken from Nikaido ( 3 8 ) . 7 the lipopolysaccharide i s essential since no mutants lacking t h i s region have been is o l a t e d (38). L i p i d A may be required for s t a b i l i z a t i o n of the outer membrane since phosphatidyl ethanol-amine, the predominant phospholipid of the outer membrane does not form vesicles r e a d i l y i n aqueous solutions (59,60). Mutants defective i n the core region are more sensitive to a n t i b i o t i c s , dyes and detergents (5,38,61-65) suggesting that the lipopolysac-charide i s involved i n the barr i e r function of the outer membrane. The hydrophobic l i p i d A portion i s embedded i n the membrane contributing, along with the phospholipids, to a central hydrophobic domain (47-49). The lipopolysaccharide i s d i s -tributed primarily on the outer surface of the membrane with the carbohydrate chains extending into the medium (45,68). The lipopolysaccharide molecules are l a t e r a l l y mobile, although t h e i r freedom of tr a n s l a t i o n i s lower than that of the phospholipids (50). This l a t e r a l mobility could account for the equilibrium between EDTA-extractable and non-extractable lipopolysac-charide (5) • Phospholipids The phospholipids of the outer membrane consist primarily of phosphatidyl ethanolamine (8l%) with small amounts of phosphatidyl glycerol (17%) and c a r d i o l i p i n (2%). The l e v e l of c a r d i o l i p i n i n c e l l s has been shown to increase upon change of growth phase from exponential to stationary (112). The fat t y acids of the phospholipids are sensitive to growth temperature, 8 becoming more saturated w i t h higher growth temperatures ( 1 1 3 ) . A phos p h o l i p i d b i l a y e r may not be the ba s i s of the outer membrane since only 25-40% of the phospholipids of the outer membrane take part i n phase t r a n s i t i o n s compared wi t h 60-80$ f o r the cytoplasmic membrane ( 1 5 9 ) . In a d d i t i o n , i t has been sug-gested that the phospholipids are d i s t r i b u t e d p r i m a r i l y on the inner l e a f l e t of the b i l a y e r , w i t h the outer surface being composed of mainly l i p o p o l y s a c c h a r i d e ( 1 6 0 ) . This i s supported by the f i n d i n g that i n t a c t c e l l s are r e s i s t a n t to p.hospholipases A2 and C unless the l i p o p o l y s a c c h a r i d e i s removed by EDTA extrac-t i o n ( 1 1 1 ) . Phospholipid has al s o been shown not to be req u i r e d f o r the s t r u c t u r a l i n t e g r e t y of the outer membrane (131) Outer membrane p r o t e i n s R e s o l u t i o n of the p r o t e i n s of the outer membrane of E. c o l i s t r a i n NRC 482 by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s r e v e a l s a l i m i t e d number of p r o t e i n bands ( F i g . 4 ) . F i g . 4 i l l u s t r a t e s the unusual behaviour that these p r o t e i n s e x h i b i t on SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . As seen i n g e l 2 , p r o t e i n s A]_, A 2, and B are not resolved on SDS-polyacryamide gels run w i t h phosphate b u f f e r (90) at a n e u t r a l pH (System 1) ( 1 2 , 1 3 , 7 9 , 8 1 , 8 2 , 8 8 ) . On the basis of t h i s observation i t was suggested t h a t the outer membrane of gram-negative b a c t e r i a i s composed of a s i n g l e major p r o t e i n w i t h a s t r u c t u r a l r o l e ( 1 2 , 1 3 , 8 9 ) . SDS-polyacrylamide gels run at a l k a l i n e pH (system 2) (79) r e s o l v e d the major p r o t e i n of the outer membrane of E. c o l i NRC 482 i n t o three bands, p r o t e i n A]_ (MW 4 4 , 0 0 0 ) , p r o t e i n A 2 (MW 3 8 , 1 0 0 ) , and p r o t e i n B (MW 33,400) as seen i n g e l 4 . c 3 Cl C2 C 4 D1 D2 E A2 1 SYSTEM F i g . 4a. E l e c t r o p h o r e s i s on 10$ polyacrylamide g e l s of pr o t e i n s of the E. c o l i outer membrane. The ge l s from l e f t to r i g h t are (1) membrane d i s s o l v e d i n dodecyl s u l f a t e - u r e a --mercaptoethanol at 37°C; (2) s o l u t i o n subsequently heated at 100°C; (3) same as (1); (4) same as (2). Electropho-r e s i s was c a r r i e d out with System 1 f o r gels (1) and ( 2 ) , and with System 2 f o r gels (3) and (4). In system 1, both b u f f e r compartments contained 0.1$ SDS i n 0.1M sodium phosphate b u f f e r , pH 7•2. In system 2, the cathodic b u f f e r compartment contained 0.1$ SDS i n 0. IM Na 2 HPOjj, pH 11.4, while the anodic b u f f e r contained 0.1$ SDS i n 0.1M NaH^PO^pH 4.1. Taken from Bragg and Hou (19). F i g . 4b. Schematic diagram of separation of outer membrane pro t e i n s of E. c o l i on 10$ polyacrylamide g e l s u s i n g Systems 1 and 2 are shown. The samples i n dodecyl s u l f a t e - u r e a - -mercaptoethanol had been heated to 100°C. 10 Similar results have been obtained with other strains of E. c o l i (88,91). Further resolving power i s obtained through the use of t h i n SDS-polyacrylamide gel slabs run with a discontinuous buffer system (83,92). Lutenberg et a l . (114) have resolved the major outer membrane proteins of E. c o l i K12 into four closely-spaced bands using a modified Laemmli system. Applica-t i o n of two dimensional gel systems (93-97) to the separation of outer membrane proteins provides a highly-resolving and sensitive method for the detection of a l l polypeptide components of the outer membrane. The widespread adoption of a two dimensional system such as that devised by O'Farrell (94), and adapted by Ames ( 9 7 ) 3 for use with membrane proteins should greatly f a c i l i -tate the comparison of results from dif f e r e n t laboratories. Confusion as to the number and molecular weights of the outer membrane proteins had not only arisen by the use of diffe r e n t SDS-polyacryamide gel systems but i t was found that the preparative conditions affected t h e i r migration on SDS-gels (79-85). The temperature and time of heating was found to pro-foundly a l t e r the pattern seen i n SDS-gels. I f the outer membrane i s s o l u b i l i z e d at 37°C and the proteins resolved on SDS-polyacrylamide gels the pattern seen i n F i g . 4, gels 1 and 3 are found. I f the sample i s heated at 100°C p r i o r to el e c t r o -phoresis the patterns i n gels 2 and 4 are seen. Protein B changes i t s apparent molecular weight from 28,500 to 33,400 upon heating. This behaviour has been designated as heat-modifiable (79). Secondly, high molecular weight aggregates of protein A are broken down into lower molecular weight species. The proteins of the outer membrane also vary greatly i n 11 t h e i r a b i l i t y to be s o l u b i l i z e d from the c e l l wall. It has already been noted that, neither Triton X-100 i n the presence of magnesium ions nor sodium l a u r y l sarcosinate could s o l u b i l i z e the proteins of the outer membrane. Extraction of the c e l l wall with Triton X-100 i n the presence of EDTA released about half of the outer membrane,protein (67). Protein B i s readily s o l u b i l i z e d from the c e l l wall by SDS at 37°C. The protein migrates as a band with an apparent molecular weight of 28,500 on SDS-polyacrylamide gels u n t i l heated at 100°C (79,81,87). Protein A i s not extracted by SDS at temperatures less than 60°C (79,80) and remains associated with the peptidoglycan. Extracts made at low temperatures w i l l therefore not contain t h i s protein (79,83,86). The tight bind-ing of protein A to the peptidoglycan has been useful i n i t s p u r i f i c a t i o n (80). Extraction of the c e l l wall at 70°C with SDS for 20 min (82) releases protein A i n the form of oligomers (8l) that have not bound t h e i r f u l l complement of SDS (80). The protein migrates a t y p i c a l l y i n SDS-polyacrylamide gels as a broad band with a molecular weight of about 60,000 (80-82). Heating the extract to 100°C res u l t s i n further SDS binding and the protein now migrates as a sharp band with a molecular weight of 36,500 (80,81). A l l the proteins of the outer membrane except the bound li p o p r o t e i n are s o l u b i l i z e d by SDS at 100°C. The l i p o p r o t e i n i s covalently linked to the peptidoglycan and i s not released u n t i l the peptidoglycan i s treated with t r y p s i n or lysozyme (44). Different nomenclatures have been applied to the proteins 12 of the outer membrane by di f f e r e n t research groups on the basis of the migration of the proteins i n SDS-polyacrylamide gels. The l i k e l y relationships of the proteins studied by other workers to the nomenclature used i n t h i s thesis are given i n Table I. Comparisons are d i f f i c u l t to make because of the d i f f e r e n t SDS-polyacrylamide gel systems i n use, and the effects of preparative conditions on the migration of the proteins. Also, the corres-ponding proteins may not be i d e n t i c a l due to differences i n st r a i n or culture conditions. Properties of the Outer Membrane Proteins Bound lip o p r o t e i n The protein responsible for the attachment of the outer membrane to the peptidoglycan i s a lipoprotein (Pig. 2) which has been extensively characterized by Braun and his coworkers (4,44). There are 250,000 copies of this protein per c e l l . It i s covalently attached to the peptidoglycan (120). The £-amino group of the C-terminal lysine residue i s linked to about every tenth diaminopimelate residue of the peptidoglycan by an amide bond (118,119). The lipoprotein i s released from the peptido-glycan by pr o t e o l y t i c digestion (117) or by lysozyme treatment (121). L i p i d i s attached to the N-terminal cysteine residue of the protein (117,119,122). Two molecules of fatty acid are e s t e r i f i e d to the gl y c e r y l side chain, while the amino group of the cysteine residue i s blocked by a fat t y acid linked by an amide bond. The ester-linked fatty acids have a composition T a b l e I N o m e n c l a t u r e s f o r t h e o u t e r membrane p r o t e i n s o f E. c o l i B r a g g and S c h n a i t m a n Henning Inouye L u t e n b e r g Hou (79) (88) and H a l l e r (120) e t a l (98) (1147 Koplow and C h a i and G o l d f i n e P o u l d s (177) (154) M a t r i x p r o t e i n (80) A. A, H e a t - m o d i f i a b l e B/B* p r o t e i n (79) F r e e l i p o p r o t e i n F (120) 3a 3b I a l b I I * IV peak 4 b c peaks 6/7 d peak 11 B C/D G 14 s i m i l a r to those of the c e l l u l a r phospholipids (45% p a l m i t i c a c i d , 11% p a l m i t o l e i c a c i d , 12% 9,10 methylenehexadecanoic a c i d , 8% 11,12 methyleneoctadecanoic a c i d and 24% c i s - v a c c e n i c a c i d ) . The amide-linked f a t t y acids are mainly p a l m i t i c a c i d (65%), p a l m i t o l e i c a c i d (11%) and c i s - v a c c e n i c a c i d (11%). The l i p o p r o t e i n has a molecular weight of about 7,000 and i t s complete s t r u c t u r e has been determined ( F i g . 2). The amino a c i d composition i s unique i n that the p r o t e i n from E. c o l i l a c k s h i s t i d i n e , p r o l i n e , g l y c i n e , phenylalanine and trytophan. C i r c u l a r dichroism studies showed that the p r o t e i n contains about 80% ^ - h e l i c a l s t r u c t u r e (44,124). The s t r u c t u r e was r e -gained a f t e r heat denaturation when the p r o t e i n , as a s o l u t i o n i n SDS, was cooled from 100°C as shown by c i r c u l a r dichroism measurements and antibody binding (44,123). The unusual r e s i s -tance of the l i p o p r o t e i n to d i g e s t i o n by t r y p s i n can be a t -t r i b u t e d to i t s high tx.-helical content (44). The amino a c i d sequence of the p r o t e i n revealed that when the p r o t e i n i s arranged i n an © c - h e l i c a l conformation, a l l the hydrophobic amino acids are al i g n e d on one side of the h e l i x (44). The aggregation of the l i p o p r o t e i n i n aqueous s o l u t i o n s may be due to the i n t e r a c t i o n of these hydrophobic regions. An a d d i t i o n a l feature of the l i p o p r o t e i n i s the r e p e t i t i v e nature of the sequence. This suggests that the gene coding f o r the l i p o p r o t e i n has a r i s e n by gene d u p l i c a t i o n (44). Free l i p o p r o t e i n Inouye has found that about two-thirds of the l i p o p r o t e i n 15 e x i s t s unattached to the peptidoglycan i n the c e l l envelope of E. c o l i (125-127). The covalently-bound l i p o p r o t e i n , released by lysozyme d i g e s t i o n could be d i s t i n g u i s h e d from the fre e form since i t contained two repeating u n i t s of peptidoglycan and there f o r e migrated more slowly i n SDS-polyacrylamide gels (120). The free form, which i s loc a t e d i n the outer membrane (128), contains a s i m i l a r composition of f a t t y acids as the bound form and has a very high o t - h e l i c a l content (127,129,271). The fr e e form of the l i p o p r o t e i n has r e c e n t l y been c r y s t a l -l i z e d i n t o s e v e r a l d i f f e r e n t forms (271,272). The c r y s t a l s are r e a d i l y formed by acetone p r e c i p i t a t i o n of the p r o t e i n from SDS s o l u t i o n s , probably due to the i n t e r a c t i o n of the hydrophobic regions of the molecule. The p r o t e i n forms a r i g i d ° c-helix, 87 A i n l e n g t h , with the f a t t y acids extending a f u r t h e r 21 A from one end of the molecule. Other gram-negative b a c t e r i a such as Salmonella typhimurium and S e r r a t i a marcesens have both free and bound forms of the l i p o p r o t e i n (44,130,132). Proteus m i r a b i l i s l a c k s both forms of t h i s p r o t e i n (44,130) during the exponential phase of growth. However, a bound l i p o p r o t e i n appears i n the s t a t i o n a r y phase of growth (267). Mat r i x p r o t e i n ( p r o t e i n A) Several groups have p u r i f i e d and c h a r a c t e r i z e d a major p r o t e i n of the outer membrane of E. c o l i which i s non-covalently bound to the peptidoglycan (80,8l,87,88,91,140-142,148,149). 5 There are approximately 1.5 X 10 molecules of p r o t e i n A i n a c e l l 16 arranged hexagonally to cover about half of the outer surface of the peptidoglycan (80). The protein i s not extracted by SDS at temperatures up to 60°C, but i s readily s o l u b i l i z e d at 100°C. This protein does not bind SDS u n t i l heated at temperatures above 60°C, accounting i n part for i t s a t y p i c a l migration on SDS-poly-acrylamide gels. The protein i s o l a t e d by Rosenbusch (80) from E. c o l i B was a single polypeptide (probably equivalent to protein A^) with a molecular weight of 36,500 and an N-terminal sequence: Ala-X-Tyr-Asx-His-Lys-Glx-Other E. c o l i strains may contain multiple forms of t h i s protein (87,91,140-142). Schnaitman has observed differences i n the major outer membrane proteins from different strains of E. c o l i (91). In addition to the matrix protein, 1, some strains contained another protein, 2 (probably k^), that migrated s l i g h -t l y faster i n SDS-polyacryiiamide gels run at alkaline pH. In strains of E. c o l i non-lysogenic for phage PA 2, protein 1 i s a predominant protein of the outer membrane (116). In cultures lysogenic for this phage, the amount of protein 1 i s reduced and protein 2 i s made i n i t s place. Protein 1 i s proposed to be the receptor for t h i s phage and protein 2 i s made to prevent superinfection (116). The synthesis of protein 2 i s also under catabolite repression (91) since c e l l s grown on carbon sources other than glucose, or i n the presence of cAMP, contain increased levels of protein 2, with a decrease i n protein 1. Both proteins 1 and 2, before and after heating, were eluted together from a column of Sephadex G-200, equilibrated with 0.1% SDS (81,88,148). Ion exchange chromatography of 17 T r i t o n X-100 e x t r a c t s c o n t a i n i n g p r o t e i n aggregates r e s u l t e d i n separation of p r o t e i n s 1 and 2 (88). A n a l y s i s of the p u r i f i e d p r o t e i n s by cyanogen bromide cleavage revealed a spectrum of peptides i n c l u d i n g p a r t i a l cleavage products. On the b a s i s of these experiments, Schnaitman concluded that p r o t e i n s 1 and 2 are d i f f e r e n t polypeptides (88). Henning's group has a l s o found that the matrix p r o t e i n from d i f f e r e n t E. c o l i s t r a i n s i s composed of m u l t i p l e polypeptide species (87,140-142). They found that the matrix p r o t e i n could be separated by e l e c t r o p h o r e s i s i n SDS-polyacrylamide gels i n t o two components, l a and l b (142). Both p r o t e i n s could be f u r t h e r separated i n t o a number of i s o e l e c t r i c species. Cyanogen bromide cleavage patterns revealed that p r o t e i n s l a and l b were es s e n t i - a l l y the same polypeptide w i t h a m o d i f i c a t i o n i n one peptide only. The p o s s i b l e correspondence of the matrix p r o t e i n s from d i f f e r e n t E. c o l i s t r a i n s has been summarized i n Table I . The matrix p r o t e i n s i s o l a t e d by Henning's group "(87,149) had a s i m i l a r amino a c i d composition to the p r o t e i n i s o l a t e d by Rosenbusch (80). The p r o t e i n i s only moderately hydrophobic and none of the cyanogen bromide fragments e x h i b i t a low p o l a r i t y ( l 4 l ) . The matrix p r o t e i n may not be i n v o l v e d i n hydrophobic i n t e r a c t i o n s i n the membrane i n contrast to membrane p r o t e i n s i s o l a t e d from other organisms (143-146). The matrix p r o t e i n has a high content of j3-structure (80) and may be r e s p o n s i b l e f o r the j2 - s t r u c t u r e seen i n the i n t a c t outer membrane (138). The ft - s t r u c t u r e of the outer membrane 18 disappears upon heating i n SDS (139) due to denaturation of the proteins by the detergent. Protein A i s not digested when c e l l envelope, c e l l wall, outer membrane or the protein A - peptidoglycan complex are treated with p r o t e o l y t i c enzymes (79,80,131,135-137). The protein when s o l u b i l i z e d i s completely degraded to small peptides by trypsin or pronase (79,80) showing that i t i s not i n t r i n s i c -a l l y resistant to digestion. The i n s e n s i t i v i t y to proteoly t i c digestion must therefore be due to the i n a c c e s s i b i l i t y of suitable peptide bonds to the proteases as a result of protein fol d i n g and protein-protein interactions. Mutants missing the matrix protein have been i s o l a t e d . Schnaitman has reported that a multiple phage-resistant mutant of E. c o l i (T4,T5,T6,T7,X) lacked the matrix protein (91). There was an increase in the amount of protein B i n this mutant, such that the phospholipid to protein r a t i o of the outer membrane was the same as i n the parent. This compensation of one protein for another i s also seen when growth conditions are altered (91). In contrast, Hancock and Reeves (103) found that a lipopolysac-charide-defective mutant of E. c o l i had a decreased amount of protein A but had a normal amount of protein B . Mutants of E. c o l i resistant to phage Tul were isolated by Henning's group (142). The mutants either lacked both proteins l a and lb or lb only, suggesting that protein lb i s the receptor for phage Tul. Heraning's group has also i s o l a t e d a mutant missing a l l the major outer membrane proteins, A^, A^ and B (98). Surprisingly, no serious defects a f f e c t i n g the i n t e g r i t y of the 19 outer membrane were observed. In a d d i t i o n , l o s s of i n d i v i d u a l major outer membrane p r o t e i n s d i d not change the f r a c t u r e faces of the outer membrane (104). The r e s u l t s obtained w i t h these mutants suggest that p r o t e i n A i s not r e q u i r e d f o r the i n t e g r i t y and perhaps normal f u n c t i o n i n g of the outer membrane. Why E. c o l i contains such high amounts of t h i s p r o t e i n i n the outer membrane i s not clear.. However, deep-rough mutants of E. c o l i and Salmonella, which lack p r o t e i n s A and B have a s e r i o u s l y d i s r u p t e d outer membrane, both morphologically and f u n c t i o n a l l y . These mutants w i l l be d i s -cussed i n the s e c t i o n concerning the b a r r i e r f u n c t i o n of the outer membrane. Heat-modifiable p r o t e i n ( p r o t e i n B) P r o t e i n B i s a major outer membrane p r o t e i n which changes i t s apparent molecular weight from 28,500 to 33,400 upon heating i n SDS-containing s o l u t i o n s (79). The same change was observed by Schnaitman f o r h i s p r o t e i n 3 (88) and Henning's group f o r t h e i r p r o t e i n I I * (87). The cause of t h i s phenomenum has been i n v e s -t i g a t e d by Schnaitman (81). He has found that there was an increase i n the i n t r i n s i c v i s c o s i t y of the p r o t e i n on heating i n SDS s o l u t i o n s . He suggested that only a f t e r heating does the p r o t e i n u n f o l d to form the r i g i d rod conformation proposed by Reynolds and Tanford (152) f o r protein-SDS complexes. I t has been shown that the heated form, B* migrates t y p i c a l l y on SDS-polyacrylamide gels (131,148) supporting Schnaitman's suggestion. Further physiochemical analyses of p r o t e i n B and the heat-20 modified B* , described i n t h i s t h e s i s , also support Schnaitman's view that the p r o t e i n unfolds upon heating. P r o t e i n B has been p u r i f i e d by a number of workers and some of i t s p r o p e r t i e s have been st u d i e d (81,87,88,110). Amino a c i d a n a l y s i s has revealed that the p r o t e i n has a molecular weight of 27,000 and i s only moderately hydrophobic (87). The p u r i f i e d p r o t e i n from E. c o l i K12 contains f i v e methionine residues and the cyanogen bromide cleavage products can be separated i n t o f i v e bands on SDS-polyacrylamide g e l s . The sum of the molecular weights of these bands i s 27,000, the same as determined by amino a c i d a n a l y s i s and close to the molecular weight of the unheated form. The true molecular weight of p r o t e i n B i s unknown. Values based on SDS-polyacrylamide g e l e l e c t r o p h o r e s i s are subject to e r r o r (204). A l s o , c a l c u l a t i o n of the minimum molecular weight by amino a c i d a n a l y s i s based on a s i n g l e amino a c i d i s not r e l i a b l e . This Is e s p e c i a l l y true of c y s t e i n e which can only be r e a d i l y detected i n hydrolysates of p r o t e i n B as c y s t e i c a c i d and i s present i n very low amounts (87). Obviously, f u r t h e r studies on t h i s p r o t e i n , p r e f e r a b l y i n d i s s o c i a t e d form, fr e e of detergent, are r e q u i r e d before the true molecular weight of t h i s p r o t e i n can be e s t a b l i s h e d with c e r t a i n t y . P r o t e i n B from E. c o l i K12 has an N-terminal alan i n e r e s i -due (87). However, three a d d i t i o n a l Edman cyc l e s d i d not release any f u r t h e r amino a c i d d e r i v a t i v e s . Henning b e l i e v e s he i s d e a l -i n g w i t h a s i n g l e polypeptide since mutants r e s i s t a n t to phage TuII* e i t h e r l a c k p r o t e i n I I or produce a p r o t e i n of a l t e r e d e l e c t r o p h o r e t i c m o b i l i t y (87,153). In contrast Schnaitman's 21 work suggests that some s t r a i n s of E. c o l i contain two s i m i l a r heat-modifiable p r o t e i n s (83). He was able to separate p r o t e i n 3 prepared from E. c o l l J-5 grown on succinate-minimal media i n t o two s p e c i e s , 3a and 3b by ion-exchange chromatography i n the presence of T r i t o n X-100 and EDTA. The pr o t e i n s gave d i f f e r e n t peptides on cyanogen bromide cleavage. The d i f f e r e n c e between Schnaitman's r e s u l t s and those of Henning may be due to the d i f f e r e n c e s i n the s t r a i n s used or growth c o n d i t i o n s as i s the case f o r p r o t e i n A. Chai and Foulds (154) have found that E. c o l i mutants ( t o l G) t o l e r a n t (157) to the b a c t e r i o c i n JF 246 lack the heat-modifiable p r o t e i n B. Ames and Nikaido (97) have confirmed t h i s by two-dimensional e l e c t r o p h o r e s i s . These mutants show an increased s e n s i t i v i t y to a n t i b i o t i c s , detergents and dyes, suggesting that the i n t e g r i t y of the c e l l envelope has been a l t e r e d . This i s i n contrast t o the r e s u l t s obtained by Henning (98) on a mutant of E. c o l i l a c k i n g both p r o t e i n s A and B which had normal r e s i s t a n c e to these agents. I t has been proposed that t o l G ( t u t ) i s the s t r u c t u r a l gene f o r t h i s p r o t e i n (153). The heat-modifiable p r o t e i n i s absent i n a mutant of E. c o l i d e f e c t i v e i n conjugation (105). This mutant i s al s o r e s i s t a n t to phage K3 and th e r e f o r e p r o t e i n B may provide the attachment s i t e f o r t h i s phage, as w e l l as f o r the p i l u s . That these are the only r o l e s of p r o t e i n B i s improbable since t h i s p r o t e i n i s present i n large amounts i n the outer membrane. Other major outer membrane p r o t e i n s Henning has i s o l a t e d a p r o t e i n ( I I I ) w i t h a molecular 22 weight of 17j000 which i s present i n major amounts i n the outer membrane (8l , l49). Only preliminary characterization has been performed. However, i t s i d e n t i t y with a lipopolysaccharide-protein i s o l a t e d by Wu and Heath (158) has been ruled out. Moldow e_t a l . (106) have p u r i f i e d some outer membrane proteins but they have not been characterized except by SDS-polyacrylamide gel electrophoresis. The outer membrane of gram-negative bacteria contains many proteins i n addition to those present i n major amounts (83,97). The only enzyme l o c a l i z e d to the outer membrane i s phospholipase A (285). Other proteins have been p u r i f i e d and th e i r function elucidated. These w i l l be discus-sed i n the following sections. Functions of the Outer Membrane Proteins Role of proteins i n the ba r r i e r function of the outer membrane. The c e l l wall of gram-negative bacteria provides a b a r r i e r against the penetration of a n t i b i o t i c s , dyes, detergents and other components (5,161-163). It also prevents the loss of proteins from the periplasmic space, the zone bound by the cyto-plasmic membrane and the outer membrane (164). The peptidogly-can alone does not act as a permeability b a r r i e r (282) suggesting that the outer membrane i s the b a r r i e r . The outer membrane must be s e l e c t i v e , allowing the passage of nutrients while excluding toxic compounds. Selection i s made on the basis of molecular si z e . Thus, pentalysine cannot penetrate, i n contrast to t e t r a l y s i n e and smaller peptides (170). The outer membrane i s impermeable to oligosaccharides with molecular weights 23 greater than 1,000 (173,282). In contrast to phospholipid b i l a y e r s , the outer membrane of wild-type c e l l s i s impermeable to hydrophobic compounds (107). Treatment of c e l l s with EDTA disrupts the ba r r i e r function, increasing the permeability of the outer membrane to quaternary ammonium compounds (165), lysozyme (166), actinomycin and other drugs (163,167)- EDTA chelates magnesium and calcium ions es s e n t i a l for the i n t e g r i t y of the outer membrane, causing re-lease of from one t h i r d to one half of the lipopolysaccharide (167,168). Rough mutants defective i n t h e i r lipopolysaccaride are more sensitive to dyes, detergents, lysozyme, and antibio-t i c s , and leak periplasmic enzymes (5,174-176,179). Although the change i n permeability has been related to the altered LPS structure, recent r e s u l t s have suggested that outer membrane proteins are also involved (86,177-179). Ames, Spudich and Nikaido (86), and Parton (178) found that heptose-deficient mutants of Salmonella had decreased amounts of the major outer membrane proteins, A-j_, A^ and B. Koplow and Goldfine (177) found a similar effect i n heptose-deficient mutants of E. c o l i where the phospholipid to protein r a t i o was increased 2.4 f o l d . These results d i f f e r from those obtained by Hancock and Reeves (103) and Wu (108) i n which defects i n the lipopolysaccharide were accompanied by a decrease i n a single major outer membrane protein. Parton (178) correlated the defects i n lipopolysaccaride with loss of outer membrane proteins. He found that a decrease i n the amount of major outer membrane proteins occurred between Rb and Rc mutants (Fig. 3). 24 Not only are the major outer membrane proteins greatly decreased i n heptose-deficient mutants, but the l e v e l of minor proteins such as the receptor for lambda phage was found to be decreased (180). This suggests that the defect i s not i n the assembly of a complex of lipopolysaccharide with the major outer membrane protein but perhaps r e f l e c t s the role of lipopoly-saccharide i n s t a b i l i z i n g the outer membrane (180). The proteins lost were not found i n the medium and so were not ex-creted (86). Hancock and Reeves (103) however concluded from t h e i r studies i n a heptose-deficient mutant that contained normal amounts of the major outer membrane protein that "lipopolysac-charide core i s not required for the presence of major outer membrane proteins i n normal amounts." The relationship between the lipopolysaccaride and major outer membrane proteins i s there-fore s t i l l undefined and further work on t h i s aspect of the outer membrane i s required. The effect of the deep-rough mutations on membrane structure has also been studied by electron microscopy and freeze-etching (181-183). A more d i s t i n c t double-track appearance of the outer membrane was observed and a stronger cleavage occurred i n the outer membrane suggesting that deep-rough mutants have a more hydrophobic zone i n t h e i r membranes then do smooth st r a i n s . Nikaido (182) suggests that i n wild-type c e l l s most of the phos-pholipid i s on the inner l e a f l e t of the b i l a y e r while the lipopolysaccharide occupies the outer surface (68,160). Loss of proteins results i n an increase i n the amount of phospholipid which now i s di s t r i b u t e d on both the inner and outer l e a f l e t of 25 the b i l a y e r (160,182). This would provide a more hydrophobic domain which would be more readi l y cleaved during freeze-etching, and would also allow the penetration of hydrophobic compounds such as a n t i b i o t i c s (107). Reconstitution studies have shown that the permeability properties of the outer membrane are due primarily to the protein components. Vesicles composed of phospholipids and lipopolysaccharide are not permeable to hydrophilic molecules as small as sucrose (173). However, addition of outer membrane proteins (172) or a complex of protein A (134), but not of the lipo p r o t e i n , to lipopolysaccharide/phospholipid vesicles resto-red the permeability properties found i n the native membrane. This suggests that protein A forms channels through the outer membrane and i s responsible for the penetration of small hydro-p h i l i c molecules through the outer membrane. Role of Outer membrane proteins i n maintaining the shape of the organism Some proteins of the outer membrane are present i n major amounts and therefore could play a st r u c t u r a l role (12,13,80,131, 184,185). Presumably t h i s would involve a repeating or regula-rly-spaced arrangement of the proteins. Protein A i s arranged i n an ordered manner on the peptidoglycan (80) and the bound li p o p r o t e i n i s also attached to the peptidoglycan regularly at about every tenth repeating disaccharide (44). There i s some evidence to suggest that the major outer membrane proteins do help maintain the shape of the bacterium. 26 Hemming's group has been able to i s o l a t e ghosts, free of peptidoglycan and cytoplasmic components, that s t i l l r e t a i n the shape of the organism (131,136,149,187). The ghosts contain phospholipid (25$), lipopolysaccharide (25-30$) and four proteins (45-50$), proteins I, I I , I I I , and IV (Table I ) . These proteins could form a repeating oligomeric structure responsible for c e l l shape, and crosslinking studies have confirmed that extensive protein-protein interactions do occur i n the outer membrane (137,186). Moreover, removal of phospholipid from the ghost membrane did not a l t e r i t s unit membrane appearence which sug-gests that a phospholipid b i l a y e r may not be the basis for thi s membrane (131). -There i s some evidence against the proposition that the proteins of the outer membrane maintain the shape of the orga-nism. A temperature sensitive shape mutant of E. c o l i contained a normal complement of outer membrane proteins but was missing a single protein of the cytoplasmic membrane (191). In addition, a mutant of E. c o l i missing a l l the major outer mem-brane proteins had a normal morphology (98). Proteus m i r a b i l i s i s rod-shaped but lacks both the bound and free forms of the lipoprotein during the exponential phase of growth (132). Obviously, the major outer membrane proteins are not the sole agents responsible for maintaining c e l l shape. Role of the outer membrane proteins i n DNA r e p l i c a t i o n The c e l l needs to coordinate the processes of DNA r e p l i c a t i o n and c e l l envelope growth i n order for c e l l d i v i s i o n to take place 27 normally. Changes i n several outer membrane proteins have been related to alterations i n DNA synthesis. Inouye (209,210) has found that a protein found i n the outer membrane of E. c o l i he c a l l s Y protein with molecular weight of 34,000 decreased i n amount whenever DNA synthesis was in h i b i t e d by n a l i d i x i c acid or thymine starvation. Another outer membrane protein with a molecular weight of 15,000 was o'verproduced under these con-ditions (211). The rate of appearance of t h i s protein was decreased by i n h i b i t i n g c e l l elongation. James has proposed that t h i s protein i s a st r u c t u r a l protein of c e l l elongation (211). It i s not known i f these changes are primary effects of the i n h i b i t i o n of DNA synthesis. The best evidence for the role of an outer membrane protein i n DNA r e p l i c a t i o n comes from the work of Gudas, James and Pardee (212). A protein which the authors c a l l protein D (M.W. 80,000) was incorporated into the outer membrane of synchronous cultures of E. c o l i towards the end of the c e l l cycle (212). This protein i s similar to one described by Churchward and Holland (213). I n h i b i t i o n of DNA synthesis delayed the appear-ance of this protein. Upon removal of the i n h i b i t i o n , protein D was synthesized, followed by a burst of DNA synthesis. Disrup-t i o n of peptidoglycan synthesis by the amidinopenicillamic acid, PL 1060 also increased the amount of thi s protein. Protein D bound to DNA i n v i t r o . The authors suggested that t h i s protein acts as an attachment s i t e for DNA to the c e l l envelope and provides a metabolic l i n k between peptidoglycan synthesis, protein synthesis and DNA i n i t i a t i o n . 28 Involvement of outer membrane p r o t e i n s i n high a f f i n i t y t r a n s p o r t systems Iron t r a n s p o r t : E. c o l i t r a n s p o r t s f e r r i c i o n as a complex wi t h c i t r a t e (214), e n t e r o c h e l i n (215) or ferrichrome (216,217). Outer membrane p r o t e i n s appear t o be i n v o l v e d i n the l a s t two systems. Hantke and Braun (218) have shown that t r a n s p o r t of i r o n by the ferrichrome system i s dependant on the ton A gene product. This gene locus was o r i g i n a l l y c l a s s i f i e d as a phage T5 receptor (219). Braun and coworkers have i s o l a t e d the r e -ceptor f o r phage T5 and have shown that t h i s p r o t e i n (M.W. 85,000), l o c a t e d i n the outer membrane, i s a l s o the bin d i n g s i t e of c o l i c i n M (220-222). Absorption of phage$80 to c e l l s was i n h i b i t e d by ferrichrome (102,224) suggesting that f e r r i c h r o m e , phages T5 and 1 80 and c o l i c i n M a l l bind to the same receptor i n the outer membrane. B a c t e r i a with a reduced a b i l i t y to u t i l i z e i r o n contained an increased l e v e l of c o l i c i n Ia receptor i n the outer membrane (277) suggesting a r o l e f o r t h i s receptor i n i r o n uptake. Enteroche-l i n , and c o l i c i n s B and D l i k e l y bind to the c o l i c i n Ia receptor (279)- Mutants r e s i s t a n t to c o l i c i n s B and I map at the ton B locus and overproduce e n t e r o c h e l i n (225,226). The r e s i s t a n c e to c o l i c i n s i s not due to competition of the excreted e n t e r o c h e l i n w i t h the c o l i c i n f o r the receptor since ton B mutants unable to synthesize e n t e r o c h e l i n (aro E) remain c o l i c i n t o l e r a n t (218). The ton B mutants are al s o d e f e c t i v e i n $ 80 i n f e c t i o n (224) and lack an outer membrane p r o t e i n necessary f o r the absorption of 80 and the uptake of e n t e r o c h e l i n . The c o l i c i n I a receptor has 29 been characterized as an outer membrane protein complex with a molecular weight of 307,000 (283). Growth of c e l l s on i r o n -deprived media results i n an increased l e v e l of two proteins (M. W. 95,000 and 85,000) of the outer membrane (100,101,268,278). These proteins may be the ton A and ton B gene products. Vitamin B12: The f i r s t step i n the transport of vitamin B12 i n E. c o l i i s binding to a receptor i n the outer membrane (227), which also acts as the binding s i t e for c o l i c i n s E^ and E^ (228) and bacteriophage BP 23 (229). The c o l i c i n E^ receptor from E. c o l i has been p u r i f i e d and characterized as a protein with a molecular weight of 60,000 (230). Maltose: The product of the lam B gene i n E. c o l i i s the receptor for bacteriophage X (231). It i s an outer membrane protein with a molecular weight of 55,000. The lam B-mutants are phage X - r e s i s t a n t and are unable to use maltose when thi s sugar i s present i n l i m i t i n g amounts (232). In addition, chemo-taxis towards maltose i s impaired i n these mutants (233) suggest-ing that the phage X receptor i s involved i n maltose transport. Organization of proteins i n the outer membrane of E. c o l i Early models describing the organization of proteins i n the c e l l envelope of E. c o l i were based on observations under the electron microscope (Pig. 5a and b). Protein granules, l i k e l y equivalent to the bound li p o p r o t e i n , were v i s i b l e on the peptido-glycan layer (207). In the model presented by Martin (208) i n 1963 (Fig. 5a) the outer membrane was v i s u a l i z e d as two non-descript layers composed of lipopolysaccharide and 30 F i g . 5a. Schematic representation of b a c t e r i a l c e l l wall struc-ture (1963). Gram-negative bacterium with complex t r i p l e -layered c e l l wall. Separation of the layers can be achieved by solvent extraction and by treatment with p r o t e o l y t i c enzymes (P). Both lysozyme (L), aided by EDTA (E) and p e n i c i l l i n (PEN) induce depolymerization of the r i g i d mucopolymer (MP), although probably to a dif f e r e n t degree and i n a diff e r e n t way. LP, lipoprotein layer; LS, lipopolysaccharide layer; RL, r i g i d layer; PG protein granula; MP, mucopolymer; CM cytoplasmic membrane; CP, cytoplasm. Taken from Martin (209). S Ps R-Ps Pr j 1 i 1 Fig.;".5b. Schematic model of the c e l l wall of E. c o l i (1967). Dimensions, shape, and arrangements of the actual single c o n s t i -tuent elements of the c e l l wall layers are s t i l l unknown. The approximate correspondence of the various components with the structure v i s i b l e i n thin section i s shown on the l e f t . The l e f t part of the diagram represents the wall of a "smooth" c e l l , i n which the polysaccharides ("S"-Ps) are assumed to be composed of a basal structure and s p e c i f i c side chains on the right wall of a "rough" form, which the polysaccharides ("R"-Ps) lack the s p e c i f i c side chains. Proteins (Pr) and polysaccharides (Ps) are considered to be di s t r i b u t e d i n a mosaic on the surface of the wall (1]_). The bulk of the l i p i d s (Lp) i s situated at the l e v e l of the L membrane, associated either with polysaccharide or possibly with proteins. Two rows of protein globular elements are drawn at the l e v e l of the G layer to indicate that i t probably contains proteins both covalently linked to the mucopeptide layer (murein, Mp) and unlinked to i t (see t e x t ) . CM, cytoplasmic membrane. Taken from DePetris (10). 31 Trypsin Lysozyme Pig. 5c. Schematic representation of the envelope of E. c o l i (1971). For s i m p l i c i t y the outer membrane are shown as simple b i l a y e r s . Arrows indicate the s i t e of action of EDTA i n the. release of proteins and lipopolysaccharide from the outer mem-brane, and the action of try p s i n i n cleavage of the murein li p o p r o t e i n . Taken from Schnaitman (67). TTTTTTObTTTTTTDDTTTT mini i i i i i i i i 0 side chain Lipid A — polor heod group \^ — hydrocarbon aj? • chain *° LPS Phospholipids Protein Divalent cation F i g . 5d. A possible arrangement of molecules i n the outer mem-brane layer (1973). In region A, LPS molecules are interspersed among phospholipid molecules reducing the e l e c t r o s t a t i c repulsion between the KDO-heptose regions of the core oligosaccharides. In region B, divalent cations reduce t h i s e l e c t r o s t a t i c repulsion, so that at least the outer l e a f l e t of the bil a y e r i s composed predominantly of LPS molecules. In region C, the protein mole-cules are thought to interact with phospholipids as well as LPS. LPS-protein i n t e r a c t i o n i s here shown as though i t involves d i -valent cations. The outer membrane i s assumed to be held i n place by the penetration of the small protein molecules, which are placed 100 A apart and are covalently linked to the peptidoglycan. Taken from Nikaido (33). 32 RELEASED BY EDTA HI I U I luiiMimmifl Hi N M . " PEPTIDOGLYCAN m AiAUA y PHOSPHOLIPID ^ 3 "UNITS" OF LIPOPOLYSACCHARIDE JOINED BY PYROPHOSPHATE BRIDGES F i g . 5c. (1974). Model o f o u t e r membrane o f g r a m - n e g a t i v e b a c t e r i a T a k e n f r o m L e i v e (61). 33 DETAIL OF MEMBRANE . ARCHITECTURE DETAIL Of MEMBRANE ARCHITECTURE TURGOR PRESSURE Pig. 5f. Schematic diagram of gram-negative c e l l envelope (1974). _ + , Free cation; -, free anion;©.©, bound cation; bound anion; adhesion point produced by ionic bonding; hydrophobic zone; - covalent bond; i, cross-linking'poly-peptide i n the peptidoglycan; ^» polysaccharide portion of cytoplasmic membrane whose function i s directed to the cytoplasm; em, enzymes associated with the cytoplasmic membrane which syn-thesize macromolecular components of the c e l l wall; ep, enzymes l o c a l i z e d i n the periplasmic zone; es, enzymes l o c a l i z e d at the c e l l surface; l p , l i p i d portion of Braun's l i p o p r o t e i n ; p, stru c t u r a l and enzymatic proteins of the outer membrane; p l , protein portion of Braun's l i p o p r o t e i n ; ps, permease; s, struc-t u r a l protein of cytoplasmic membrane. Taken from Costerton, Ingram and Cheng ( 5 ) . 3 4 CHANEL F i g 5 g . Schematic i l l u s t r a t i o n of the outer membrane structure (1975). A superhelix made of six <* -helices i s shown to be i n -serted into the outer membrane and to span the f u l l 75A -thick membrane. The three hydrocarbon chains attached at the top of each molecule are f l i p p e d over, hanging down from the top, and are anchored i n the l i p i d b i l a y e r of the outer membrane. At the bottom (carboxyl-terminal ends of the lipoproteins) of the as-sembly, two molecules are linked to the peptidoglycan layer, as shown by small bars. The peptidoglycan layer i s i l l u s t r a t e d by rectangular blocks (for the glycan chains) and small bars (for the peptide portions) which are cross- l i n k i n g the glycan chains. Phospholipids forming the l i p i d b i l a y e r are shown by hydrophilic, open, c i r c u l a r heads and hydrophobic, hatched, long t a i l s . Channel opening of 7- and 8-membered assemblies are also i l l u -strated on the surface of the outer membrane. Taken from Inouye (120). • Free calion - Free anion • Bound cation © Bound anion S Adhesion point produced by ionic bonding •iv:'. Hydrophobic zone Cross-linking polypeptide in the peptidoglycan Polysaccharide portion ol peptidoglycan Enzymatically active protein Phospholipid Lipopolysaccharide Lipopolysaccharide (schematic) bp Binding protein 5 rrrf 35 cc Capsular carbohydrate cp Capsular protein ec Enzymes associated with the cytoplasmic membrane whose function is directed to the cytoplasm em Enzymes associated with the cytoplasmic membrane which synthesize macro-molecular components of the cell wall ep Enzymes localized in the periplasmic zone es Enzymes localized at the cell surface lp P Braun's lipoprotein Structural and enzymatic proteins of the outer membrane ps Permease Structural protein of cytoplasmic membrane TURGOR PRESSURE TURGOR PRESSURE F i g . 5h. Schematic diagram of the gram-negative c e l l envelope (1975). Taken from Costerton and Cheng (62). 36 F i g . 5 i . M o l e c u l a r model o f t h e o u t e r membrane (1975). See t a b l e I f o r p r o t e i n n o m e n c l a t u r e . T a k e n f r o m Inouye (120). 37 l i p o p r o t e i n . De Petris (10) i n 1967 suggested that the outer membrane i s composed of lipopolysaccharide, l i p i d s and proteins arranged i n a mosaic pattern. In this model (Pig. 5b) the carbohydrate chains of the lipopolysaccharide protude from the external surface of the membrane. The protein v i s u a l i z e d i n the g l area may be the matrix protein A since De Petris found that t h i s protein was non-covalently attached to the peptidoglycan. The s i m p l i f i e d scheme proposed by Schnaitman (67) i n 1971 (Fig. 5c) i s based on the Davson-Danielli model (205). The proteins are organized on the inner and outer surfaces of a phospholipid b i l a y e r . The hydrophobic l i p i d A portion of the lipopolysaccharide interacts with the hydrophobic core of the membrane and the hydrophilic carbohydrate chains extend into the medium. The bound li p o p r o t e i n l i n k s the outer membrane to the peptidoglycan. Models for the outer membrane which are based on studies of the lipopolysaccharide have been presented (Fig. 5 d and e). These models arrange the lipopolysaccharide and phospholipid molecules into a bi l a y e r and emphasize the role played by diva-lent cations i n s t a b i l i z i n g the outer membrane. The lipopolysac-charide molecules are arranged i n regions corresponding to EDTA-releasable and non-releasable f r a c t i o n s . The protein components are presented as globules dissolved i n the bil a y e r as proposed by Singer and Nicholson (240). The bound l i p o p r o t e i n i s v i s u a l i z e d as penetrating only part way through the outer membrane. Costerton, Ingram and Cheng (5) have presented a more detailed model (Pig. 5f) i l l u s t r a t i n g e s s e n t i a l l y the same features. 38 Inouye (120,130) has proposed that bound and free l i p o -proteins form channels through the outer membrane. In t h i s model (Pig. 5g), six li p o p r o t e i n molecules, two bound and four free, are arranged i n a superhelix with a hydrophilic channel 12.5 A i n diameter i n the centre. The assembly i s 76 A i n l e -ngth and could span the 7 5 A thick outer membrane. Braun has proposed (123) that the lip o p r o t e i n spans only part of the thickness of the outer membrane i n contrast to the proposal by Inouye (133)- This conclusion was drawn from exten-sive immunological studies with antibodies directed against various forms of the li p o p r o t e i n . In wild-type c e l l s , the l i p o -protein was unable to react with antibodies to i t , but became accessible i n c e l l s with a defective lipopolysaccharide (123). This does not necessarily .contradict Inouye's proposal since the carbohydrate chains of the lipopolysaccharide may r e s t r i c t access of the antibody to the lipoprotein which extends just through the membrane (Fig. 5g). A model (Fig. 5h) based on Inouye's superhelix, but not allowing the lipoprotein to penetrate the membrane, was recen-t l y proposed by Costerton and Cheung (101). This arrangement of the li p o p r o t e i n i s very unlikely since a l l the hydrophobic amino acid residues on the outside of the helix would be i n contact with the water of the periplasmic space. A more detailed model postulated recently by Inouye (120) i s i l l u s t r a t e d i n Fig . 51. The matrix protein, 4 (equivalent to protein A) i s associated with the peptidoglycan (80). Protein 7 (protein 8) i s exposed at the external surface of the 39 outer membrane (137)• P r o t e o l y t i c d i g e s t i o n of the outer membrane w i l l r e s u l t i n the h y d r o l y s i s of the exposed part of p r o t e i n B to leave a r e s i s t a n t fragment embedded i n the membrane (79,82,131,135,136,149). Fragments with a molecular weight of 25,000 and 20,000 are produced by the a c t i o n of t r y p s i n and pronase r e s p e c t i v e l y . P r o t e i n 11, the l i p o p r o t e i n i s arranged as a s u p e r h e l i x that penetrates the membrane. Tr y p s i n r e l e a s e s t h i s complex from the peptidoglycan (117,121) while treatment of the outer membrane wit h pronase r e s u l t s i n d i g e s t i o n of the l i p o p r o t e i n (79). Results obtained r e c e n t l y suggest that the model proposed by Inouye ( F i g , 5i) i s no longer adequate. P r o t e i n A l i k e l y serves as a receptor f o r bacteriophage (41,103,142) suggesting that i t i s exposed on the outer surface of the membrane. This i s confirmed by the f i n d i n g that p r o t e i n A could form channels through l i p o p o l y s a c c h a r i d e / p h o s p h o l i p i d v e s i c l e s (134) and there-fore spans the membrane. In a d d i t i o n , Nakae (134) found that the l i p o p r o t e i n d i d not form channels through these v e s i c l e s . Treatment of i s o l a t e d outer membrane preparations with p r o t e o l y t i c enzymes have suggested that the p r o t e i n s of the outer membrane are arranged asymmetrically (79). As already discussed, p r o t e i n A i s r e s i s t a n t to d i g e s t i o n while a p o r t i o n of p r o t e i n B i s exposed. Bragg and Hou al s o found that p r o t e i n s D, E and F were r e a d i l y digested and were the r e f o r e exposed at the surface of the membrane. P r o t e i n s C 2 , C^ ' and D 2 were not digested s i g n i f i c a n t l y . The r e l a t i o n s h i p s between r e s u l t s obtained w i t h i s o l a t e d outer membrane preparations such as outer membrane, c e l l 40 wall and c e l l envelope and intact c e l l s i s not clear. Schindler and Teuber (238) found no l a b e l l i n g of c e l l envelope proteins when intact c e l l s of Salmonella typhimurium were treated with dansyl chloride i n phospholipid micelles i n order to be nonpenetrating. In contrast, a l l polypeptides could be l a b e l l e d by dansylation of i s o l a t e d c e l l envelopes. The d i f -ference could not be due to interference by the carbohydrate portion of the lipopolysaccharide since a deep-rough mutant of Salmonella which had only KDO l e f t i n the lipopolysaccharide also did not react with dansyl chloride. The authors suggest that t h i s was because there were no free amino groups on the surface of the c e l l . Lactoperoxidase-catalyzed iodination of exposed tyrosine residues did not result i n extensive l a b e l l i n g of outer membrane proteins i n intact c e l l s . A single protein band (M.W., 13,000) i n Salmonella (236) and a phospholipoprotein (M.W. 16,000) i n Pseudomonas (266) were iodinated by t h i s technique. The lack of more general l a b e l l i n g may be accounted for by i n -h i b i t i o n of the iodination reaction by the c e l l s . Gow, Parton and Wardlaw (237) have shown that bovine serum albumin, which i s r e a d i l y iodinated by t h i s technique, was not l a b e l l e d when mixed with intact c e l l s of Bordetella pertussis. The polypeptides of the outer membrane of intact c e l l s were not readily crosslinked through the formation of d i s u l -fide bonds, catalyzed by the CuSO^-O-phenanthroline reagent (137). However, in order to.be crosslinked, the proteins must be accessible to the reagent and have suitably disposed, free 41 s u l f h y d r y l groups. Some outer membrane p r o t e i n s of deep-rough mutants could be c r o s s l i n k e d by t h i s technique, suggesting that the l i p o p o l y s a c c h a r i d e p r o t e c t s the outer membrane p r o t e i n s from the reagent. The p r o t e i n s of ghosts and c e l l envelopes could also be r e a d i l y c r o s s l i n k e d . The p r o t e i n s c r o s s l i n k e d by t h i s technique presumably are arranged on the outer surface of the ghost membrane. About t h i r t y d i f f e r e n t polypeptides were c r o s s l i n k e d , however, none of the major outer membrane pr o t e i n s (A,B or P) were i n v o l v e d . Henning (186) has shown that treatment of i n t a c t c e l l s w ith dimethyl imidoesters r e s u l t s i n extensive c r o s s l i n k i n g of pr o t e i n s I ( A x ) , I I * (B) I I I ( l i k e l y D ) and IV (P). The a b i l i t y of the imidoesters to react with the amino groups, which con-t r a s t s with r e s u l t s obtained w i t h dansyl c h l o r i d e Is probably due to the a b i l i t y of the est e r s to penetrate the membrane. The r e s u l t i n g sacs were the same shape as c e l l s and were r e s i s -tant to d i s r u p t i o n by b o i l i n g 1% SDS. Ghosts, con t a i n i n g these four p r o t e i n s plus some higher molecular weight p r o t e i n s could be s i m i l a r l y c r o s s l i n k e d . Trypsin treatment of ghosts, which cleaved a p o r t i o n of p r o t e i n B, precluded formation of these d e t e r g e n t - r e s i s t a n t sacs. On the basis of these r e s u l t s , Henning has suggested that extensive p r o t e i n - p r o t e i n i n t e r a c t i o n s occur i n the outer membrane and that the model proposed by Capaldi and Green (239) may more adequately describe t h i s mem-brane than the f l u i d mosaic model of Singer and Nicholson (240). A number of problems are s t i l l unresolved. Many outer membrane p r o t e i n s must be exposed on the outer surface of the c e l l s i n c e , as noted p r e v i o u s l y , these p r o t e i n s serve as recep-42 t o r s f o r phage, c o l i c i n s , e t c . The i n a b i l i t y to detect ex-posed p r o t e i n s i n i n t a c t c e l l s may be due to l i m i t a t i o n s of the technique used or perhaps because of i n t e r f e r e n c e by the carbo-hydrate chains of the l i p o p o l y s a c c h a r i d e . Both aspects of t h i s problem are explored i n t h i s t h e s i s . In a d d i t i o n , the s p a t i a l r e l a t i o n s h i p s of the i n d i v i d u a l outer membrane p r o t e i n s to one another w i l l be studied. B i o s y n t h e s i s and assembly of outer membrane p r o t e i n s B i o s y n t h e s i s The p r o t e i n s of the outer membrane are synthesized on ribosomes l o c a t e d i n the cytoplasm or on the inner aspect of the cytoplasmic membrane. However, the synthesis of envelope and cytoplasmic p r o t e i n s d i f f e r e d i n t h i s s e n s i t i v i t y to ribosome-d i r e c t e d a n t i b i o t i c s (120,241). Envelope p r o t e i n synthesis was more r e s i s t a n t to kasugamycin and puromycin than cytoplasmic p r o t e i n s y n t h e s i s , while the reverse i s true f o r t e t r a c y c l i n e and sparsomycin. Chloramphenicol i n h i b i t e d both envelope and cytoplasmic p r o t e i n synthesis to the same extent (241,242). However, no degradation of incorporated inner and outer mem-brane p r o t e i n s occurred under these c o n d i t i o n s (276). Furthermore, i n h i b i t i o n patterns of the synthesis of i n d i v i d u a l envelope p r o t e i n s d i f f e r e d . The synthesis of the l i p o p r o t e i n and a major p r o t e i n molecular weight of 38,000 were very r e s i s t a n t to the a c t i o n of c e r t a i n a n t i b i o t i c s . The d i f f e r e n c e between the s e n s i t i v i t y of envelope LEAF 43 OMITTED IN PAGE NUMBERING. 44 p r o t e i n and cytoplasmic p r o t e i n synthesis has been a t t r i b u t e d (120) to a number of p o s s i b l e causes. There i s evidence f o r a d i f f e r e n c e i n mRNA molecules. The s t a b i l i t y of rriRNA's f o r envelope p r o t e i n s was determined from the e f f e c t of r i f a m p i c i n on t h e i r b i o s y n t h e s i s (241). The mRNAs f o r the outer membrane pr o t e i n s are more s t a b l e ( h a l f - l i f e , 5.5 mins) than those f o r the synthesis- of inner membrane and cytoplasmic p r o t e i n s (243). S i m i l a r r e s u l t s were found i n E. c o l i m i n i c e l l s which lacked DNA but i n which p r o t e i n synthesis was s e n s i t i v e to chloramphenicol but r e s i s t a n t to r i f a m p i c i n . In these c e l l s , the mRNAs had h a l f - l i v e s of 40-80 minutes and coded f o r the outer membrane p r o t e i n s . The d i f f e r e n c e between outer membrane p r o t e i n synthesis and inner membrane and cytoplasmic p r o t e i n synthesis may a l s o be due to d i f f e r e n t i a t i o n s of the ribosome p o p u l a t i o n . P u r i f i e d r i b o -somes of E. c o l i were found to be heterogeneous with respect to the content of ribosomal p r o t e i n s (244 ,245). However Randall and Hardy (246) examined the polysomes cont a i n i n g the sta b l e mRNAs f o l l o w i n g r i f a m p i c i n treatment•and concluded that there was no d i f f e r e n c e between the p r o t e i n composition of the ribosomes s y n t h e s i z i n g outer membrane p r o t e i n s and those s y n t h e s i z i n g t o t a l p r o t e i n s . Another p o s s i b l e explanation f o r the d i f f e r e n c e could be the compartmentalization of the ribosomes. For example, ribosomes s y n t h e s i z i n g a l k a l i n e phosphatase, or peri p l a s m i c en-zyme are asso c i a t e d w i t h the cytoplasmic membrane (247). The b i o s y n t h e s i s of the two forms of the l i p o p r o t e i n has been studied i n greatest d e t a i l (44,120). The l i p o p r o t e i n does 45 not contain h i s t i d i n e and i s the only envelope protein synthe-sized by a h i s t i d i n e auxotroph i n the absence of th i s amino acid (248). Tetracycline and chloramphenicol were highly i n -h i b i t o r y to lipoprotein synthesis under conditions of h i s t i d i n e starvation. Rifampicin, however had l i t t l e e f f e c t , confirming the stable nature of the mRNA. This stable mRNA has been isol a t e d and translated i n both an E. c o l i c e l l - f r e e system (249) and a wheat-germ system (250). The mRNA contained two species which were 230 and 250 nucleo-tides long. Since a sequence of 180 bases are required for t r a n s l a t i o n into a protein of 58 amino acids, 50-70 bases must be untranslated. A single protein characterized as the poly-peptide portion of the l i p o p r o t e i n was formed i n the wheat-germ system. A second protein with about twice the molecular weight of the l i p o p r o t e i n was also synthesized. How th i s was accomplished with the mRNA molecules previously characterized i s not clear, however, the large product may be a precursor normally found i n the c e l l . The complete biosynthesis of the lipoprotein requires the attachment of the l i p i d moiety. L i t t l e i s known about th i s process except that the c i s d i o l group i s derived from g l y c e r o l and that attachment of the diglyceride occurs after synthesis of the apoprotein (270). This i s consistant with the finding that the ester-linked fatty acids are similar to those found i n the c e l l u l a r phospholipids (122). The a v a i l a b i l i t y of the l i p i d -free protein formed i n v i t r o and E. c o l i mutants altered i n the bound lip o p r o t e i n (279) should be of considerable use i n esta-46 b l i s h i n g the steps involved i n the biosynthesis of the unique l i p i d portion of t h i s molecule. The free form of the lipoprotein i s the precursor of the bound form and thi s process i s not i n h i b i t e d by chloramphenicol, amino acid starvation or carbonyl cyanide m-chlorophenyl-hydrazone, an energy uncoupler (126). No li p o p r o t e i n -mucopeptides are found i n the cytoplasmic membrane and the lipop r o t e i n i s incorporated into newly-made murein at discrete si t e s (251). The maximum rate of incorporation of the l i p o -protein Into the outer membrane occurs at the time of septation (252). In addition, a mutant defective i n coupling outer mem-brane invagination with septation contained a decreased amount of bound lipoprotein and a corresponding increased l e v e l of the free form.(109). The authors suggest "that the morphogenetic defect may re s u l t from a defect i n formation of covalent bonds between the free lipoprotein of the outer membrane and the murein of the nascent septum". Assembly of the proteins of the outer membrane C e l l d i v i s i o n involves the coordination of DNA r e p l i c a t i o n , septation and c e l l envelope growth (32,253,255). C e l l wall growth requires coordinated synthesis and assembly of peptido-glycan, phospholipid, lipopolysaccharide and protein. A defect i n thi s coordination may result i n the production of morphological mutants (192-203). Coordination of phospholipid and membrane protein synthesis has been studied i n a gl y c e r o l auxotroph mutant of E. c o l i (256). 47 Upon glycerol deprivation, t h i s mutant continued soluble and envelope protein synthesis causing the protein to phospholipid r a t i o to increase by 60$. The authors concluded that the bio-synthesis of inner and outer membrane phospholipids and proteins are not t i g h t l y coupled. This i s supported by the finding that i n h i b i t i o n of J3-ketoacylthioesterase by cerulenin did not prevent the incorporation of the X phage receptor into the outer membrane (257). In addition, no differences i n the polypeptide composition of the outer membrane was observed i n fatty acid auxotrophs of E. c o l i supplemented with various fatty acids (284) . Reconstitution The reassembly of the i s o l a t e d components of b i o l o g i c a l membranes into a form i d e n t i c a l to the native membrane w i l l aid in the understanding of the role of the components i n the membrane (258). Membranes are self-assembling systems (259) that can be reformed from dissociated components upon removal of the s o l u b i l i z i n g agent, which i s usually a detergent (260). Reconstitution can be monitored by electron microscopy (258,261). A second measurement of reconstitution i s the restoration of function i n the reassembled membrane (258) as has been accompli-shed with the components of oxidative phosphorylation (258,262, 263). De Pamphilis (72) dissociated p u r i f i e d outer membranes with Tri t o n X-100 i n the presence of EDTA and found that the sol u b i -l i z e d material reassembled into vesicles upon removal of the 48 detergent i n the presence of magnesium ions. Bragg and Hou (79) have s o l u b i l i z e d the outer membrane with SDS and recovered reformed membranes upon removal of the detergent by d i a l y s i s . The reconstituted membranes had a similar morphology, density and chemical composition to that of the native membrane. More s i g n i f i c a n t l y , the proteins appeared to be arranged i n a similar manner i n the native and reformed membranes as judged by pronase digestion. A similar study has been reported by Sckizawa and Fukai (264). Nakamura and Mizushima (265) have separated the protein, lipopolysaccharide and phospholipid components of the outer membrane and have studied the role of the p u r i f i e d components i n the reassembly of membrane v e s i c l e s . Removal of detergent by d i a l y s i s i n the presence of magnesium ions was essential for ' ve s i c l e formation. Although lipopolysaccharide alone reassem-bled into a trilaminer structure, phospholipid was required for ves i c l e formation. Protein from the outer membrane but not the cytoplasmic membrane was reincorporated into the membranous v e s i -cles. Recent studies (115) have shown that reassembly of the outer membrane on the peptidoglycan requires the presence of the bound lip o p r o t e i n and the matrix protein A. Nikaido and coworkers have studied the permeability pro-perties of reformed v e s i c l e s . Vesicles composed of phospho-l i p i d and lipopolysaccaride were not permeable to molecules as small as sucrose (173)- Protein was required for penetra-t i o n of small oligosaccharides (172) with protein A being responsible for the formation of a channel through the 49 lipopolysaccharide/phospholipid v e s i c l e (134). The reconsti-tution technique may also help define the nature of the interactions between outer membrane components, and the use of defective components (eg. lipopolysaccharide) i n reconstitution systems may lead to an understanding of the function of the altered component. 49b T h e s i s P r o j e c t The unique f e a t u r e s o f the outer membrane suggest t h a t i t may not be a t y p i c a l membrane, making i t an i n t e r e s t i n g system f o r study. Two major p r o t e i n s of the outer membrane, the mat r i x p r o t e i n , A, and the h e a t - m o d i f i a b l e p r o t e i n , B, were p u r i f i e d and c h a r a c t e r i z e d s i n c e l i t t l e was known about the p r o p e r t i e s of the outer membrane p r o t e i n s , except f o r the bound l i p o p r o t e i n . P r o t e i n B changes i t s apparent molecular weight upon h e a t i n g i n SDS-containing s o l u t i o n s . The reason f o r t h i s change was i n v e s t -i g a t e d by comparing p r o t e i n s B and the m o d i f i e d form, B*, by a number of p h y s i c a l techniques. The o r g a n i z a t i o n o f the p r o t e i n s i n w i l d - t y p e and n u t r i t i o n a l l y or g e n e t i c a l l y - a l t e r e d membranes was s t u d i e d by p r o t e o l y t i c d i g e s t i o n , c o v a l e n t l a b e l l i n g , c r o s s -l i n k i n g and r e c o n s t i t u t i o n s t u d i e s . A model f o r the o r g a n i z a t i o n of the p r o t e i n s i n t h i s unique membrane i s presented. 50 METHODS AND MATERIALS Reagents A l l chemicals were reagent grade. The sources of the chemicals were: acrylamide, bis(N,N-methylenebisacrylamide, Bio-Gel P-150 and Dowex AG1-X4 were purchased from BioRad Labora-t o r i e s ; sodium dodecyl sulfate ( s p e c i a l l y pure), B r i t i s h Drug Houses Ltd.; ©t -chymotrypsin, chymotrypsinogen, bovine serum albumin, egg-white lysozyme, equine hemoglobin, human 5<-globulin, phenylmethylsulfonyl Fluoride, pronase, r i b o f l a v i n , r i b o f l a v i n - 5 ' -•phosphate, Tri t o n X-100, t r y p s i n , Calbiochem; Cheng-Chin polya-mide layer sheets were obtained from Gallard-Schlesinger Chemical Corp.; 2-mercaptoethanol, N,N,N',N'-tetramethylethylenediamine, phenylisothiocyanate, Eastman Kodak Company; D 20,ICN Pharma-ceuticals Inc.; glutaraldehyde ( h i s t o l o g i c a l grade), Matheson, Coleman and B e l l ; deoxyribonuclease, N u t r i t i o n a l Biochemicals Corp.; d i t h i o b i s (succinimdyl propionate), Pierce Chemical Company; Sephadex G-100 and Sepharose 6-B, Pharmacia; egg-white lysozyme, ovalbumin, Sigma Chemical Company. Fluorescamine (Hoffmann-La Roche) was a g i f t from Dr. E.P.M. Candido of the Department of Biochemistry, University of B r i t i s h Columbia. Bac t e r i a l strains E. c o l i s t r a i n 482 of the culture c o l l e c t i o n of the National Research Council of Canada was used for the majority of the work described i n t h i s t h e s i s . E. c o l i s t r a i n JE 1011 (F~,Thr~,Leu~, Trp",His~,B 1~,Lac~,Gal~,Xyl~,M+l~,Str^ and i t s heptose-deficient 51 mutant, NS-1 were used In studies on l i p o p o l y s a c c h a r i d e -d e f e c t i v e c e l l s . The b a c t e r i a were maintained on agar s l a n t s on a medium co n t a i n i n g 3 g of Try p t i c a s e Soy medium D i f c o , 0.33 g of yeast e x t r a c t and 1.5 g of agar per 100 ml. Growth con d i t i o n s (143) E. c o l i s t r a i n 482 was r o u t i n e l y grown on a minimal medium of 0.4% glucose and s a l t s (0.7% KgHPO^, 0.3% KHPO^, 0.02% MgSOij.-7H203 0.05% sodium c i t r a t e , 0.002% F e r r i c c i t r a t e and 0.1% (NH^) 2 S04. In experiments studying the e f f e c t of the carbon source, glucose was replaced by e i t h e r 0.4% g l y c e r o l , 1.4% disodium succinate or 0.7% sodium acetate. When 0.5% casein amino acids were used, glucose and (NH i|) 2 SO^ were omitted. C e l l s were also grown on a complex medium made by d i s s o l v i n g 17-5 g of Penassay medium (Difco) or 30 g of Trypt i c a s e Soy medium (Difco) i n 1 1 of water. E. c o l i JE 1011 and NS-1 were grown on Penassay medium. D i s t i l l e d water was used to prepare a l l media which were s t e r i l i z e d f o r 1\5 min i n an autoclave before use. The normal procedure f o r growing 4 1 batches of c e l l s was as f o l l o w s . A c u l t u r e tube c o n t a i n i n g 10 ml of medium was in o c u l a t e d from a n u t r i e n t agar s l a n t and incubated at 37°C f o r 24 h. This l i q u i d c u l t u r e was used to i n o c u l a t e a f u r t h e r 200 ml of medium contained i n a 1S1 f l a s k . The f l a s k c u l t u r e was grown at 37°C i n e i t h e r a R e c i p r o c a l Water Bath Shaker (New Brunswick S c i e n t i f i c Co. Inc.) at 100 cycles per min or a C o n t r o l l e d Environment Incubator (New Brunswick S c i e n t i f i c Co. Inc.) at 200 r e v o l u t i o n s per min overnight. The 200 ml f l a s k c u l t u r e was used to i n o c u l a t e 4 1 of medium. The b a c t e r i a 52 were grown at. 37"C with vigorous aeration provided by forcing water-saturated a i r through a sintered glass sparger. C e l l growth was monitored by measuring the absorbance of the culture at 420 nm and the c e l l s were harvested at the appropriate phase of growth by centrifugation at 4,000 x g for 15 min at 4°C. Cells were either used immediately or stored frozen at -20°C. C e l l Fractionation Preparation of Triton-extracted c e l l envelopes ( c e l l wall) (79) Unless stated otherwise, a l l operations were performed at 0-4°C. Ce l l s (50 g, wet weight) were suspended to a volume of 160 ml with 10 mM Tris-HCl, pH 7-5, containing 10 mM MgCl*. A /2 / few c r y s t a l s of DNase were added and the c e l l s were broken by passage through a pre-cooled French Pressure C e l l at 20,000 p lbs/inch . The lysed c e l l suspension was centrifuged at 4,000 x g for 10 min to remove unbroken c e l l s . The r e s u l t i n g supernatant was then centrifuged at 120,000 x g for 1 h to sedi-ment c e l l envelopes. The c e l l envelope f r a c t i o n was extracted with 1.5$ Triton X-100 i n 0.1 M Tris-HCl, pH 8.0, containing 35 mM MgCl 2 at 22°C for 1 h by suspending the p e l l e t to a v o l -ume of 160 ml and agitating vigorously on a wrist-shaker. This resulted i n s o l u b i l i z a t i o n of the inner membrane. The c e l l wall f r a c t i o n consisting of outer membrane and peptido-glycan, was sedimented by centrifugation at 120,000 x g for 1 h. The r e s u l t i n g p e l l e t was washed with 0.1 M Tris-HCl buffer, . pH 8.0 containing. 35 mM MgCl 2 and resedimented at 120,000 x g for 1 h. 53 Sequential e x t r a c t i o n of c e l l w a l l The c e l l w a l l f r a c t i o n , derived from 50 g (wet weight) of c e l l s was suspended to 160 ml of 0.5% SDS and incubated at 37°0 f o r 1 h. The SDS-extracted c e l l w a l l was sedimented at 120,000 x g f o r 1 h at 15°C to give "SDS e x t r a c t 1". The p e l l e t was re e x t r a c t e d w i t h 80 ml of 1% SDS at 100°C f o r 15 min. Pep-t i d o g l y c a n was removed by c e n t r i f u g a t i o n at 120,00 x g f o r 1 h at 15°C to y i e l d "SDS e x t r a c t 2". E x t r a c t 1 was used i n the p u r i f i c a t i o n of p r o t e i n B. Prepar a t i o n of p r o t e i n A-peptidoglycan complexes (80) C e l l envelopes, prepared from 50 g (wet weight) of c e l l s , were suspended to 160 ml of 2% SDS and incubated at 60°C f o r 1 h. The i n s o l u b l e peptidoglycan was sedimented by c e n t r i f u g a t i o n at 120,000 x g f o r 1 h at 15°C. The e x t r a c t i o n step was repeated once and the r e s u l t i n g p r o t e i n A-peptidoglycan complex was washed four times w i t h an appropriate b u f f e r to remove r e s i d u a l SDS. Preparation and l y s i s of spheroplasts (155) C e l l s were harvested i n the mid-exponential phase of growth (A 2 + 2 0 , 5 . 0 ) . The c e l l s were washed once with 0.18 M NaCl, 40 ml per g (wet weight) of c e l l s . The washed c e l l s were suspended at 22°C to a concentration of 1 g per 40 ml i n 30 mM T r i s - H C l , pH 8.0 c o n t a i n i n g 20% (w/v) sucrose and the suspension was s t i r r e d gently f o r 20 min. Lysozyme (5 mg/g of c e l l s ) was then added as a s o l u t i o n i n d i s t i l l e d H 20 and the suspension was s t i r r e d f o r an a d d i t i o n a l 30 min at 22°C. The suspension was 54 d i l u t e d r a p i d l y by pouring i n t o 10 volumes of 0.2 mM MgCl^, con-t a i n i n g a few c r y s t a l s of DNase. The decrease i n t u r b i d i t y was monitored at 420 nm u n t i l a constant absorbance was reached. The suspension of lysed c e l l s was ce n t r i f u g e d at 15°C at 4,000 x g f o r 5 min to remove i n t a c t c e l l s , and then c e n t r i f u g e d at 15,000 x g f o r 30 min at 4°C to obta i n the pr e p a r a t i o n of sphero-p l a s t s used i n t h i s t h e s i s . Outer membrane was prepared from the spheroplast membranes by e x t r a c t i o n of the inner membrane with 1.5$ T r i t o n X-100 i n 0.1. M T r i s - H C l , c o n t a i n i n g 35 mM MgCl 2 at 22°C f o r 1 h. Membrane was recovered by c e n t r i f u g a t i o n at 120,00 x g f o r 1 h at 4°C. Release of outer membrane from spheroplasts (77,78) C e l l s were grown to mid-exponential phase of growth. The harvested c e l l s (ca. 3-5 g, wet weight) form 1 1 of c u l t u r e were suspended to 35.5 ml wi t h d i s t i l l e d water i n a 1 1 f l a s k cooled i n ice-water. The f o l l o w i n g a d d i t i o n s of i c e - c o l d reagents were added i n order and allowed to incubate f o r the i n d i c a t e d times: (a) 20 ml of 0.1 M T r i s - H C l , pH 8.3 f o r 5 min, (b) 18 ml of 2 M sucrose f o r 5 min, (c) 3-5 ml of 1$ EDTA, PH 7.0 f o r 2 min, and (d) 3-5 ml of 0.5$ lysozyme f o r 2 min. The suspension was warmed to 30°C and kept at that temperature f o r 1 h. The spheroplasts were removed by c e n t r i f u g a t i o n at 20,000 x g f o r 30 min at 40°C. The supernatant, c o n t a i n i n g the released outer membrane, was then c e n t r i f u g e d at 120,000 x 55 o g f o r 1 h a t 4 C t o s e d i m e n t t h e o u t e r membrane. The membranes were washed w i t h e i t h e r 0.2 M t r i e t h a n o l a m i n e b u f f e r , pH 8.5 o r 0.1 M T r i s - H C l , pH 8.0, c o n t a i n i n g 35 mM M g C l 2 and t h e n c e n t r i f u g e d as b e f o r e . The membrane p e l l e t was washed f o u r t i m e s w i t h one o f t h e above b u f f e r s t o remove c o n t a m i n a t i n g l y s o z y m e . T h i s p r e p a r a t i o n o f o u t e r membrane was u s e d i n c r o s s -l i n k i n g and p r o t e o l y t i c d i g e s t i o n s t u d i e s . S e p a r a t i o n o f i n n e r and o u t e r membrane by s u c r o s e d e n s i t y c e n -t r i f u g a t i o n (14,35,156) The o u t e r membrane w h i c h had been r e l e a s e d f r o m t h e s p h e r o -p l a s t s was a n a l y s e d by s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . T h i s t e c h n i q u e s e p a r a t e s t h e i n n e r and o u t e r membranes o f gram-n e g a t i v e b a c t e r i a . The o u t e r membrane d e r i v e d f r o m 1 1 o f m i d - e x p o n e n t i a l phase c u l t u r e was s u s p e n d e d t o 1 ml i n 0.1 M EDTA, pH 7-5. The samples (1 ml) were a p p l i e d t o t h e t o p o f 30-55% s u c r o s e g r a d i e n t s i n 5 mM EDTA, pH 7.5 and c e n t r i f u g e d a t 51,500 x g f o r 20 h i n a SW 25.1 r o t o r a t 4°C. At t h e end o f t h e r u n , t h e b o t t o m o f e a c h t u b e was p i e r c e d and f i f t e e n 2 ml f r a c t i o n s were c o l l e c t e d . The a b s o r b a n c e a t 280 nm, t h e r e f r a c -t i v e i n d e x , and t h e p r o t e i n c o n t e n t o f e a c h t u b e was mea s u r e d . P u r i f i c a t i o n o f o u t e r membrane p r o t e i n s P u r i f i c a t i o n o f p r o t e i n B D e t e r g e n t was removed f r o m SDS e x t r a c t 1 by d i a l y s i s a g a i n s t s e v e r a l changes o f d i s t i l l e d w a t e r a t room t e m p e r a t u r e f o r 24 h and t h e n a t 4°C f o r 2 t o 5 d a y s . The d i a l y z e d s o l u t i o n was 56 then f r e e z e - d r i e d and s t o r e d at -20°C. In a t y p i c a l e x p e r i -ment, 40 mg of e x t r a c t 1 was d i s s o l v e d i n 10 ml of 0.1 M sodium phosphate b u f f e r , pH 7-2, c o n t a i n i n g 1% SDS and 0.1% 2-mercaptoethanol and incubated at 37°C f o r 1 h. The c l e a r s o l u t i o n was a p p l i e d to a column o f Sephadex G-100 (5 x 40 cm) connected i n s e r i e s to a column of Sepharose 6B of the same dimensions. The p r o t e i n s were e l u t e d at room temperature with 0.1 M sodium phosphate b u f f e r , pH 7.2, c o n t a i n i n g 1% SDS. F r a c t i o n s (10 ml) were c o l l e c t e d and the absorbance measured at 280 nm. F r a c t i o n s were a l s o assayed f o r t h e i r content of pro-t e i n and l i p o p o l y s a c c h a r i d e . Column f r a c t i o n s (50 and 100 ^1) were incubated at 37°C and 100°C with added urea t o 4 M concen-t r a t i o n and 2-mercaptoethanol and run on 10% polyacrylamide g e l s c o n t a i n i n g 0.1% SDS. The f r a c t i o n s c o n t a i n i n g p r o t e i n B were pooled, d i a l y z e d a g a i n s t d i s t i l l e d water and l y o p h i l i z e d . The p r o t e i n was d i s -s o l v e d i n 10 ml of 0.1 M sodium phosphate b u f f e r , pH 7-2, cont-a i n i n g .1% SDS' and 0.1% 2^-mercaptoethanol, heated at 100°C f o r 15 min, and r e a p p l i e d to the double column system. The f r a c t i o n s from the column were examined as before and those c o n t a i n i n g p r o t e i n B* were pooled, d i a l y z e d a g a i n s t d i s t i l l e d water and l y o p h i l i z e d . The p u r i f i e d p r o t e i n was subjected to amino a c i d a n a l y s i s , N-terminal a n a l y s i s and cyanogen bromide cleavage. P u r i f i c a t i o n of p r o t e i n B f o r r e c o n s t i t u t i o n P r o t e i n B i s o l a t e d as d e s c r i b e d above i s i n the heat-4 m o d i f i e d form, B*. For the p r e p a r a t i o n of unmodified p r o t e i n B, 57 f r e e of phos p h o l i p i d and l i p o p o l y s a c c h a r i d e , the scheme was modified as f o l l o w s . The c e l l envelope f r a c t i o n was prepared as described. The inner membrane, l i p o p o l y s a c c h a r i d e and phos-p h o l i p i d were ext r a c t e d by 2% T r i t o n X-100 i n 10 mM Hepes b u f f e r , pH 7.4, conta i n i n g 5 mM EDTA at 0°C f o r 30 min ( 6 7 , 8 l ) . The e x t r a c t e d membrane was recovered by c e n t r i f u g a t i o n at 120,000 x g f o r 1 h. The e x t r a c t i o n was repeated once and the r e s u l t i n g p e l l e t was washed twice with d i s t i l l e d water at 0°C. P r o t e i n B was ex t r a c t e d from t h i s p e l l e t by treatment with 0.5% SDS at 37°C f o r 1 h and f u r t h e r p u r i f i e d by g e l f i l t r a t i o n i n 1% SDS i n the double column system of Sephadex G-100 (5 x 40) and Sepharose 6B (5 x 40). In experiments where p r o t e i n s B and B* were to be compared, samples were prepared by d i v i d i n g a s o l u t i o n c o n t a i n i n g p r o t e i n B i n t o two equal p a r t s and heat-ing one h a l f at 100°C f o r 15 min. P u r i f i c a t i o n of p r o t e i n A P r o t e i n A was ext r a c t e d from the p r o t e i n A-peptidoglycan complexes by treatment with 80 ml of 2% SDS at 100°C f o r 15 min per o r i g i n a l 50 g (wet weight) of c e l l s . The peptidoglycan was removed by c e n t r i f u g a t i o n at 120,00 x g f o r 1 h at 15°C. The SDS e x t r a c t was e i t h e r d i a l y z e d against d i s t i l l e d water to remove the detergent and f r e e z e - d r i e d , or concentrated 5 - f o l d by u l t r a f i l t r a t i o n through a PM 10 u l t r a f i l t e r i n a Model 420 c e l l (Amicon Cor p o r a t i o n ) . A s o l u t i o n of p r o t e i n A (10 ml at 1-5 mg/protein/ml) was a p p l i e d to the double column g e l f i l -t r a t i o n system described i n the p u r i f i c a t i o n of p r o t e i n B and 58 the protein was eluted with 0.1 M sodium phosphate buffer, pH 7.2, containing 1% SDS. In some cases, a single column (1 x 45 cm) of Bio-Gel P-150, equilibrated with the same buffer was used. The fractions containing protein A were pooled, dialyzed against d i s t i l l e d water and freeze-dried. The p u r i f i e d protein was subjected to amino acid analysis and cyanogen clea-vage . • P u r i f i c a t i o n of protein Bp, the pronase-resistant fragment derived from protein B The Triton-extracted c e l l envelope preparation was digested with pronase at an enzyme: protein r a t i o of 1:25 for 2 h at 37°C. The digested membrane was recovered by centrifugation for 1 h at 120,00 x g and extracted with 0.5% SDS at 37°C for 1 h. This extraction step removed- a l l the proteins from the outer membrane except proteins A and Bp. The extracted membrane was recovered by centrifugation at 15°C and re-extracted with 1% SDS at 100°C for 15 min. The r e s u l t i n g extract was freed of peptidoglycan by centrifugation at 120,000 x g for 1 h at 15°C and l y o p h i l i z e d a f t e r removal of the detergent by d i a l y s i s against d i s t i l l e d water at 22°C. Proteins A and Bp, contained in t h i s extract were readily separated by gel f i l t r a t i o n i n a Bio-Gel P-150 column (1 x 45 cm), equilibrated with 0.1 M sodium phosphate buffer, pH 7-2, containing 1% SDS, or i n the double column system of Sephadex G-100 and Sepharose 6B already des-cribed. Fractions containing protein Bp were pooled, dialyzed against d i s t i l l e d water and l y o p h i l i z e d . The amino acid com-59 position of the p u r i f i e d protein was determined. Preparation of lipopolysaccharide ( l 6 l ) Ten grams (wet weight) of c e l l s was suspended to 20 ml with d i s t i l l e d water and held at 65°C. Twenty ml of 90% o phenol preheated to 65 C was added with vigorous mixing and the mixture was kept at t h i s temperature-for 15 min. After cooling to about 10°C i n an ice bath, the emulsion was centrifuged at 4,000 x g for 10 min, which resulted i n the formation of three layers, a water layer, a phenol layer and an insoluble residue at the interphase. The water phase was removed and the phenol layer and the insoluble residue were treated at 65°C with ano-ther 20 ml portion of d i s t i l l e d water. The water extracts were combined and washed by shaking with an equal volume of 90% phenol. The water extract was dialyzed for 3-4 days at 4°C against several changes of d i s t i l l e d water (8 1) to remove phenol and low molecular weight material. The extract was then dialyzed against 4 1 of 0.01 M Tris-acetate buffer, pH 7-5, containing 0.1 M NaCl for 2 h at room temperature. RNase (500 ,Mg) was added to the extract and the absorbance at 260 nm was measured both inside and outside the d i a l y s i s sac. When no further change was observed, a further 500 ug of RNase was added. The extract was then dialysed against 4 1 of 0.1 M MgSO^ for 2 h at room temperature. DNase (100 ug) was added and the absorb-ance at 260 nm was monitored. An additional 100 ug of DNase was added when no further change i n absorbance was observed. Two volumes of ice- c o l d ethanol were added and the solution was 60 stored at -20~C overnight. F i n a l l y , the solution was c e n t r i -fuged at 20,000x g for. 30 min i n sealed stainless steel tubes, "the p e l l e t resuspended i n d i s t i l l e d water and freeze-dried. The"dried lipopolysaccharide was stored at -20°C u n t i l used i n reconstitution studies. Small-scale preparation of t o t a l outer membrane proteins (110, 179) The procedure used to prepare the outer membrane proteins from 1 g (wet weight) of c e l l s was similar to that described for the preparation of Triton-extracted c e l l envelope. The c e l l s were suspended to a volume of 10 ml with 10 mM Tris-HCl, ph 7-5, containing 10 mM MgCl^ and broken by passage through a French Pressure C e l l . The c e l l envelope f r a c t i o n was prepared by centrifugation and then extracted with 15 ml of 1.5% Triton X-100 i n 0.1 M Tris-HCl, pH 8.0 containing 35 mM MgCl 2 at 22°C. The c e l l wall was recovered by centrifugation at 120,000 x g for 1 h and extracted with 5 ml of 1% SDS at 100°C for 15 min. The insoluble peptidoglycan was removed from the solution of outer membrane proteins by centrifugation at 100,000 x g for 1 h at 15°C. The s o l u b i l i z e d proteins were subsequently analyzed by SDS-polyacrylamide gel electrophoresis. Digestion of membrane preparations with p r o t e o l y t i c enzymes The c e l l s (4 g, wet weight) or c e l l envelopes derived from this weight of c e l l s , were suspended to 60 ml i n 0.1 M Tris-HCl, pH 8.0, containing 35 mM MgCl^sand warmed to 37°C. The pro-61 t e o l y t i c enzyme at an enzyme : protein r a t i o of 1:25 was added and the suspension was incubated at 37°C for 2 h. Samples (15 ml) were removed at timed i n t e r v a l s , 0.75 ml of a phenylmethyl-sulfonyl f l u o r i d e solution (7mg/ml i n ethanol) was added immediately, and the solution was cooled i n i c e . The digested 2 c e l l s were broken i n a French Pressure c e l l at 20,000 lb/inch and the c e l l envelope f r a c t i o n was recovered by centrifugation under the usual conditions. The inner membrane was removed from the c e l l envelope by Triton extraction at 22°C. The outer mem-brane proteins were s o l u b i l i z e d from the Triton-extracted c e l l envelope by 1$ SDS at 100°C for 15 min and analyzed by SDS-polyacrylamide gel electrophoresis. Crosslinking of outer membrane proteins. Membrane samples were suspended at about 1 mg protein/ml of 0.2 M triethanolamine buffer, pH 8.5. Various amounts of DSP, from a freshly prepared solution at 20 mg/ml i n dimethyl sulfoxide were added to 100 J J I portions of membrane preparation at 22°C. The reaction was allowed to proceed for 30 sec and then excess 1 M Tris-HCl buffer, pH 8.5, was added i n a volume equal to the volume of DSP added. F i n a l l y , 2-9 volumes of a solution containing 1$ SDS, 10$ g l y c e r o l and 0.005$ Bromophenol Blue i n 0.0625 M Tris-HCl, pH 6.8 (SDS-electrophoresis sample buffer without 2-mercaptoethanol) was added and the solution was heated at 100°C for. 3 min to s o l u b i l i z e the proteins. Membrane samples i n 0.2 M triethanolamine buffer, pH 8.5 were also crosslinked at 22°C by 0.05-1.0$ glutaraldehyde 62 ( v / f i n a l volume of s o l u t i o n ) . Removal of SDS from p r o t e i n samples (144-146) D i a l y s i s : SDS could be removed from p r o t e i n samples by d i a l y s i s against d i s t i l l e d water. The usual proced-ure-was an . i n i t i a l d i a l y s i s step against 100 volumes of d i s t i l l e d water at 22°C f o r 24 h. The d i s t i l l e d water was replaced d a i l y and d i a -l y s i s was continued at 4°C f o r 2 to 5 days. Not a l l of the SDS could be removed from the p r o t e i n by t h i s method since the pre-sence of detergent could be detected even a f t e r extensive d i a l y -s i s . Acetone p r e c i p i t a t i o n : P r o t e i n was r e a d i l y p r e c i p i t a t e d from SDS s o l u t i o n s by a d d i t i o n of 9 volumes of i c e - c o l d acetone f o l -lowed by storage of the mixture at -20°C f o r 24 h. P r o t e i n was recovered by c e n t r i f u g a t i o n at 10,000 x g f o r 20 min at 4 C i n sealed s t a i n l e s s s t e e l tubes. Ion exchange: Dowex AG1-X4 was converted to the acetate form by s e q u e n t i a l e q u i l i b r a t i o n w i t h 2 N sodium hydroxide, 4 N ace-t i c a c i d , and 50 mM T r i s - a c e t a t e b u f f e r , pH 7-5• The r e s i n was washed e x t e n s i v e l y with d i s t i l l e d water between each e q u i l i b r a -t i o n step. The r e s i n was dispensed i n t o disposable p i p e t t e s to give 1 ml s e t t l e d bed volume, and e q u i l i b r a t e d w i t h 50 mM T r i s -acetate b u f f e r , c o n t a i n i n g 6 M urea. P r o t e i n samples (1 ml) i n SDS s o l u t i o n s were made 6 M i n urea by a d d i t i o n of s o l i d urea, a p p l i e d to the column, and el u t e d at 22 C with 2 ml of the f i n a l e q u i l i b r a t i o n b u f f e r . The columns were discarded a f t e r each 63 use . Cyanogen bromide cleavage of proteins (l48 ,28l) Freeze-dried samples containing ca_. 1 mg of protein were dissolved i n 1.5 ml of 70$ formic acid. Cyanogen bromide (5-100 f o l d excess over protein by weight) was added as a s o l i d or as a solution (1 g/10 ml) i n 70$ formic acid. The solution was stored i n the dark for 24 h at 22°C. Twenty volumes of d i s t i l -led water were added and the sample was freeze-dried, and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. A n a l y t i c a l techniques SDS-polyacrylamide tube gel electrophoresis - system 1 (79,90) Reagents: 0.1 M sodium phosphate buffer pH 7.2, containing 0.1$ SDS Cyanogum 4 l g e l l i n g agent (a mixture of 95$ w/w acrylamide and 5$ w/w bisacrylamide Ammonium persulfate TEMED Sample solvent (1$ of SDS, 4 M urea, 0.1$ 2-mercaptoethanol and 0.01$ Bromophenol Blue Procedure: A 10$ polyacrylamide gel was ...formed by dissolving 1.0 g of Cyanogum to 10 ml i n sodium phosphate buffer. This solution was deaerated by suction with a water pump and then 10 of TEMED and 10 mg of ammonium persulfate were added. After the ammonium persulfate had dissolved, the gel solution was d i s -64 pensed i n t o e i g h t g l a s s tubes (7-5 x 0.5 cm) which were plugged at the bottom with polythene c l o s u r e s . The g e l sur f a c e was o v e r l a i d with d i s t i l l e d water and the ge l s were l e f t to p o l y -merize f o r 1 h at room temperature. A f t e r p o l y m e r i z a t i o n was complete the water o v e r l a y was removed and the tubes were p l a c e d i n a Shandon g e l e l e c t r o p h o r e s i s apparatus. Sodium phosphate b u f f e r was added to the upper and lower r e s e r v o i r s and the sam-p l e (10-100 y u l ) was l a y e r e d on the g e l s u r f a c e with a m i c r o p i p -e t t e . E l e c t r o p h o r e s i s towards the anode was c a r r i e d out at 22°C at a constant cu r r e n t of 10 mA per tube u n t i l the dye f r o n t was about 0.5 cm from the bottom of the g e l s (2.5 h ) . The g e l s were then removed f o r s t a i n i n g . SDS-polyacrylamide tube g e l e l e c t r o p h o r e s i s at an a l k a l i n e pH-System 2 (79) P o l y a c r y l a m i d e g e l s were prepared as alre a d y d e s c r i b e d . The upper r e s e r v o i r b u f f e r was 0.1 M sodium phosphate b u f f e r , pH 11.4, c o n t a i n i n g 0.1% SDS and the lower r e s e r v o i r b u f f e r was 0.1 M sodium phosphate b u f f e r , pH 4.2, c o n t a i n i n g 0.1% SDS. E l e c -t r o p h o r e s i s was c a r r i e d out as d e s c r i b e d f o r System 1, except that a nylon mesh around the bottom of the g l a s s tube was r e q u i r e d to prevent the g e l from s l i p p i n g from the tube. P r e p a r a t i o n of samples of SDS-polyacrylamide tube g e l e l e c t r o -p h o r e s i s P r e e z e - d r i e d p r o t e i n samples were d i s s o l v e d i n the sample s o l v e n t (1% SDS 0.1% 2-mercaptoethanol and 4 M urea) at concen-65 t r a t i o n s of p r o t e i n of 0.1 to 2 mg/ml. So l u t i o n s c o n t a i n i n g p r o t e i n were d i l u t e d w i t h 1 volume of a 2X concentrated sample so l v e n t . Unless stated otherwise, a l l samples f o r e l e c t r o -phoresis were heated at 100°C f o r 5 min. S t a i n i n g polyacrylamide gels f o r p r o t e i n The polyacrylamide gels were removed from the tubes and the dye f r o n t was marked with black I n d i a ink ap p l i e d with a f i n e needle. The gels were placed i n f i l t e r e d s t a i n i n g s o l u t i o n (0.25$ Coomassie Blue i n 25$ methanol, 10$ a c e t i c acid) and heated at 60°C f o r the f o l l o w i n g times: 5$ g e l , 30 min, 7-5$ g e l , 40 min; 10$ g e l , 50 min and 12.5$ g e l , 60 min. The gels were destained at 60°C wit h 2-3 successive changes of 10$ a c e t i c a c i d f o r 1 h each change. F i n a l l y , the gels were kept i n f r e s h 10$ a c e t i c a c i d overnight. The gels were stored i n 10$ a c e t i c a c i d and then scanned at 550 nm i n a G i l f o r d Model 240 Spectro-photometer equipped w i t h a l i n e a r t r a n s p o r t e r . The R.^  of a stained p r o t e i n band was c a l c u l a t e d as: R _ distance migrated by p r o t e i n band f distance migrated by Bromophenol Blue S t a i n i n g polyacrylamide gels f o r carbohydrate (167) Carbohydrate was detected i n polyacrylamide gels by the p e r i o d i c a c i d - S c h i f f base r e a c t i o n . The gels were st a i n e d by se q u e n t i a l immersion i n : (a) 12.5$ t r i c h l o r o a c e t i c a c i d f o r 30 min, (b) d i s t i l l e d water f o r 1 min, 66 (c) 1% p e r i o d i c a c i d in. 3% a c e t i c a c i d f o r 50 min, (d) d i s t i l l e d water, o v e r n i g h t , (e) P u s h s i n - s u l f i t e s t a i n f o r 50 min i n the dark, (f ) 0.5% m e t a b i s u l f i t e f o r 10 min, three times, and (g) d i s t i l l e d water, o v e r n i g h t . The g e l s were s t o r e d i n 10% a c e t i c a c i d . The P u s h s i n - s u l f i t e s t a i n was prepared by d i s s o l v i n g 160 mg o f potassium metabi-s u l f i t e and 0.21 ml of concentrated HC1 i n 20 ml of d i s t i l l e d water. B a s i c F u s h s i n dye (80 mg) was added and the s o l u t i o n was s t i r r e d g e n t l y f o r 2 h. The s o l u t i o n was allowed to stand f o r a f u r t h e r 2 h, when a small amount of Darco c h a r c o a l (grade G-60) was added and the s o l u t i o n was f i l t e r e d w i t h i n 15 min. The c o l o u r l e s s dye was s t o r e d at 4°C and was usable u n t i l the s o l u t i o n turned pink. The 0.5% m e t a b i s u l f i t e s o l u t i o n was prepared j u s t before use. Discontinuous SDS-polyacrylamide g e l e l e c t r o p h o r e s i s - L a e m m l i system (83,92) Reagents: S e p a r a t i n g g e l b u f f e r , 4 X concentrated (1.5 M T r i s -HCl, pH 8.8, c o n t a i n i n g 0.4% SDS) S t a c k i n g g e l b u f f e r , 4 X concentrated (0.5 M T r i s - H C l , pH 6.8, c o n t a i n i n g 0.4% SDS) R e s e r v o i r b u f f e r , 5 X concentrated (0.125 M T r i s and 0.960 M g l y c i n e , pH 8.3) Sample b u f f e r (0.0625 M T r i s - H C l , pH 6.8, c o n t a i n i n g ]% SDS, 1% 2-mercaptoethanol and 10% g l y c e r o l ) Acrylamide (30%. (w/v) acrylamide and 0.8% (w/v) b i s a c r y l a m i d e i n d i s t i l l e d water) 67 TEMED Ammonium persulfate (10% (w/v) aqueous solution, prepared fresh d a i l y . Only high quality SDS was used ("specially pure" grade from B r i t i s h Drug House Ltd.). The pH of the reservoir was not adjusted, but the solution of Tr i s and glycine (ammonia-free) consistantly gave a pH of 8.3 when dil u t e d . The protocol used to prepare the diff e r e n t separating gel concentration i s given below: Volume (ml) per 15 ml solution % gel Separating gel buffer D i s t i l l e d water Acrylamide TEMED 10% persulfate 7.5 3.75 7.50 3.75 15 u l 75 u l 9.0 3.75 6.75 4.50 15 u l 75 u l 10 .0 3-75 6.25 5.00 15 u l 75 u l 12.0 3.75 5.25 6.00 15 u l 75 u l 15.0 3.75 3.75 7.50 15 u l 75 u l Stacking gel solution (4% acrylamide) was prepared by dis s o l v i n g 0.2 g of Cyanogum i h a mixture of 1.25 ml of stock stacking gel buffer and 3-75 ml"of d i s t i l l e d water. TEMED (10 ul) and 10% ammonium persulfate (15 ul) were then added to the solution. Electrophoresis was carried out using a Bio-Rad Model 220 68 s l a b g e l a p p a r a t u s . G e l s were f o r m e d between two g l a s s p l a t e s , s e p a r a t e d by a 0.75 mm t h i c k s p a c e r , g r e a s e d w i t h a s m a l l amount o f C e l l o S e a l ( F i s h e r C h e m i c a l C o . ) . The p l a t e s were mounted on t h e c e n t r e c o r e o f t h e a p p a r a t u s and t h e b o t t o m was s e a l e d w i t h t h e s e a l i n g b a r . A t e n o r t w e n t y s l o t f o r m e r was i n s e r t e d between t h e p l a t e s and d e a e r a t e d s e p a r a t i n g g e l s o l u t i o n (15 ml) was added w i t h a P a s t e u r p i p e t t e . The s l a b " s a n d w i c h " was f i l l e d t o 1 cm below t h e s l o t f o r m e r and was t h e n o v e r l a i d w i t h t - b u t a n o l . The g e l was a l l o w e d t o p o l y -m e r i z e a t 22°C f o r 30 min and t h e n t h e s l o t f o r m e r was removed. The t - b u t a n o l was a b s o r b e d on a p i e c e o f f i l t e r p a p e r and t h e g e l s u r f a c e was washed t w i c e w i t h d i l u t e d (4-fold) s e p a r a t i n g g e l b u f f e r . The s l o t f o r m e r was r e i n s e r t e d and d e a e r a t e d s t a c k i n g g e l s o l u t i o n (5 ml) was added t o f i l l t h e g e l " s a n d -w i c h " . No o v e r l a y s o l u t i o n was r e q u i r e d and p o l y m e r i z a t i o n was a l l o w e d t o p r o c e e d a t 22°C f o r 30 min. The s l o t f o r m e r was t h e n removed c a r e f u l l y and t h e sample s l o t s were washed t w i c e w i t h d i l u t e d (4-fold) s t a c k i n g g e l b u f f e r . D i l u t e d r e -s e r v o i r b u f f e r was added t o t h e e l e c t r o p h o r e s i s chambers (1.5 1 i n t h e b o t t o m and 300 ml i n t h e t o p ) . The samples (5-25 u l i n a 20 s l o t g e l , 10-100 u l i n a 10 s l o t g e l ) were a p p l i e d t h r o u g h a f i n e p i e c e o f p o l y t h y l e n e t u b i n g a t t a c h e d t o a m i c r o -p i p e t t e . C o l d t a p w a t e r was c i r c u l a t e d t h r o u g h t h e c e n t r e c o r e o f t h e a p p a r a t u s . E l e c t r o p h o r e s i s was c a r r i e d out a t a c o n -s t a n t c u r r e n t o f 30 mA p e r g e l s l a b u n t i l t h e dye f r o n t was 1 cm f r o m t h e b o t t o m o f t h e g e l (1.5 h ) . 69 Staining of polyacrylamide slab gels After electrophoresis, the gel "sandwich" was immersed i n a large pan of water. The plates were separated and the gel was transferred to a Pyrex dish (8.5 x 8.5 x 2 inches deep) and 300 ml of staining solution (0.05$ Coomassie Blue i n 25$ i s o -propanol, 10$ acetic acid, f i l t e r e d before use) was added. The gels were stained at 22°C for 1 h. The staining solution was removed by suction and was replaced by 300 ml of 10$ acetic acid. The gel was stored i n this solution overnight. F i n a l l y , the gel was washed with several changes of fresh 10$ acetic acid u n t i l the background was clear. Analysis of DSP-crosslinked proteins by two-dimensional SDS-gel electrophoresis Crosslinked products were analyzed i n a two-dimensional SDS-polyacrylamide gel system using a Bio-Rad Model 220 slab gel apparatus. The gel solutions were prepared as already described. Crosslinked proteins were f i r s t resolved on a 7-5$ polyacrylamide slab g e l , 0.75 mm thick. A 3 mm wide s t r i p from th i s gel was placed on top of a previously-formed second dimension gel and embedded i n warm (45°C) 1% agarose, 2$ 2-mercaptoethanol i n f o u r - f o l d diluted stacking gel buffer. The agarose was allowed to cool for 5 min. The time taken from stopping the f i r s t dimension electrophoresis to s t a r t i n g the second dimension did not. exceed 15 min. The second dimension gel was stored overnight,, covered with a layer of f o u r - f o l d 70 d i l u t e d s t a c k i n g g e l b u f f e r c o n t a i n i n g 10%. 2 - m e r c a p t o e t h a n o l . The b u f f e r o v e r l a y was removed j u s t b e f o r e i n s e r t i o n o f t h e f i r s t d i m e n s i o n s t r i p . W i t h t h e s e c o n d d i m e n s i o n g e l , a c o n -s t a n t c u r r e n t o f 40 mA was a p p l i e d u n t i l t h e dye was 1 cm f r o m t h e b o t t o m o f t h e g e l (2.5 h ) . The t w o - d i m e n s i o n a l g e l s were s t a i n e d w i t h C o o m a s s i e B l u e as d e s c r i b e d f o r 0.75 mm t h i c k s l a b g e l s e x c e p t t h e s t a i n i n g t i m e was 2 h. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e p r e s e n c e o f u r e a R e a g e n t s : G e l b u f f e r (0.375 M T r i s - H C l , pH 8.7, c o n t a i n i n g 6 M u r e a ) R e s e r v o i r b u f f e r (10 mM T r i s , 80 mM g l y c i n e , pH 8.3) Sample b u f f e r (50 mM T r i s - H C l , pH 6.8, c o n t a i n i n g 6 M u r e a , 1% 2 - m e r c a p t o e t h a n o l and 0.01% Bromo-p h e n o l B l u e ) TEMED Ammonium p e r s u l f a t e P r o c e d u r e : A 5% p o l y a c y l a m i d e g e l was f o r m e d by d i s s o l v i n g 0.5 g o f Cyanogum t o 10 ml i n t h e g e l b u f f e r . The s o l u t i o n was de-a e r a t e d and 10 u l TEMED and 10 mg o f ammonium p e r s u l f a t e were added. The g e l s o l u t i o n was d i s p e n s e d i n t o e i g h t g l a s s t u b e s and t h e g e l s u r f a c e was o v e r l a i d w i t h d i s t i l l e d w a t e r . The g e l s were l e f t t o p o l y m e r i z e f o r 1 h at 22°C b e f o r e t h e w a t e r o v e r l a y was removed and t h e g e l s p l a c e d i n a Shandon g e l e l e c -t r o p h o r e s i s a p p a r a t u s . The r e s e r v o i r b u f f e r was added t o t h e u p p e r and l o w e r chambers and t h e samples were a p p l i e d t o t h e g e l s u r f a c e w i t h a m i c r o p i p e t t e . E l e c t r o p h o r e s i s was c a r r i e d 71 out at 22 C at a constant current of 3 mA per tube u n t i l the dye front was 0.5 cm from the bottom of the gel (2 h). Determination of molecular weight by gel f i l t r a t i o n Gel f i l t r a t i o n was performed at 22°C using a column (1 x 37 cm) of Sepharose 6B equilibrated with 0.1 M sodium phosphate buffer, pH 7-2, containing 1$ SDS. Standard proteins were dissolved at 1 mg/ml. i n the column buffer containing 0.1$ 2-mercaptoethanol and heated at 100°C for 5 min before application to the column. The elution positions of the proteins was determined by assaying a l l fractions (1 ml) for protein by the method of Lowry et_ a l . (171) and by subjecting a l l fractions to analysis by SDS-polyacrylamide electrophoresis. The void vo-lume (V0) and t o t a l volume (V ) of the column was determined from the elution positions of Blue Dextran, and sodium chloride (detected by ionic strength) or triethanolamine (detected by the Lowry protein assay) respectively. A c a l i b r a t i o n curve was obtained by p l o t t i n g log.: (molecular weight) versus K , l u a v where V - V K = e ° av — t o Sucrose density gradient centrifugation of proteins B-and B* Sucrose density gradient centrifugation was performed on 5-20$ sucrose gradients prepared i n 1 mM Tris-acetate buffer, pH 7.0,. containing either 0..1, 0.2, or 0.3$ SDS. Samples (0.20 ml), containing ca. 1 mg protein/ml were layered on top of 5.0 72 ml gradients and centrifuged i n a Spinco SW 50-L rotor at o 45,000 rpm for 22 h at 20 C. After the run, 20 ten-drop fractions were collected from the top of the gradient by pump-ing a 60% sucrose solution, containing Blue Dextran, through the bottom of the tube. The r e f r a c t i v e index and the protein content of each f r a c t i o n were measured. Sedimentation co-e f f i c i e n t s and p a r t i a l s p e c i f i c volumes were calculated as described i n the Appendix. Measurement of binding of SDS to proteins The amount of SDS bound to protein B and B* was determined by equilibrium d i a l y s i s . Protein samples (1 ml) were dialyzed at 22°C against 1 1 of 1 mM Tris-acetate buffer, pH 7-0, con-taining various concentrations of SDS. The amount of SDS i n -side and outside the d i a l y s i s membrane was determined as described by Hayashi (169). Measurement of i n t r i n s i c v i s c o s i t y I n t r i n s i c v i s c o s i t e s were measured with a Cannon-Manning viscometer, immersed i n a water bath thermostated to 25± 0.01° C. The method used to calculate the i n t r i n s i c v i s c o s i t y i n the Appendix. Spectral measurements U l t r a v i o l e t spectra were taken at 22°C with a Perkin-Elmer model 356 Dual wavelength Double Beam Spectrophotometer. Ci r c u l a r dichroism and o p t i c a l rotatory dipersion measurements 73 were made at 25^C with a J a s c o J - 2 0 Automatic re c o r d i n g Spectropolarimeter. Amino a c i d a n a l y s i s The p r o t e i n sample (0.5-2 mg) was placed i n a 13 x 100 mm Pyrex t e s t tube and 0.5-1.0 ml of 6 N HC1 was added. The tube was sealed under vacuum and the s o l u t i o n was heated at 110°C f o r 24, 48 or 72 h. A f t e r h y d r o l y s i s , the sample was d r i e d under vacuum and then d i s s o l v e d i n 100-500 u l of d i s t i l l e d water. Samples were analyzed on a s i n g l e column of Aminex A-5 r e s i n using sodium c i t r a t e b u f f e r s , on a Beckman Model 120.C amino a c i d analyzer or on an instrument constructed by Mr. J . Durgo of the Department of Biochemistry, U n i v e r s i t y of B r i t i s h Columbia. The content of amino acids was determined by c a l c u l a t i n g the peak areas f o r each amino a c i d . Determination of cyste i n e (176) Reagents: Sodium phosphate b u f f e r (0.1 M sodium phosphate, pH 8.0, c o n t a i n i n g 2% SDS and 0.05% EDTA) DTNB (40 mg of 5 , 5 ' - d i t h i o b i s (2-nitrobenzoic acid) i n 10 ml of 0.1 M sodium phosphate b u f f e r , pH 8.0 Procedure: The sample cont a i n i n g 0.01-0.04 ^moles of p r o t e i n was d i s s o l v e d i n 6 ml of sodium phosphate b u f f e r and 0.1 ml of DTNB s o l u t i o n was added to each of two three ml p o r t i o n s . The colour was allowed to develop f o r 15 min at 22°C and then the absorbance was measured at 410 nm against a reagent blank. A standard curve was also constructed using c y s t e i n e . 74 Amino-terminal analysis Protein samples were dissolved to 'ca. 1 mg/ml i n 0.5 M NaHCO^, pH 9-8, containing 1$ SDS and 0.5 volumes of a dansyl chloride solution (5 mg/ml i n acetone) was added. The dansy-l a t i o n reaction was allowed to proceed for 20 min at 37°C, then the protein was precipitated by addition of 2 volumes of i c e -cold 20$ t r i c h l o r o a c e t i c e acid. The protein was recovered by centrifugation at 33000 x g for 5 min. The prec i p i t a t e was washed once with 1 N HC1 to remove dansylic acid. The sample was hydrolyzed i n 6 HC1 at 105°C for 6 h i n an evacuated glass tube and then evaporated to dryness i n a heated vacuum desicca-tor. The residue was dissolved i n 50$ aqueous pyridine (v/v) and applied to one side of a 5 x 5 cm polyamide plate. A stan-dard mixture of dansyl-amino acids was applied on the reverse side of the plate. The sample was resolved by chromatography in Solvent I (1.5$ (v/v) aqueous formic acid), dried and exami-ned under u l t r a v i o l e t l i g h t . The plate was turned 90° and subjected to chromatography i n a second dimension with Solvent II (benzene-acetic acid, 9:1)» dried and examined under u l t r a -v i o l e t l i g h t . Fluorescent spots were i d e n t i f i e d by comparison to the standard mixture of dansyl-amino acids. Preparation of dansyl-amino acids Amino acids (10 mM) i n 0.5 M NaHCO^ buffer, pH 9.8 were mixed with an equal volume of dansyl chloride i n acetone such that there was-.a 5-fold molar excess of dansyl chloride. The reaction was carried out for. 30 min at 37°C and then terminated 75 by a d d i t i o n of 1/30 volume of 88% formic a c i d . This mixture was used d i r e c t l y f o r s p o t t i n g on the chromatogram. Determination of p r o t e i n P r o t e i n was determined by the method of Lowry e_t a l . (171) , except samples c o n t a i n i n g membrane preparations were made 1% i n SDS and heated at 100°C f o r 15 min before assaying f o r p r o t e i n . Bovine serum albumin was used t o construct a standard curve. Determination of carbohydrate (273) Samples ( 1 ml co n t a i n i n g 0-1 mg carbohydrate/ml) were mixed with 50 p.1 of 80% phenol and then 2.5 ml concentrated . H 2S0i| was added w i t h r a p i d mixing. The absorbance of the so-l u t i o n was measured at 490 nm a f t e r 30 min. Glucose (0.25 umoles/ml) gave an absorbance of 1.0. Determination of l i p o p o l y s a c c h a r i d e (274,275) Reagents: 0.04 N HIO^ i n 0.125 N H SO^ 3% sodium a r s e n i t e i n 0.5 N HC1 0.3% t h i o b a r b i t u r i c a c i d Procedure: Samples (0.2 ml, conta i n i n g 0^10 nmoles of 3-keto-2-deoxyoctonicv acid) were mixed w i t h 0.25 ml of HIO^ s o l u t i o n and incubated at 22°C f o r 20 min. Sodium a r s e n i t e s o l u t i o n (0.5 ml) was added w i t h shaking and the s o l u t i o n was allowed to stand f o r 2 min. T h i o b a r b i t u r i c a c i d s o l u t i o n (2 ml) was then added and the s o l u t i o n was heated at 100°C f o r 20 min. The 76 absorbance of the s o l u t i o n was measured, at 548 nm. An absorbance of 1.9 was obtained with 100 nmoles of 3-keto-2-deoxyoct onic a c i d . Determination of phosphate i n p r o t e i n samples (280) Reagents: 10$ Mg(NO^) 2 • H~20 i n ethanol Ascorbic-molybdate reagent (1 part 10$ a s c o r b i c a c i d to 6 parts of 0.42$ ammonium molybdate-R^O i n 1 N H 2S0^j prepared fresh) 1 N HC1 Procedure: Samples (0.01-0.05 ml c o n t a i n i n g 0-50 nmoles) were placed i n 13 x 100 mm Pyrex tubes and mixed w i t h 0.05 ml of the magnesium n i t r a t e reagent. The mixture was evaporated with r a p i d shaking to a white ash over a strong flame u n t i l the brown fumes had disappeared. A f t e r the tube had cooled, 0.3 ml of 1 N HC1 was added. The tube was capped with a marble and heated i n a b o i l i n g water bath f o r 15 min to hydrolyze to i n o r g a n i c phosphate any pyrophosphate formed i n the ashing pro-cedure. Ascorbic-molybdate mixture (0 .7 ml) was added to tube and a f t e r 20 min at 45°C the absorbance of the s o l u t i o n was read at 660 nm against a blank s o l u t i o n . An absorbance of 0.24 was obtained with 10 nmoles of phosphate as Na 2HP0^ 77 RESULTS Use of various c e l l fractions A number of di f f e r e n t c e l l fractions were used during the course of the research described i n this t h e s i s . Figure 6 i l l u s t r a t e s diagramatically the procedures used to prepare these c e l l fractions and the selective extraction of outer mem-brane proteins by SDS at di f f e r e n t temperatures. Spheroplasts: Cells could be lysed after lysozyme-EDTA treatment to produce spheroplasts, composed of inner and outer membranes. Outer membrane was prepared from spheroplasts by s o l u b i l i z a t i o n of the inner membrane with T r i t o n i n the presence of Mg . Outer membrane was also released during spheroplast formation and could be prepared by centrifugation without detergent treatment. These preparations served as controls i n studies on the organization of proteins i n the outer membrane. Envelope: Spheroplast preparations were not a convenient source of material for the large scale i s o l a t i o n of outer mem-brane proteins and therefore c e l l wall preparations (Triton-extracted c e l l envelope) were used for t h i s purpose. The outer membrane proteins (ca. 5 mg protein/g wet weight of c e l l s ) were prepared from the c e l l wall f r a c t i o n by s o l u b i l i z a t i o n with SDS at 100°C leaving an insoluble residue of peptidoglycan and covalently-bound l i p o p r o t e i n . The proteins could also be d i f f e r e n t i a l l y extracted from the :cell wall by SDS at lower temperatures. Thus, extraction at. 37°C to give extract 1 c e l l s • lysozyme-EDTA .<- OM <j- IM s p h e r o p l a s t A o l u b i l i z e d X IM T r i t o n - M g OM e x t r a c t 1 ( p r o t e i n B) F r e n c h p r e s s OM r e l e a s e d OM s o l u b i l i z e d IM c e l l w a l l SDS, 37 p r o t e i n s A t o F SDS, 100 F i g . 6. S c h e m a t i c r e p - e x t r a c t 2 r e s e n t a t i o n o f p r o c e d u r e s u s e d t o p r e p a r e c e l l f r a c t i o n s and t o s o l u b i l i z e o u t e r membrane p r o t e i n s . OM, o u t e r membrane; IM, i n n e r membrane; PG, p e p t i d o g l y c a n ; LP, l i p o p r o t e i n . SDS, 100 p r o t e i n s B t o F P r o t e i n oo SDS, 60° \mm\. p r o t e i n s A-PG complex SDS, 100 bound LP PG 79 (2 mg p r o t e i n / g wet weight of c e l l s ) released p r i m a r i l y p r o t e i n B. This e x t r a c t was used i n the p u r i f i c a t i o n of p r o t e i n B. A f u r t h e r e x t r a c t i o n at" 100°C to give e x t r a c t 2 (3 mg p r o t e i n / g wet weight of c e l l s ) s o l u b i l i z e d the remaining outer membrane p r o t e i n s , except f o r the bound l i p o p r o t e i n . E x t r a c t i o n of the c e l l w a l l with SDS at 60 C removed a l l p r o t e i n s except p r o t e i n A and the bound l i p o p r o t e i n . P r o t e i n A could be subsequently s o l u b i l i z e d by SDS at 100°C and t h i s e x t r a c t was used i n the p u r i f i c a t i o n of p r o t e i n A. The p u r i f i c a t i o n and p r o p e r t i e s of pr o t e i n s A and B are presented i n the f i r s t part of the r e s u l t s s e c t i o n . The o r g a n i z a t i o n of the pr o t e i n s i n the outer membrane was studied by p r o t e o l y t i c d i g e s t i o n , covalent labelling;.;>.and• cross-l i n k i n g . The r e s u l t s obtained w i t h the d i f f e r e n t preparations were compared to r u l e out gross a r t i f a c t u a l rearrangement of the p r o t e i n s during p r e p a r a t i o n of the c e l l f r a c t i o n s . The r e s u l t s of these studies are presented i n the second part of t h i s s e c t i o n . P u r i f i c a t i o n and p r o p e r t i e s of the outer membrane p r o t e i n s Major p r o t e i n s of the outer membrane The p r o t e i n s of the outer membrane were e x t r a c t e d from the. c e l l w a l l by 1% SDS at 100°C f o r 15 min and then r e s o l v e d by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s using d i f f e r e n t b u f f e r systems ( P i g . 7 ) . E l e c t r o p h o r e s i s i n phosphate b u f f e r at pH 7.2 d i d not r e s o l v e the major outer membrane p r o t e i n s A and B (-scan. 1). 80 A / B P i g . 7. R e s o l u t i o n o f o u t e r membrane p r o t e i n s by S D S - p o l y a c r y -l a m i d e g e l e l e c t r o p h o r e s i s . S c an 1, 10$. p o l y a c r y l a m i d e g e l r u n i n s y s t e m 1; s c a n 2, 10$. g e l r u n i n s y s t e m 2; s c a n 3,12.5$ g e l r u n i n t h e Laemmli s y s t e m . A l l samples were h e a t e d a t 100°C f o r 5 min. G e l s were s t a i n e d w i t h C o o m a s s i e B l u e and s c a n n e d a t 550 nm. 81 The apparent molecular weight of t h i s band was 40,00.0. The major outer membrane p r o t e i n s were r e a d i l y separated i n t o three c l o s e l y spaced bands, A ± (MW 44,000), A 2 (MW. 38,000) and B (MW 33,400) by e l e c t r o p h o r e s i s at an a l k a l i n e pH. This g e l system (System 2 of Bragg and Hou (79)) was used r o u t i n e l y to res o l v e these p r o t e i n s . The use of the discontinuous e l e c t r o p h o r e s i s system designed by Laemmli (92) f u r t h e r r esolved the other outer membrane p r o t e i n s . However, p r o t e i n s A and A^ migrated as a s i n g l e band with a molecular weight of 37',000. The f a s t e s t m i g r a t i n g band seen i n a l l the g e l scans ( p r o t e i n P) i s the fre e form of the l i p o p r o t e i n (MW 7,200). The e f f e c t of heating the p r o t e i n samples i n SDS on the migra t i o n of outer membrane p r o t e i n s i s shown i n F i g . 8. Outer membrane prepared from spheroplasts by T r i t o n X-100 ex-t r a c t i o n i n the presence of Mg** was heated i n SDS-electropho-o r e s i s sample b u f f e r at 37 C f o r 1 h (scan 1). The sample i n o scan 2 was heated at 100 C f o r 5 min before a p p l i c a t i o n to the g e l . As seen, the method of sample prep a r a t i o n profoundly a l t e r e d the p a t t e r n seen i n the SDS-polyacrylamide g e l s . Heat-ing caused the depolymerization of high molecular weight aggregates c o n s i s t i n g p r i m a r i l y of p r o t e i n A and a l s o changed the m o b i l i t y of p r o t e i n B. The e f f e c t of heating on p r o t e i n B was studied f u r t h e r since the change i n apparent molecular weight might be due to a conformational change that might be f u n c t i o n a l l y s i g n i f i c a n t . Pig. 8. Effect of heating on the migration of outer membrane proteins i n SDS-polyacrylamide gels run i n system 2. Scan 1, outer membrane s o l u b i l i z e d at 37°C for 1 h i n SDS-electrophoresis buffer, scan 2, sample heated at 100°c for 5 min. 82 83 Heat-modifiability of protein B Protein B was readily s o l u b i l i z e d from the c e l l wall by extraction with 0.5% SDS at 37°C for 1 h. Sixty percent of the protein i n t h i s extract (extract 1) was protein B, with an apparent molecular weight of 28,500 as determined by SDS-polyacrylamide gel electrophoresis (Fig. 9 A ). Upon heating a solution containing 1% SDS, 0.1% 2-mercaptoethanol and 4 M urea, protein B was converted to a form B* (MW 33,400). This behaviour i s designated as heat-modifiable. The mobility of a l l other proteins i n extract 1 was unaffected by the heat t r e a t -ment (Fig. 9 A ) . In addition, the presence or absence of 2-mercaptoethanol had no effect on the migration of proteins B or B*. Measurement of peak areas of the gel scans showed a quantitative conversion of protein B to B*, proceeding more rapidly at higher temperatures (Fig. 9B). No change was detected a f t e r a 2 h incubation of the sample at HO C, while o heating at 100 C resulted i n a rapid conversion. The rates of conversion (k) are plotted as a function of r e c i p r o c a l temperature i n F i g . 10. The activation energy for t h i s process i s 36.5 k^al per mole. Protein B* did not revert back to form B on cooling or after prolonged storage. These results suggest that protein B was i r r e v e r s i b l y denatured upon heating. The behaviour of protein B on gel f i l t r a t i o n was changed by heating i n the presence of SDS (Fig. 11). The large peak (fr a c t i o n 71) absorbing at 280 nm was Triton X-100. This was 84 Hours Distance -cm F i g . 9. E f f e c t o f h e a t i n g on m i g r a t i o n o f p r o t e i n B i n S D S - p o l y a c r y l a m i d e g e l s . A: D e n s i t o m e t e r s c a n n i n g t r a c e s o f g e l s . Samples were h e a t e d a t t h e i n d i c a t e d t e m p e r a t u r e s f o r 20 min. B: Time c o u r s e o f c o n v e r s i o n o f p r o t e i n B t o B* a t v a r i o u s t e m p e r a t u r e s . P i g . 10. A r r h e n i u s p l o t f o r t h e c o n v e r s i o n o f p r o t e i n B t o B*. The r a t e o f c o n v e r s i o n , K, i s d e f i n e d as t h e change i n t h e p e r c e n t a g e o f p r o t e i n B* p e r min. 85a Fi g . 11. Effect of heating on the elution position of protein B from a column (5 x 40 cm) of Sephadex G-100 equilibrated with 0.IM sodium phosphate buffer, pH 7.2, containing 1% SDS. The freeze-dried sample of extract . 1 was dissolved i n 10 ml of column buffer containing 0.1% 2-mercaptoethanol and heated at 37°C for 1 h (sample 1, 40 mg proteins) or 100°C for 15 min (sample 2, 20 mg protein) and then applied to the column. Fractions (10 ml) were collected and analyzed by SDS-polyacryla-mide gel electrophoresis. 86 5 0 7 0 9 0 F r a c t i o n n u m b e r 87 shown by comparing the u l t r a v i o l e t spectrum of the peak f r a c t i o n ( F i g . 12,. scan 1) with T r i t o n X-100 d i s s o l v e d i n the column b u f f e r ( F i g . 12, scan 2). The amount of T r i t o n i n the peak f r a c t i o n ( F i g . 11, p r o f i l e 1) was about 0.4 mg/ml ( F i g . 13). The e l u t i o n p o s i t i o n of p r o t e i n B, i n d i c a t e d by the arrows i n F i g . 11, changed upon heating e x t r a c t 1 i n the presence of SDS. Incubation of the sample at 37°C f o r 1 h r e s u l t e d i n the e l u -t i o n of p r o t e i n B at f r a c t i o n 58. Heating at 100°C caused p r o t e i n B to e l u t e at f r a c t i o n 54. P r o t e i n B i n f r a c t i o n 58 of p r o f i l e 1 migrated as a band w i t h a molecular weight of 28,500 on SDS-polyacrylamide g e l s , while the p r o t e i n i n f r a c t i o n 54 of p r o f i l e 2 migrated as the heat-modified form B*, w i t h a molecular weight of 33,400. The a b i l i t y t o d i s t i n g u i s h p r o t e i n B from the heat-modified form B* by g e l f i l t r a t i o n suggests that the increase i n molecular weight observed i n SDS-gels was not due t o an a r t i f a c t of e l e c t r o p h o r e s i s . P u r i f i c a t i o n of p r o t e i n B* The a l t e r e d e l u t i o n p o s i t i o n of p r o t e i n B a f t e r heating suggested that p r o t e i n B could be p u r i f i e d by g e l f i l t r a t i o n i n the presence of SDS. E x t r a c t 1 was resolved i n t o w e l l -separated p r o t e i n peaks by g e l f i l t r a t i o n i n a column (5 x 40 cm) of Sephadex G-100 connected i n s e r i e s to a column of Sepharose 6B of the same dimensions, i n the presence of SDS ( F i g 14A). The l a r g e s t peak absorbing at 280 nm contained T r i t o n . P r o t e i n B could be separated from contaminating T r i t o n , most of the l i p o p o l y s a c c h a r i d e and the other p r o t e i n s of the outer membrane Pig. 12. U l t r a v i o l e t spectrum of Triton X-100 in_0.1M sodium phosphate buffer, pH 7.2, con-taining 1% SDS. Scan 1, spectrum of f r a c t i o n 71 of extract 1 resolved by gel f i l t r a t i o n (Fig 11, p r o f i l e 1); scan 2, spectrum of 0.05$ Triton X-100 i n column buffer. 88 2 4 0 2 8 0 3 2 0 W a v e l e n g t h - n m 89 g. 13- S t a n d a r d c u r v e f o r t h e d e t e r m i n a t i o n T r i t o n X-100 i n 0.IM sodium p h o s p h a t e b u f f e r , 2, c o n t a i n i n g 1% SDS by a b s o r b a n c e a t 280 nm. 90 OJ u c ro F r a c t i o n F i g . 14. Separation of p r o t e i n s of e x t r a c t 1 by g e l f i l t r a t i o n i n a column (5 x 40 cm) of Sephadex G-100 connecting i n s e r i e s to a column of Sepharose 6B of the same dimensions and i n the presence of 1% SDS. A: g e l f i l t r a t i o n of e x t r a c t 1 without p r i o r heat-treatment of sample. The f r a c t i o n s under the bar con-t a i n e d only p r o t e i n B. B: g e l f i l t r a t i o n of h a l f of p r o t e i n B from separation A f o l l o w i n g heating at 100°C f o r 15 min. The f r a c t i o n s under the bar contained only B*. The concent r a t i o n of 2-keto - 3-deoxyoctonic a c i d (KDO) i s expressed as nmoles/0.2 ml sample. The absorbance of the f r a c t i o n s was measured at 280 nm. F r a c t i o n volume, 10 ml. V , v o i d volume of double column system. 0 91 by t h i s method. P r o t e i n B contained i n f r a c t i o n s 90 to 96 migrated as a s i n g l e band of molecular weight 28,500 on SDS-polyacrylamide g e l s . When p r o t e i n B was heated at 100°C f o r 15 min i n SDS and rerun i n the double column system, i t eluted i n f r a c t i o n s 80 to 88 ( P i g . 14B). The p r o t e i n i n these f r a c -t i o n s gave a s i n g l e band of molecular weight 33, 400 on SDS-polyacrylamide g e l s . The heating procedure enabled r e s o l u t i o n of pure p r o t e i n B* from small amounts of contaminating p r o t e i n s and l i p o p o l y s a c c h a r i d e which co-eluted w i t h p r o t e i n B. Amino a c i d composition The amino a c i d a n a l y s i s of e x t r a c t s 1 and 2 are given i n Table I I . More vigorous co n d i t i o n s were re q u i r e d to obta i n e x t r a c t 2 than e x t r a c t 1, suggesting that the pr o t e i n s of e x t r a c t 2 were more t i g h t l y associated i n the membrane. This d i f f e r e n c e was not r e f l e c t e d i n the amino a c i d compositions which were s i m i l a r . The amino a c i d composition of p r o t e i n B i s given i n Table I I I . P r o t e i n B had a high content of p r o l i n e and a c i d i c a c i d s . L i t t l e c y s t e i n e was found and the p r o t e i n was only moderately hydrophobic. Cyanogen bromide cleavage Treatment of e i t h e r p r o t e i n B or B* with cyanogen bromide produced the same two fragments which had molecular weights of about 18,000 .and 15,000 on SDS-polyacrylamide gels ( P i g . 15). The r e s u l t s presented show the most complete cleavage that was obtained. G e n e r a l l y , numerous other bands with molecular 92 Table II Amino acid composition of SDS extracts Amino Acid 1 Extract 2 Lys 5.35 5.60 His 1.50 0.90 Arg 4.90 11.7 4.30 10.8 Asp 11.80 16.70 Glu 9 . 4 0 21.2 10.70 27.4 Val 6.35 6.30 Met 1.90 1.70 H e 4.15 3.00 Leu 7.30 6.20 Tyr 4.30 5.20 Phe 6.00 30.0 3.50 25.9 Thr 6.05 6.30 Ser 5.25 6.60 Pro 4.75 1.50 Gly 10. 80 9.30 Ala 10.20 12.30 JgCys Trace 37.1 Trace 36.0 Values are expressed as moles percent. The data are grouped and summed as basic, a c i d i c , hydrophobic and neutral residues. Duplicate analyses were performed after hydrolysis i n 6 N HC1 at 105°C for 25 h. Cysteine was determined as cysteic acid and trypto-phan was not measured. T a b l e I I I Amino a c i d c o m p o s i t i o n o f p u r i f i e d p r o t e i n s Amino A c i d P r o t e i n B P r o t e i n Bp P r o t e i n A From g l u c o s e -grown c e l l s (A-^) From CAA-grown c e l l s ( A 2 ) L y s 6.2 5.6 6.4 6.9 H i s 2.2 3.2 0.4 0.4 A r g 4.5 12.9 3.8 12.6 3.3 10.1 3.6 Asp n:.i .\ 10.9 15.4 15.7 G l u 8.7 19.8 9.6 20.5 8.2 23.6 . 7.8 V a l 7.6 6.0 6.4 5.7 Met 2.3 1.7 0.9 0.7 H e 5.4 5.2 4.2 4.5 L e u 7.8 7.8 8.1 7.4 T y r 4.6 6.3 6.9 7.3 Phe 3.1 3.9 6.5 6.9 T h r 6.0 30.8 5.9 30.9 5.9 33.0 5.8 S e r 4.5 5.2 3.6 4.4 P r o 5.4 3.7 1.4 1.2 G l y 10.9 11.6 12.6 12.6 A l a 9.3 9.1 9.5 9.1 hCys 0.5 0.6 0.3 0.3 36.6 10 .9 23-5 32.5 36.1 33.3 33.4 V a l u e s a r e e x p r e s s e d as moles p e r c e n t . The d a t a a r e g r o u p e d and summed as b a s i c , a c i d i c , h y d r o p h o b i c and n e u t r a l r e s i d u e s . D u p l i c a t e a n a l y s e s were p e r f o r m e d a f t e r h y d r o l y s i s i n 6N HC1 a t 105°C f o r 24 h. C y s t e i n e was d e t e r m i n e d as c y s t e i c a c i d . A b b r e v i a t i o n : CAA, c a s e i n amino a c i d s . 94 2 3 Distance - c m P i g . 15. Cyanogen b r o m i d e c l e a v a g e o f p r o t e i n B. The l o w e r g e l s c a n shows t h e s e p a r a t i o n o f t h e c l e a v a g e p r o d u c t s o f p r o t e i n B i n a 10% SDS-p o l y a c r y l a m i d e g e l r u n w i t h s y s t e m 1. G e l s were s t a i n e d w i t h C o o m a s s i e B l u e and s c a n n e d a t 550 nm. G e l s c a n s o f p r o t e i n s B and B* a r e g i v e n f o r r e f e r e n c e . 95 weights of. 30,00.0 to 18,000 were produced. The protein i n the faster migrating peak was probably heterogeneous since the amino acid composition suggests that there are about five methionine residues i n protein B. Amino-terminal analysis Amino-terminal analysis of protein B and B* f a i l e d to. produce an amino-terminal dansyl derivative (Fig. 16). However, the amino-terminal lysine of lysozyme could be de-termined under the same conditions. The nature of the blocking group was not investigated further. Physical properties of proteins B and B* The r e s u l t s presented above are consistant with proteins B and B* being the same polypeptide. However, the reason for the change i n the apparent molecular weight of protein B upon heating i n solutions containing SDS was not clear. Protein B was therefore p u r i f i e d free of phospholipid and lipopolysaccha-ride and the difference between the unmodified form, B, and the heat-modified form, B * , i n SDS was investigated by a number of physical techniques. Protein B i n SDS extract 1 was shown to increase i t s appa-rent molecular weight upon heating (Fig. 9). P u r i f i e d protein B when heated i n 1% SDS changes i t s migration i n SDS-polyacryl-amide gels run i n the Laemmli system (Fig. 17). Protein B was converted to form B* with an increase i n the apparent molecular weight from 29,500 to. 34,700 .(Fig. 18, panel 1). 96 F i g . 16. A m i n o - t e r m i n a l a n a l y s i s o f p r o t e i n s B and Bp, t r a c i n g s o f t h e f l u o r e s c e n t s p o t s o b t a i n e d a f t e r t w o - d i m e n s i o n a l c h r o m a t o g r a p h y o f t h e d a n s y l a t e d d e r i v a t i v e s on p o l y a m i d e s h e e t s ( 5 x 5 cm) a r e shown. The d i r e c t i o n o f c h r o m a t o g r a p h y i s i n -d i c a t e d by t h e l a r g e numbers b e s i d e t h e p l a t e s . S, s t a n d a r d d a n s y l amino a c i d s : 1, p r o l i n e ; 2, v a l i n e ; 3> m e t h i o n i n e ; 4, s e r i n e ; 5, i s o l e u c i n e ; 6, p h e n y l a l a n i n e ; 7, g l y c i n e ; 8, a r g i n i n e ; 9, a s p a r t i c a c i d . L, B and Bp, f l u o r e s c e n t d e r i v a -t i v e s f o r m e d by d a n s y l a t i o n o f l y s o z y m e , p r o t e i n B,and p r o t e i n Bp r e s p e c t i v e l y : 10, d a n s y l i c a c i d ; 11, d a n s y l amine; 12, E - d a n s y l l y s i n e ; 13, b i s (o<,E) d i d a n s y l l y s i n e ; 14, 0 - d a n s y l t y r o s i n e . 96a F i g . 17. SDS-polyacrylamide g e l e l e c t r o -phoresis of p u r i f i e d p r o t e i n B. Scan A; sample heated at 37°C f o r 20 min. Scan B; i d e n t i c a l sample heated at 100°C f o r 5 min. Gels were 12.5% polyacrylamide, run i n the Laemmli system (92). The gels were stained f o r p r o t e i n with Coomassie Blue and scanned at 550 nm using a G i l f o r d Model 240 spec-trophotometer. The arrow i n d i c a t e s d i r e c t i o n of mi g r a t i o n toward the anode. 98 F i g . 18. Determination of the molecular weights of proteins B and B* by SDS-polyacrylamide gel electrophoresis (panel 1) and by gel f i l t r a t i o n i n the presence of SDS (panel 2). Gels (12.5% acrylamide) were run i n a discontinuous buffer system, stained with Coomassie Blue, and scanned at 550 nm. Gel f i l t r a t i o n was performed i n a column (1 x 45 cm) of Sepharose 6B, e q u i l i -brated with 0.1 M sodium phosphate buffer, pH 7-2, containing 1% Standard proteins, 1, bovine serum albumin; 2, oval-SDS ^ , - - _-_ , bumin; 3, chymotrypsinogen'A chain; 4, &-lactoglobulin; 5, haemoglobin; 6, lysozyme; 7, chymotrypsin B chain; 8, chymo-trypsin C chain, were heated at 100° for 5 min i n sample buffer containing 0.1% 2-mercaptoethanol. 99 The molecular weights of proteins B and B* were also determined by gel f i l t r a t i o n i n a column of Sepharose 6B equilibrated with 1% SDS in 0.1 M sodium phosphate buffer, pH 7.2 (Fig. 18, panel 2). When compared to standard proteins run under i d e n t i c a l conditions, protein B was eluted i n a v o l -ume corresponding to a molecular weight of 29,000, while the molecular weight of protein B* was 42,600. Thus, the true molecular weight of protein B i s s t i l l i n doubt. Schnaitman has found that the i n t r i n s i c v i s c o s i t y of protein B increased on heating from 28 to 35 cc/g, when mea-sured at SDS concentration below the c r i t i c a l m i cellar concentra-ti o n ( 8 l ) . He suggested that heating caused unfolding of protein B with an increased binding of SDS to the protein mole-cule. This experiment was repeated at concentrations of SDS above the c r i t i c a l micellar concentration to ensure complete saturation of the protein with SDS. The results are presented i n F i g . 19. The i n t r i n s i c v i s c o s i t y of protein B increased on heating from 28.5 to 34 cc/g i n agreement with Schnaitman's data. In order to check the v a l i d i t y of these measurements, the i n t r i n s i c v i s c o s i t i e s of three proteins, BSA, ovalbumin and lysozyme, previously characterized by Reynolds and Tanford (152) were measured. The values determined for these proteins, 59, 32 and 8 cc/g, respectively, agree with those found previously. Calculation of a x i a l r a t i o s (a/b) for proteins B and B* using the Simha relationship (see Appendix) and assuming a pro-late e l l i p s o i d with a hydration of 0.9 g/g protein at a binding l e v a l of 1.4 g of SDS per g of protein (152,286) gave values of 100. 60 KBSA S"30 cr B r<0 B k L o m g p r o t e i n / m l B* at 25°C i n n M S ^ n ^ M 3 ( % P / c ) o f Proteins B and 7 ? I S l n ° ' ^ SDS> 0-1 M sodfBm phosphate buffer, PH 7.2. Reduced v i s c o s i t i e s for standard proteins were deter 101 7-5 and 9-0 for proteins B and B* respectively. These corres-pond to f r i c t i o n a l r a t i o s ( f / f ) of 1.4 and 1.5 for proteins B and B*. Using a molecular weight of 27,000 for the polypeptide chain as determined by Garten e_t al_. from amino acid analysis and summation of the molecular weights of the peptides formed on cleavage with cyanogen bromide (87), absolute values for the dimensions of the protein-SDS complex could be determined. The protein-SDS complex of protein B had e l l i p s o i d axes of 117 x 16 angstrom units while the protein B* complex had dimensions of 136 x 15 angstrom units. The difference between proteins B and B* could be due to the increased asymmetry, an increased l e v e l of SDS binding, or a combination of both. To distinguish between these p o s s i b i l i -t i e s we have measured the amount of detergent bound to proteins B and B*. The results of SDS binding by equilibrium d i a l y s i s (Pig. 20) are presented i n Pig. 21. Both proteins bound ca. 0.5g of SDS per gram of protein at concentrations of SDS up to 0.1%. The amount of SDS bound to proteins B and B* above the c r i t i c a l micellar concentration (ca. 0.2% i n 1 mM Tris-acetate buffer, pH 7.0), was determined by c a l c u l a t i n g (see Appendix) the molecular weight of the protein-detergent complex. The following equation can be applied to the protein-SDS complex (287): s = M(T-vp) (1) 1 0 2 Q 2 5 5 0 1 0 0 2 0 0 H o u r s F i g . 2 0 . . Equilibrium d i a l y s i s of protein B. Protein samples ( 1 ml) were dialyzed at 22°C against 1 1 of 1 mM Tris-acetate buffer, pH 7 . 0 , containing 0 . 2 0 $ (o), 0 . 1 0 $ ( • ) , 0 . 0 4 8 $ ( o ) and 0 . 0 0 8 $ ( » ) f i n a l concentration of SDS. Since the curves obtained for protein B* were indistinguish-able from those shown for protein B, these points are not plotted. 102a F i g . 21. Binding of SDS to proteins B (•) and B* O ) at 22°C i n 1 mM Tris-acetate buffer, pH 7.0. Binding was determined by equilibrium d i a l y s i s except for the points indicated by the open symbols which were calculated from the molecular weights of the protein-SDS complexes as described i n the text. 104. where M, , R S , and s are the molecular weight, p a r t i a l spec-i f i c volume, Stokes radius, and sedimentation c o e f f i c i e n t , res-pectively, of the protein-SDS complex. The Stokes r a d i i of proteins B and B* were determined by gel f i l t r a t i o n i n the presence of 1% SDS (see Appendix). A plot of Stokes radius versus e r f c ~ 1 k d (288,289) i s presented i n Pig. 22. The values of Stokes r a d i i for the standard proteins were taken from Reynolds and Tanford (152). The Stokes radius of protein B increased from 50 to 63 angstrom units upon conver-sion to form B*. Sedimentation c o e f f i c i e n t s for proteins B and B* were determined by sucrose density gradient centrifugation at SDS concentration from 0.1 to 0.3%- The p a r t i a l s p e c i f i c volumes of proteins B and B* were determined by sucrose density gradient centrifugation i n H20 and i n D 2 0-containing gradients (292). Assuming that the proteins bind the same amount of detergent i n H20 and D 2 0, the p a r t i a l s p e c i f i c volume ( ) may be calculated (see Appendix) from: SH^H - 1 (2) V = SD^D P D fHnH - p H  SD*D where subscripts H and D re f e r to values measured i n H20 and i n D 2 0, s i s the measured sedimentation-coefficient i n a solution of density., p, and *\ , the v i s c o s i t y determined at the h a l f -distance of t r a v e l , r a v g . Results, obtained at 0.170, 0.2%, 104a F i g . 22. Chromatography of proteins B and B* on Sepharose 6B i n 1% SDS, 0.1 M sodium phosphate buffer, pH 7.2. Arrows indicate the elution positions of proteins B and B*. The Stokes r a d i i of the standard proteins were taken from Reynolds and Tanford (152). Stokes radius (R s) i s plotted as a function of the inverse error function com-plement (erfc-^K^) (289). Standard proteins: 1, BSA; 2, ovalbumin; 3,J3 - l a c t o g l o b u l i n ; 4, hemiglobin; 5, lysozyme. 105 106 0.3% SDS are given In Figs. 23, 24 and 25. The S values c. 0 , W for proteins B and B* at dif f e r e n t concentrations of SDS were calculated (seeAppendix) from: s20,w - s T , m ^ < 2 ° - w ) ( 3) t\20,w U - V T . T . ) The v i s c o s i t i e s (^ ) of standard sucrose solutions i n and i n D^ O buffers, containing the specified concentrations of SDS, were measured at 20°C with a Cannon-Manning viscometer. Den-s i t i e s (f>) for the same solutions were determined by weighing 5.0 or 10.0 ml of the solution at 23°C. The determined values for sedimentation c o e f f i c i e n t and p a r t i a l s p e c i f i c volumes are presented i n Table IV. The s o n T T values are the means of the c d U , w values obtained i n H 20 and i n D 20 gradients. For the calc u l a t i o n of the molecular weight of the protein-SDS complex at concentrations of SDS above the c r i t i c a l micellar concentration, the s o n value at 0.3$ SDS was taken for both d U , W proteins B and B*. Substituting these values and the determined values for R s into equation 1:gave molecular weights of 77,000 and 84,000 for the SDS complexes of proteins B and B*, res-pectively. The difference i n molecular weight between the protein-SDS complex and the protein alone must be due to the bound detergent. Using a value of 27,000 for the molecular weight of protein B (87), the amount of SDS bound by proteins B and B* above the c r i t i c a l micellar concentration w a s i # 8 5 and 2 ...1 g per g of protein respectively. That there i s only a small difference i n the amount of SDS F i g . 23. C e n t r i f u g a t i o n o f p r o t e i n s B and B* i n 5 t o 20% s u c r o s e g r a d i e n t s p r e p a r e d i n 0.1% SDS, 1 mM T r i s - a c e t a t e b u f f e r , pH 7.0. P a n e l A: g r a d i e n t p r e p a r e d i n H P 0 , p a n e l B: g r a d i e n t p r e p a r e d i n D 2 0 . The s u c r o s e c o n c e n t r a t i o n o f e a c h f r a c t i o n was d e t e r m i n e d f r o m t h e r e f r a c t i v e i n d e x . F i g . 24. C e n t r i f u g a t i o n of p r o t e i n s B and B* i n 5 to 20% sucrose gradients prepared i n 0.2% SDS , 1 mM T r i s - a c e t a t e b u f f e r , pH 7-0. Panel A: gradient prepared i n H^Oj panel B: grad-i e n t prepared i n DpO. The sucrose concentra-t i o n of each f r a c t i o n was determined from the r e f r a c t i v e index. F r a c t i o n n u m b e r F i g . 25. C e n t r i f u g a t i o n o f p r o t e i n s B and B* i n 5 t o 20% s u c r o s e g r a d i e n t s p r e p a r e d i n 0.3% SDS, 1 mM T r i s - a c e t a t e b u f f e r , pH 7.0. P a n e l A: g r a d i e n t p r e p a r e d i n H2O, p a n e l B: g r a d -i e n t p r e p a r e d i n D2O. The s u c r o s e c o n c e n t r a -t i o n o f e a c h f r a c t i o n was d e t e r m i n e d f r o m t h e r e f r a c t i v e i n d e x . 601 110 Table IV P a r t i a l -specific volumes and sedimentation c o e f f i c i e n t s of proteins B and B* at diff e r e n t concentrations of SDS SDS (% w/v) B* s -, 0.1 0.772 3.40 0.753 2.73 0.2 0.776 2.78 0.772 2.20 0.3 0.793 2.83 0.797 2.40 I l l bound to proteins B and B* i s further substantiated by t h e i r p a r t i a l s p e c i f i c volumes. The p a r t i a l s p e c i f i c volumes of both proteins B and B* increased with increasing SDS concentra-tions (Table IV) i n d i c a t i n g that more detergent was bound at higher SDS concentrations as has been found with other proteins (290j291j293). However, above the c r i t i c a l micellar concentra-t i o n there was no s i g n i f i c a n t difference between the p a r t i a l s p e c i f i c volumes of proteins B and B*. At a l l SDS concentra-tions examined, the sedimentation c o e f f i c i e n t of protein B was always higher than form B* (Table IV). There was no obvious relationship between the sedimentation c o e f f i c i e n t and the concentration of SDS as has also been observed by Nelson (294). The similar size of the protein-SDS complexes of proteins B and B* and the r e l a t i v e l y small differences i n the amount of detergent bound indicates that the apparent increased molecular weight of protein B compared to form B* i s not due to increased binding of detergent but rather i s due to the increased asym-metry of the protein B* molecule. This would account for the higher Stokes radius and the lower sedimentation c o e f f i c i e n t of protein B*. A protein on unfolding w i l l become more susceptible to degradation by p r o t e o l y t i c enzymes (204). We therefore ex-amined the s u s c e p t i b i l i t y of proteins B and B* to digestion by pronase i n 1% SDS. As can be seen from F i g . 26, protein B* was degraded more rapidly than protein B when a pronase to protein r a t i o of 1:200. was used. These re s u l t s suggest that protein B* i s indeed more unfolded than protein B. 111a F i g . 26. D i g e s t i o n of p r o t e i n s B and B* by pronase i n 1% SDS, 10 mM T r i s - H C l b u f f e r , pH 7.5. The p r o t e i n s were incubated at 37°C at a protease to p r o t e i n r a t i o of 1:200. Sam-ples were removed at i n t e r v a l s up t o two hours and d i g e s t i o n was stopped by adding a 100-fold excess of phenylmethanesulfonyl f l u o -r i d e followed by heating at 100°C f o r 5 min i n the presence of 0 .1% a. -mercaptoethanol. Samples were made 4 M i n urea and then exami-ned by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . The g e l s were st a i n e d f o r p r o t e i n w i t h Coomassie Blue and scanned at 550 nm using a G i l f o r d Model 240 spectrophotometer. The r e l a t i v e areas of the peak corresponding to p r o t e i n B* were determined by weighing. 113 Since the results presented above have indicated that a marked shape change occurs upon heating, o p t i c a l methods were employed to determine the effect on protein conformation.-In a l l samples examined, protein B* had an increased u l t r a -v i o l e t absorption at 275 nm over protein B (Fig. 27). This was most c l e a r l y seen i n the difference spectra between proteins B* and B shown i n the lower panel of F i g . 27. The increased absorption due to aromatic amino acids indicates that there was an unfolding of the polypeptide chain upon heating. ORD and CD spectroscopy have been extremely useful i n studies of protein conformation i n b i o l o g i c a l membranes and i n detergent solutions (152,295). The ORD and CD spectra (Fig. 28, panel A) of the two proteins did not d i f f e r greatly but both had a form that i s t y p i c a l of protein-SDS complexes with a negative trough at about 233 nm (152). Reynolds and Tanford (152) have found that increased binding of SDS decreases the magnitude of t h i s trough. Thus, the i d e n t i c a l nature of the spectra for proteins B and B* supports the finding that these proteins did not d i f f e r greatly In the amount of bound detergent. The CD spectra for proteins B and B* i n 0.2$ SDS are shown i n panel B. Protein B was s l i g h t l y more o p t i c a l l y active i n the 250-300 nm region of the CD spectrum while only small differences could be seen i n the 190-250 nm region. This suggests that protein B* has a more disordered structure than protein B. The CD spectra are consistent with a h e l i c a l con-formation for the protein-SDS complex. As Tanford (287) has suggested, small differences i n o p t i c a l properties of protein-113a Pig. 27. Panel A: u l t r a v i o l e t absorption spectra of proteins B and B* at 22°C i n 0.2% SDS , 1 mM Tris-acetate buffer, pH 7-0. The scan for protein B* i s displaced upwards by 0.1 absorbance unit. Protein concentra-t i o n , 0.7 mg/ml. Panel B: u l t r a v i o l e t difference spectrum of protein B* versus B. The same samples from panel A were used. 1 1 4 i 300 260 W a v e l e n g t h - n m 340 F i g . 28. Panel A: ORD spectra of proteins B and B* at 25°C i n 0.2$ SDS, 1 mM Tris-acetate buffer, pH 7.0. For c l a r i t y the spectrum of protein B i s displaced downwards by 300 degrees. Protein concentration, 0.7 mg/ml. Panel B: CD spectra of proteins B and B*. The spectra of protein B* are displaced 30 and 3000 degrees cm2 decimole -! upwards i n the near uv and far uv regions, respectively. A 1 cm c e l l was used i n the near uv region, while a O i l mm pathlength was used i n the far uv region. The same samples as i n the ORD spectra were used. 116 detergent complexes may not r e f l e c t the extent o f conformation-a l change t h a t might occur upon h e a t i n g i n SDS-containing s o l u t i o n s . The r e s u l t s above i n d i c a t e that h e a t i n g of p r o t e i n B i n SDS-containing s o l u t i o n s causes u n f o l d i n g of the p o l y p e p t i d e chain without•a s i g n i f i c a n t change i n the amount of detergent bound. SDS i s r e q u i r e d to s o l u b i l i z e p r o t e i n B from the mem-brane, however, i t i s not c l e a r i f i t i s r e q u i r e d f o r the conversion of p r o t e i n B t o form B*. T h i s problem was i n v e s t -i g a t e d as f o l l o w s . Weber and Kuter (144) have shewn that complete removal of SDS from p r o t e i n may be accomplished by i o n exchange i n the presence o f 6M urea i n 0.05 M T r i s - a c e t a t e b u f f e r (pH 7-8). SDS was removed from both p r o t e i n s B and B* t o a l e v e l un-d e t e c t a b l e by the methylene blue assay by passage through a sm a l l column of t h i s r e s i n . T h i s assay d e t e c t s both f r e e and bound SDS (169). P r o t e i n B, f r e e of SDS, was heated i n the T r i s - u r e a b u f f e r at 100°C f o r up to 15 min. Samples were removed at 0, 2, 5> and 10 min and were made to a c o n c e n t r a t i o n of Ifo i n SDS immediately. The samples were examined f o r the extent of co n v e r s i o n of p r o t e i n B to form B* under these con-d i t i o n s by s u b m i t t i n g the samples t o SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s without f u r t h e r h e a t i n g . As seen i n F i g . 29, scans 1-4, p r o t e i n B was p r o g r e s s i v e l y converted t o form B* upon h e a t i n g i n the absence of SDS. T h i s change was c o r r e l a t e d with an i n c r e a s e d l i g h t a b s o r p t i o n i n the u l t r a v i o l e t r e g i o n s i m i l a r to that p r e v i o u s l y observed when h e a t i n g was c a r r i e d out i n the I f 6 a P i g . 2 9 . E f f e c t o f h e a t i n g p r o t e i n B i n t h e a b s e n c e o f SDS. S D S - p o l y a c r y -l a m i d e g e l e l e c t r o p h o r e s i s was c a r r i e d o u t as d e s c r i b e d by Weber and O s b o r n ( 9 0 ) . G e l s were s t a i n e d and s c a n n e d as d e s c r i b e d i n F i g . 17 . The e x p e r i -ment was p e r f o r m e d as d e s c r i b e d i n t h e t e x t . 118 presence of SDS. In order to test for the reversal of t h i s conversion the remainder of the heated mixture was allowed to cool at 22°C for 4 8 h. Samples removed at various time in t e r v a l s showed no reconversion of form B* to protein B (Pig. 29; scans 5-8). Moreover removal of SDS from the protein B*-SDS complex did not r e s u l t i n reformation of protein B. These studies i n d i c -ate that bulk SDS i s not required for the conversion of protein B to form B* and that the i r r e v e r s i b l e nature i s not due to the binding of large amounts of SDS. However, the possible i n -volvement of a few molecules of SDS s t i l l remaining bound to the protein, but undetectable by the detergent assay method, cannot be ruled out. It appears that protein B, as extracted from the outer membrane of E. c o l i by 0.5$ SDS as 37°C, contains some native structure and that t h i s structure i s lost upon heating. This process w i l l occur i n the absence of SDS and involves the un-foldin g of the polypeptide chain. Although further binding of SDS may occur to a small extent i t i s unlikely that the appa-rent increase i n the molecular weight i s due to a greater amount of bound detergent. E f f e c t of growth conditions on the outer membrane proteins Schnaitman (91) reported that the synthesis of protein 2 (Table I) was under catabolite repression. The ef f e c t of the carbon source on the formation of the proteins of the outer mem-brane of E.' c o l i NRC 482 was studied. C e l l s were grown on 1 1 9 i n o r g a n i c s a l t s medium c o n t a i n i n g g l u c o s e , g l y c e r o l , s u c c i n a t e or a c e t a t e , or on c a s e i n amino a c i d s or t r y p t i c a s e soy b r o t h and harvested d u r i n g the e x p o n e n t i a l and s t a t i o n a r y phases of growth ( P i g . 3 0 ) . The outer membrane p r o t e i n s were i s o l a t e d and r e s o l v e d on SDS-polyacrylamide g e l s ( P i g . 3 D . P r o t e i n A-^  predominated over p r o t e i n A 2 i n c e l l s grown on media c o n t a i n i n g glucose ( P i g . 3 1 , Qlc and TSB). The l e v e l o f p r o t e i n A 2 r e -l a t i v e to p r o t e i n A -j_ i n c r e a s e d i n c e l l s grown on g l y c e r o l , s u c c i n a t e , a c e t a t e and c a s e i n amino a c i d s ( P i g . 3 1 ) . T h i s suggests t h a t p r o t e i n A 2 i s e q u i v a l e n t t o Schnaitman's p r o t e i n 2 . P u r i f i c a t i o n o f p r o t e i n A. P r o t e i n A i s t i g h t l y a s s o c i a t e d with the p e p t i d o g l y c a n . E x t r a c t i o n o f the c e l l envelope with SDS at 60°C leaves an i n s o l u b l e r e s i d u e o f p r o t e i n A bound to the p e p t i d o g l y c a n ( F i g . 6 ) . The p r o t e i n can be e x t r a c t e d from t h i s complex with SDS at 1 0 0°C. In c e l l s grown on glucose both p r o t e i n s A-^  and A^ were a s s o c i a t e d with the p e p t i d o g l y c a n , p r o t e i n A-^  being the predominant p r o t e i n (ca. 15%) ( F i g . 3 2 , upper scan ) . In c e l l s grown on c a s e i n amino a c i d s , where p r o t e i n A 2 predominated (ca. 15%), again both p r o t e i n s A-j_ and A 2 were a s s o c i a t e d with the p e p t i d o g l y c a n ( F i g . 3 2 , second scan ) . E x t r a c t s of p r o t e i n A, d e r i v e d from glucose and c a s e i n amino acid-grown c e l l s were f u r t h e r p u r i f i e d by g e l f i l t r a t i o n i n the presence of SDS ( F i g . 3 3 ) f o r amino a c i d a n a l y s i s and cyanogen bromide cleavage. The e x t r a c t c o n t a i n i n g p r o t e i n A from glucose-grown c e l l s was c o n s i s t a n t l y found to c o n t a i n more carbohydrate than the p r o t e i n 119a F i g . 30. Growth of E. c o l i NRC 482 on d i f f e r e n t carbon sources at 37°C with vigorous aeration. G l c 3 glucose; Gly, g l y c e r o l ; Succ, succinate; CAA, casein amino acids: TSB, trypticase soy broth; Acet, acetate. Arrows indicate the time at which the c e l l s were harvested. 120 F i g . 31. E f f e c t o f growth c o n d i t i o n s on o u t e r membrane p r o t e i n s . Scans o f S D S - p o l y a c r y l a m i d e g e l s o f o u t e r membrane p r o t e i n s p r e p a r e d f r o m c e l l s grown on g l u c o s e , G l c ; g l y c e r o l , G l y ; s u c c i n a t e , S u c c ; c a s e i n amino a c i d , CAA; t r y p t i c a s e soy b r o t h , TSB and a c e t a t e , A c e t , and h a r v e s t e d d u r i n g t h e e x p o n e n t i a l (e) and s t a t i o n a r y ( s ) p h a s e s o f g r o w t h . G e l s were s t a i n e d w i t h Coomassie B l u e and s c a n n e d a t 550 nm. A b s o r b a n c e 1ST A b s o r b a n c e A b s o r b a n c e 123a Pig. 32. Cyanogen bromide cleavage of proteins A-^  and A 2- The two lower gel scans show the separation of the cleavage products. Gel scans of the preparations of proteins A^ and Ap are given for reference. SDS-gels [12.5% acrylamide.). were run with a phosphate buffer system at an alkaline pH, stained with Coomassie Blue, and scanned at 550 nm. 124 125 F r a c t i o n N u m b e r F i g . 33. Gel f i l t r a t i o n chromatography of proteins A and A on a column of Bio Gel P150 ( 1 x 45 cm) equilibrated with 0.1M sodium phosphate buffer, pH 7.2, containing 1% SDS. Samples ( 1 ml) were dissolved to 3 mg protein/ml i n column buffer containing 0.1% 2-mercaptoethanol and heated at 100°C for 5 min before applying to the column. Fractions (0.9 ml) from the column were collected and assayed for protein (•) and carbohydrate (*). V O , void volume. 126 A extract from casein amino acid-grown c e l l s . The bulk of carbohydrate was found not to co-elute with the protein (Pig. 33) and i s therefore l i k e l y a contaminant s o l u b i l i z e d from the peptidoglycan. Amino acid composition of proteins A-]_ and A 2 The amino acid compositions of the protein A fractions from c e l l s grown on glucose (mainly protein A^) and on casein amino acids (mainly protein A 2) are shown i n Table I I I . The compositions of proteins A-^  and A 2 were very similar which i s i n agreement with t h e i r similar d i s p o s i t i o n i n the outer membrane. Like protein B, protein A was enriched i n acidic amino acids, was only moderately hydrophobic, and contained l i t t l e cysteine. Proteins A and B are c l e a r l y d i f f e r e n t polypeptides as i n d i -cated by the differences i n the content of h i s t i d i n e , methio-nine, tyrosine, phenylalanine and proline residues. Cyanogen bromide cleavage of protein A Cyanogen bromide cleavage of preparations enriched i n proteins Ai_ and A 2 gave similar cleavage products as determined by SDS-polyacrylamide gel electrophoresis (Fig. 3 2 , lower two scans). The migration positions of the peptides derived from proteins A-]_ and A 2 were i d e n t i c a l , however the amount of poly-peptide i n each peak was d i f f e r e n t . This was probably due to incomplete cleavage of the protein since the sum of the molecu-l a r weights of the cleavage products exceeded the molecular weight of protein A. Since the cyanogen bromide cleavage 127 p a t t e r n s and t h e amino a c i d c o m p o s i t i o n s o f p r o t e i n s Aj_ and A 2 were s i m i l a r , t h e y may be two forms o f t h e same p o l y p e p t i d e . O r g a n i z a t i o n o f p r o t e i n s i n t h e o u t e r membrane The o r g a n i z a t i o n o f t h e p r o t e i n s i n t h e o u t e r membrane was examined by p r o t e o l y t i c d i g e s t i o n , c o v a l e n t l a b e l l i n g and c r o s s l i n k i n g t e c h n i q u e s w h i c h have been s u c c e s s f u l l y a p p l i e d t o th e s t u d y o f o t h e r membrane s y s t e m s (234,235,296-298). The o u t e r membrane o f E . c o l i i s a u s e f u l s y s t e m f o r t h e s t u d y o f membrane s t r u c t u r e b e c a u s e o f t h e r e l a t i v e l y s i m p l e p r o t e i n com-p o s i t i o n . I n a d d i t i o n , t h e components o f t h e o u t e r membrane can be a l t e r e d n u t r i t i o n a l l y o r by m u t a t i o n and t h e e f f e c t o f t h e a l t e r a t i o n on membrane s t r u c t u r e can be s t u d i e d . E f f e c t o f p r o t e o l y t i c enzymes on t h e p r o t e i n s o f t h e o u t e r membrane i n i n t a c t c e l l s T r e a t m e n t o f i n t a c t c e l l s w i t h p r o n a s e , t r y p s i n o r chymo-t r y p s i n d i d n o t a l t e r t h e p r o t e i n p r o f i l e s s e e n i n P i g . 34 f o r c e l l s grown on g l u c o s e o r on c a s e i n amino a c i d s . S i n c e t h e c a r b o h y d r a t e c h a i n s o f t h e l i p o p o l y s a c c h a r i d e m ight be r e s t r i c t i n g t h e a c c e s s i b i l i t y o f t h e o u t e r membrane p r o t e i n s t o t h e p r o t e o l y t i c enzymes, t h e c e l l s were a l s o p r e t r e a t e d w i t h 5 mM EDTA f o r 30 min. T h i s r e s u l t e d i n t h e l o s s o f o n e - t h i r d o f t h e l i p o p o l y s a c c h a r i d e f r o m t h e o u t e r membrane ( P i g . 35). T h i s l o s s was n o t a c c o m p a n i e d by t h e l o s s o f any o f t h e m a j o r p r o t e i n s f r o m t h e o u t e r membrane, n o r were t h e p r o t e i n s any more s u s c e p t i b l e t o p r o n a s e d i g e s t i o n . Thus, t h e EDTA-1 2 7 a F i g . 34. Control scans of SDS-gels i n •'. studies on the effect of pr o t e o l y t i c d i -gestion on the outer membrane proteins i n c e l l s . Coomassie Blue stained SDS-polyacrylamide gels of the outer membrane proteins prepared from c e l l s grown on glucose (scan.l) or casein amino acids (scan 2) were scanned at 550 nm. The alkaline buffer system was used for electrophoresis. A b s o r b a n c e 129 Minutes F i g . 35. Release-i-of lipopolysaccharide from c e l l s by EDTA. Cells were suspended (lg/ 8 ml) i n 0.IM Tris-HCl buffer, pH 8.0, containing either 10 mM MgCl2 (control, open symbols) o 5 mM EDTA (closed symbols) and incubated at 37°C for 30 min. Samples (1 ml) were removed, MgCl2 was added (10 mM, f i n a l concentration) to the EDTA-treated samples and the c e l l s wer recovered by centrifugation at 10,000 x g for 5 min at 4°c. The absorbance at 260 nm and lipopolysaccharide content of the supernatant were determined. Cel l s for pronase diges-t i o n were resuspended (1 g/10 ml) i n 0.1 M Tris-HCl buffer, pH 8.0, containing 35 mM MgCl ?. 130 r e l e a s a b l e f r a c t i o n o f t h e l i p o p o l y s a c c h a r i d e c a n n o t be r e s -p o n s i b l e f o r t h e r e s i s t a n c e o f t h e o u t e r membrane p r o t e i n s i n i n t a c t c e l l s t o d i g e s t i o n by p r o n a s e . The p o s s i b l e p r o t e c t i o n o f t h e o u t e r membrane p r o t e i n s by t h e n o n - r e l e a s a b l e f r a c t i o n o f t h e l i p o p o l y s a c c h a r i d e was examined i n a h e p t o s e - d e f i c i e n t mutant o f E . c o l i . S t r a i n NS-1 l a c k s t h e c o r e r e g i o n o f t h e l i p o p o l y s a c c h a r i d e and, as p r e v i o u s l y shown (179), c o n t a i n s d r a s t i c a l l y r e d u c e d amounts o f p r o t e i n s A and B ( F i g . 36, sample 2) when compared t o t h e p a -r e n t s t r a i n , J E 1011 (sample 1). T r e a t m e n t o f i n t a c t c e l l s o f b o t h t h e p a r e n t and t h e mutant w i t h p r o n a s e r e s u l t e d i n t h e l o s s o f o n l y a few h i g h e r m o l e c u l a r w e i g h t p r o t e i n s f r o m t h e o u t e r membrane ( s a m p l e s 3 and 4). When i s o l a t e d c e l l w a l l s , c o n s i s t -i n g o f o u t e r membrane and p e p t i d o g l y c a n , p r e p a r e d f r o m t h e s e s t r a i n s were t r e a t e d w i t h p r o n a s e t h e r e was a marked change i n t h e p r o t e i n p r o f i l e o f t h e o u t e r membrane ( s a m p l e s 5 and 6). P r o t e i n A was r e s i s t a n t t o d i g e s t i o n b u t p r o t e i n B was c l e a v e d t o a p r o n a s e - r e s i s t a n t f r a g m e n t , p r o t e i n Bp, w i t h a m o l e c u l a r w e i g h t o f about 20,000. The s m a l l e r amounts o f t h e s e p r o t e i n s i n t h e mutant b e h a v e d s i m i l a r l y . The l o s s o f p r o t e i n s A and B, and t h e m o d i f i e d l i p o p o l y s a c c h a r i d e s t r u c t u r e , do n o t a l t e r t h e a r r a n g e m e n t o f t h e o u t e r p r o t e i n s i n t h e o u t e r membrane. E f f e c t o f p r o t e o l y t i c enzymes on t h e p r o t e i n s i n i s o l a t e d membrane p r e p a r a t i o n s I n c o n t r a s t t o t h e r e s u l t s w i t h whole c e l l s , p r o t e a s e t r e a t m e n t o f e n v e l o p e p r e p a r a t i o n s r e s u l t e d i n e x t e n s i v e d i g e s -131 B, F i g . 36. E f f e c t of a l t e r a t i o n i n l i p o p o l y s a c c h a r i d e s t r u c -ture on the d i g e s t i o n of outer membrane p r o t e i n s by pronase. Outer membrane p r o t e i n s were resolved by sodium dodecyl s u l f a t e g e l e l e c t r o p h o r e s i s on 0.75 mm t h i c k 12% p o l y a c r y -lamide s l a b s . 1, 2, outer membrane p r o t e i n s from untreated c e l l s of JE 1011 and hepto s e - d e f i c i e n t mutant NS01, r e s -p e c t i v e l y ; 3j 4, outer membrane p r o t e i n s from pronase-t r e a t e d c e l l s of JE 1011 and NS-1, r e s p e c t i v e l y ; 5, 6, outer membrane p r o t e i n s from pronase-treated c e l l w a l l s of JE 1011 and NS-1, r e s p e c t i v e l y . 132 t i o n o f c e r t a i n o u t e r membrane p r o t e i n s . The e n v e l o p e p r e p a r a t i o n f o l l o w i n g p r o t e o l y t i c d i g e s t i o n was e x t r a c t e d w i t h T r i t o n X-100 i n t h e p r e s e n c e o f M g 2 + i n o r d e r t o remove p r o -t e i n s o f t h e i n n e r membrane. The o u t e r membrane p r o t e i n s were t h e n s o l u b i l i z e d f r o m t h e e x t r a c t e d p r e p a r a t i o n w i t h 1% SDS a t 100°C f o r e x a m i n a t i o n by p o l y a c r y l a m i d e g e l e l e c t r o p h o -r e s i s . The r e s u l t s were c o n f i r m e d u s i n g i s o l a t e d o u t e r mem-b r a n e s p r e p a r e d f r o m s p h e r o p l a s t s by t h e method o f M i z u s h i m a and Yamada (77) i n o r d e r t o e l i m i n a t e t h e p o s s i b i l i t y t h a t T r i t o n X-100 had removed p r o t e i n s o r d i g e s t i o n p r o d u c t s f r o m t h e o u t e r membrane. The k i n e t i c s o f d i g e s t i o n by p r o n a s e o f t h e o u t e r membrane p r o t e i n s i n e n v e l o p e s p r e p a r e d f r o m g l u c o s e - g r o w n c e l l s a r e shown i n F i g . 37. The r a t e s o f d i g e s t i o n o f t h e d i f f e r e n t p r o t e i n s v a r i e d g r e a t l y . B o t h p r o t e i n s A-^  and A 2 were r e s i s -t a n t t o d i g e s t i o n . I n c e l l s grown on c a s e i n amino a c i d s i n w h i c h p r o t e i n k^ was p r e d o m i n a n t , t h e same r e s i s t a n c e t o d i g e s -t i o n was o b s e r v e d . The l o s s o f p r o t e i n B o c c u r r e d w i t h t h e c o n c o m i t t a n t p r o d u c t i o n o f a p r o n a s e r e s i s t a n t f r a g m e n t , p r o t e i n Bp. T h i s s u g g e s t s t h a t p r o t e i n B i s p a r t i a l l y e x p o s e d f r o m t h e membrane. The f r a g m e n t must be p r o t e c t e d by i t s a r r a n g e m e n t i n t h e membrane. P r o t e i n s o f t h e C group w h i c h were r e a d i l y d e g r a d e d a r e p r o b a b l y a t t h e s u r f a c e o f t h e o u t e r membrane. The p l a t e a u s e e n f o r p r o t e i n F, t h e f r e e f o r m o f t h e l i p o p r o t e i n may i n d i c a t e t h a t t h e r e a r e two p o p u l a t i o n s o f t h i s p r o t e i n i n t h e membrane.' The k i n e t i c s o f d i g e s t i o n o f t h e o u t e r membrane p r o t e i n s by t r y p s i n ( F i g . 38) and c h y m o t r y p s i n ( F i g . 39) were F i g . 37. K i n e t i c s o f d i g e s t i o n o f o u t e r membrane p r o t e i n s by p r o n a s e . C e l l e n v e l o p e was i n c u b a t e d a t 37°C w i t h p r o n a s e a t an e n z y m e : p r o t e i n r a t i o o f 1:25. Samples were removed a t i n t e r v a l s and t h e o u t e r membrane p r o t e i n s were p r e p a r e d . The p r o t e i n s were r e s o l v e d by S D S - g e l e l e c t r o p h o r e s i s and q u a n t i t a t e d f r o m a b s o r b a n c e s c a n s o f t h e s t a i n e d g e l s by w e i g h i n g . The p r o t e i n s were i d e n t i f i e d as i n F i g . 32. P r o t e i n Bp i s t h e p r o n a s e - r e s i s t a n t f r agment d e r i v e d f r o m p r o t e i n B. Pig. 38. Kinetics of digestion of outer membrane proteins by trypsin. C e l l envelope was incubated at 37°C with trypsin at an enzymeiprotein r a t i o of 1:25. Samples were removed at int e r v a l s and the outer membrane proteins were prepared. The proteins were resolved by SDS-gel electrophoresis and quan-t i t a t e d from absorbance scans of the stained gels by weighing. The proteins were i d e n t i f i e d as i n Pig. 32. Protein i s the tryp s i n - r e s i s t a n t fragment derived from protein B. Pig. 39. Kinetics of digestion of outer membrane proteins by chymotrypsin. C e l l envelope was incubated at 37°C with chymotrypsin at an enzyme: protein r a t i o of 1:25- Samples were removed at inte r v a l s and the outer membrane proteins were re-solved by SDS-gel electrophoresis and quantitated from absorbance scans of the stained gels by weighing-;' The proteins were i d e n t i f i e d as i n Fi g . 32. Protein B c i s the chymotrypsin-re s i s t a n t fragment derived from protein B. 135 136 s i m i l a r to those obtained with pronase. However, the f r a g -ments derived from p r o t e i n B by t r y p s i n and chymotrypsin treatment both had a molecular weight of 25,000. In a d d i t i o n , the k i n e t i c s of d i g e s t i o n of p r o t e i n D 2 suggests that t h i s p r o t e i n may not be a c c e s s i b l e at the surface of the membrane. L a b e l l i n g of outer membrane p r o t e i n s with fluorescamine The exposure of p r o t e i n s at the surface of membranes can also be determined by covalent l a b e l l i n g with non-penetrating reagents (235). Fluorescamine reacts r a p i d l y with amino groups to give a f l u o r e s c e n t l a b e l on p r o t e i n molecules (299). I t has been used to l a b e l the surface p r o t e i n s of BHK c e l l s (300) where i t s high r e a c t i v i t y r e s u l t s i n the l a b e l l i n g only of exposed p r o t e i n s . We have confirmed that cytoplasmic p r o t e i n s are not l a b e l l e d when i n t a c t c e l l s of E. c o l i are tr e a t e d with flurescamine. L a b e l l i n g of c e l l envelope com-ponents i n both E. c o l i JE 1011 and i t s h e p t o s e - d e f i c i e n t mutant NS-1 were found a f t e r t r e a t i n g i n t a c t c e l l s with f l u o -rescamine ( F i g . 40). I s o l a t e d outer membrane or c e l l w a l l (outer membrane-peptidoglycan) preparations were a l s o t r e a t e d w i t h f l u o r e s c a -mine. The p r o t e i n s were then e x t r a c t e d w i t h SDS and examined by polyacrylamide g e l e l e c t r o p h o r e s i s . As shown i n F i g . 4 l , although a l l of the outer membrane p r o t e i n s could be l a b e l l e d by t h i s reagent, the extent of r e a c t i o n v a r i e d . The bands migrating around the dye f r o n t which are not ass o c i a t e d with a p a r t i c u l a r p r o t e i n were not i d e n t i f i e d , but one of these 136a F i g . MO. Slab gel electrophoresis of fluorescamine-labelled intact c e l l s of E. c o l i JE 1011 (Samples 1-5) and i t s heptose-deficient mutant NS-1 (Samples 6-10). C e l l s were suspended (l^.g/10 ml) i n 0 . 2M triethano-lamine buffer, pH 8.5 and treated with various levels of f;'luorescamine at 22°C. After 15 sec the samples (100/4'1) were s o l u b i l i z e d by the addition of 2 volumes of SDS-electro-phoresis buffer followed by heating at 100°C for 3 min. Samples (10/*l) 1 and 6 , control; 2 and 7 , 100 j u g f luorescamine; 3 and 8 , 250 Mg f luorescamine; 4 and 9 , 500 Mg f •luo-rescamine. Samples (25A*0 5 and 10; 500 M g fluorescamine. The slab gel (12% acrylamide) was photographed under u l t r a v i o l e t l i g h t (up-per photograph( and then- stained for protein with Coomassie Blue (lower photograph). 137 138 2 3 4 5 6 7 8 9 F i g . 4 l . Slab gel electrophoresis of fluorescamine-labelled outer membrane proteins. Samples (100/*1) of c e l l wall (samples 1-5) and outer membrane (samples 6-10) preparations containing about 1 mg/ml of protein i n 0.2 M triethanolamine buffer, pH 8.5, were treated with various lev e l s of f l u o r e s -camine at 220C After 15 sec the samples were s o l u b i l i z e d by the addition of 2 volumes of 1% SDS i n the electrophoresis sample buffer and heated at 100°C for 3 min. Samples 2 and 6, control; 3 and 7, 50 A*- g fluorescamine; 4 and 8, 100 A* g fluorescamine; 5 and 9, 500 A* g fluorescamine. Samples 1 and 10 are control samples stained with Coomassie Blue. Samples 2-9 were photographed under u l t r a v i o l e t l i g h t . The concentration of polyacrylamide gel was 12$. 139 products i s probably l a b e l l e d phosphatidylethanolamine. P r o t e i n B reacted more r e a d i l y w i t h the reagent than p r o t e i n A although both p r o t e i n s were present i n about equal amounts i n the membrane. This r e s u l t confirms that obtained by pronase which suggests that p r o t e i n B i s more exposed at the membrane surface than p r o t e i n A. A s s o c i a t i o n of oligomers of p r o t e i n A with the peptidoglycan P r o t e i n A i s t i g h t l y a s s ociated with the peptidoglycan. In c e l l s grown on glucose both p r o t e i n s and A 2 were a s s o c i -ated w i t h the peptidoglycan, p r o t e i n A^ being the predominant p r o t e i n ( F i g . 42, scan 1, l e f t ) . In c e l l s grown on casein amino a c i d s , where p r o t e i n A 2 predominated, again both p r o t e i n s A-j_ and A 2 were a s s o c i a t e d w i t h the peptidoglycan ( F i g . 42, scan 1, r i g h t ) . D i g e s t i o n of these p r o t e i n A-peptidoglycan complexes, wi t h ronase d i d not r e s u l t i n any l o s s of p r o t e i n s A-j_ or A 2 as s i m i l a r p r o t e i n p r o f i l e s to those seen i n F i g . 42, scans 1, ere obtained. I n t a c t peptidoglycan was not re s p o n s i b l e f o r the r e s i s t a n c e of these p r o t e i n s t o d i g e s t i o n s i n c e p r e i n c u -b a t i o n of the complex wi t h lysozyme ( F i g . 42, scans 2) p r i o r to the a d d i t i o n of pronase d i d not r e s u l t i n d i g e s t i o n ( F i g . 42, scans 3). Since t h i s • r e s i s t a n c e to d i g e s t i o n was l o s t on ex-t r a c t i o n of the p r o t e i n s i n t o SDS, i t appeared that i n t e r a c t i o n between d i f f e r e n t molecules of p r o t e i n A might account f o r t h i s phenomonen. Therefore, an attempt was made to i s o l a t e o l i g o -mers of p r o t e i n A. P w P i g . 42. Resistance of proteins A-j_ and A2 to pronase d i g e s t i o n . P r o t e i n A - peptidoglycan complexes were prepared from c e l l s grown on glucose ( l e f t panel) or casein amino acids ( r i g h t p a n e l ) . The proteins were extracted from the complexes with 1% SDS at 100° a f t e r the f o l l o w i n g treatments: 1, none; 2, complexes with lysozyme (40/Mg/mg p r o t e i n ) ; 3 } complexes pret r e a t e d with lysozyme and then with pronase (1 mg/25 mg p r o t e i n A). The p r o t e i n s were resolved by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s and stained with Coomassie Blue. The stained gels were scanned at 550 nm. 141 E x t r a c t i o n o f t h e p r o t e i n A - p e p t i d o g l y c a n complex w i t h c h a o t r o p i c a g e n t s s u c h as 6 M u r e a , d i d n o t s o l u b i l i z e any p r o t e i n a t 37°C, a l t h o u g h a t 100°C p r o t e i n A was c o m p l e t e l y removed f r o m t h e p e p t i d o g l y c a n (Pig. - . 4 3 ) . The p r o t e i n i n t h e s e e x t r a c t s was p r e s e n t as a monomer as shown by g e l f i l -t r a t i o n ( F i g . 44) and e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e g e l s i n t h e p r e s e n c e o f 6 M u r e a ( F i g . 44). S i m i l a r r e s u l t s were o b t a i n e d w i t h 4 M g u a n i d i n i u m h y d r o c h l o r i d e o r 4 M g u a n i d i n i u m t h i o c y a n a t e . P r e t r e a t m e n t o f t h e p r o t e i n A - p e p t i d o g l y c a n complex w i t h l y s o z y m e at room t e m p e r a t u r e , f o l l o w e d by t h e a d d i t i o n o f u r e a t o a c o n c e n t r a t i o n o f 6 M, p r o d u c e d a complex o f p r o t e i n , p o s -s i b l y s t i l l a s s o c i a t e d w i t h f r a g m e n t s o f p e p t i d o g l y c a n , w h i c h was e x c l u d e d f r o m a column o f S e p h a r o s e 6B e q u i l i b r a t e d w i t h 50 mM T r i s - a c e t a t e b u f f e r , pH 7-5, c o n t a i n i n g 6 M u r e a ( F i g . 45). The complex y i e l d e d monomers o f p r o t e i n A when h e a t e d a t 100°C f o r 5 min. I s o l a t e d o u t e r membrane p r e p a r e d f r o m s p h e r o p l a s t s (77) was c o m p l e t e l y s o l u b i l i z e d by 1% SDS a t room t e m p e r a t u r e a l t h -ough i t was l i t t l e a f f e c t e d by 6 M u r e a . P r o t e i n A e x i s t e d as o l i g o m e r s i n t h e d e t e r g e n t e x t r a c t as shown by g e l f i l t r a t i o n i n t h e p r e s e n c e o f sodium d o d e c y l s u l f a t e ( F i g . 46, l o w e r p r o f i l e ) . P r o t e i n A was e l u t e d i n f r a c t i o n s 49-51 as j u d g e d by SDS-p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s o f h e a t e d samples f r o m e a c h column f r a c t i o n . T h i s e l u t i o n p o s i t i o n c o r r e s p o n d s t o a m o l e c u l a r w e i g h t o f 60,00.0.. However, t h i s v a l u e i s p r o b a b l y l o w e r t h a n t h e t r u e m o l e c u l a r w e i g h t o f t h e o l i g o m e r s i n c e 141a F i g . 4.3. SDS-polyacrylamide gel scans of protein A extracted from protein A-peptido-glycan complexes by 1% SDS at 100°C for 15 min (scan 1), 6 M urea at 100°c for 15 min (scan 2), 6 M urea at 37°C for 1 h (scan 3). Peptidoglycan was removed by centrifugation at 100,000 x g for 1 h at 15°C A l l sam-ples for electrophoresis were made 1% i n SDS and 6 M i n urea and heated at 100°C for 5 min. Gels (10$ acrylamide) were run i n a phosphate buffer system at neutral pH, stained with Coomassie Blue and scanned at 550 nm. 142 A U c CO -Q o n < 1 c m I H 142a P i g . 4 4 . E x t r a c t i o n of p r o t e i n A-peptido-glycan complexes, prepared from glucose-grown c e l l s , with 6 M urea i n 50 mM T r i s - a c e t a t e b u f f e r , pH 7-5 at 100°C f o r 1 5 min. Peptidoglycan was removed by c e n t r i f u g a t i o n at 120,000 x g f o r 1 h at 15°C. Panel 1, scan of a 5% polyacrylamide g e l run i n the presence of 6 M urea i n a T r i s b u f f e r system. The g e l was s t a i n e d with Coomassie Blue and scanned at 550 nm. The d i r e c t i o n of m i g r a t i o n was towards the anode. Panel 2 shows the e l u t i o n p r o f i l e of p r o t e i n A from a column (1 x 37 cm) of Sepharose 6B, e q u i l i b r a t e d with 50 mM T r i s -a c e t a t e , pH 7.5 S c o n t a i n i n g 6 M urea. The sample (1 ml) i n the column b u f f e r (1 mg p r o t -ein/ml) was a p p l i e d to the column and f r a c t i o n s (o.9 ml) were c o l l e c t e d and the p r o t e i n content determined (•). The e l u t i o n p o s i t i o n s of Blue Dextran (Vo), bovine serum albumin (BSA) and ovalbumin (Oval) are i n d i c a t e d . 143 F r a c t i o n N u m b e r P i g . 45. G e l f i l t r a t i o n o f p r o t e i n A o l i g o m e r s . P r o t e i n A - p e p t i d o g y l c a n complexes d e r i v e d f r o m g l u c o s e - g r o w n (A]_) and c a s e i n amino a c i d - g r o w n ( A 2 ) c e l l s were d i g e s t e d w i t h l y s o z y m e (10 /\g/mg p r o t e i n ) f o r 15 h at 37°C i n 50 mM T r i s - a c e t a t e b u f f e r , pH 7-5- U r e a was added t o a c o n c e n t r a -t i o n o f GM and t h e sample (1 m l , c o n t a i n i n g c a . 1 mg o f p r o t e i n ) was a p p l i e d t o a column (1 x 37 cm) o f S e p h a r o s e 6B, e q u i l i b r a t e d w i t h T r i s - G M u r e a b u f f e r . F r a c t i o n s (0.9 ml) were c o l l e c t e d and th e p r o t e i n c o n t e n t d e t e r m i n e d . A b s o r b a n c e was measured a t 500 nm. Open sy m b o l s , sample i n c u b a t e d a t 37°C f o r 1 h p r i o r t o a p p l i c a t i o n t o column; c l o s e d s y m b o l s , sample h e a t e d a t 100°C f o r 15 min p r i o r t o a p p l i c a t i o n t o column. 144a F i g . 46. Gel f i l t r a t i o n of p r o t e i n s s o l u b i l i z e d from i s o l a t e d outer membrane by SDS at 22°C (lower p r o f i l e ) and at 100°C (upper p r o f i l e ) . The pr o t e i n s were resolved on a column of Sepharose 6B (1 x 37 cm) e q u i l i b r a t e d with 0.1 M sodium phosphate b u f f e r , pH 7*2. F r a c t i o n s (0.9 ml) were c o l l e c t e d and the p r o t e i n content was determined by the method of Lowry et_ a l . (171). A, (A)x, B and B* i n d i c a t e the e l u t i o n p o s i -t i o n s of p r o t e i n A oligomer, p r o t e i n B, and heat-modified p r o t e i n B, as deter-mined by a n a l y s i s of the f r a c t i o n s by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . 146 p r o t e i n A does not r e a d i l y b i n d sodium d o d e c y l s u l f a t e (80) and so wou l d have a s m a l l e r S t o k e s r a d i u s compared t o t h e m o l e c u l a r w e i g h t m a r k e r s w h i c h w o u l d b i n d up t o 1.4 g d e t e r g e n t / g p r o t e i n (152,290). M o r e o v e r , t h e m o l e c u l a r w e i g h t m a r k e r s w o u l d be r e t a r d e d t o some e x t e n t on t h e column due t o t h e asymmetry o f t h e i r SDS complexes (152). P r o t e i n B m i g r a t e d as t h e n o n - h e a t m o d i f i e d f o r m ( f r a c t i o n s 60-62) w i t h a m o l e c u l a r w e i g h t o f 25,000. When t h e e x t r a c t was h e a t e d a t 100°C p r i o r t o c h r o m a t o g r a p h y p r o t e i n B was c o n v e r t e d t o t h e h e a t - m o d i f i e d f o r m ( p r o t e i n B*) and e l u t e d a t a p o s i t i o n c o r r e s p o n d i n g t o a m o l e c u l a r w e i g h t o f 33,400 ( F i g . 43, u p p e r c u r v e ) . O l i g o m e r s o f p r o t e i n A were d i s a g g r e g a t e d i n t o t h e monomer and e l u t e d i n f r a c t i o n s 56 and 57 c o r r e s p o n d i n g t o a m o l e c u l a r w e i g h t o f 43,000. The m o l e c u l a r w e i g h t d e t e r m i n e d by S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s was 37,000. P r o t e i n A, as monomeric s u b u n i t s i n SDS o r u r e a was com-p l e t e l y s e n s i t i v e t o d i g e s t i o n by p r o n a s e . Removal o f t h e d e n a t u r a n t by d i a l y s i s a g a i n s t 10 mM T r i s - H C l b u f f e r , pH 7.5, c o n t a i n i n g 20 mM MgCl2, i n t h e p r e s e n c e o r a b s e n c e o f p e p t i d o -g l y c a n , d i d n o t r e s t o r e r e s i s t a n c e t o d i g e s t i o n . T h u s , t h e c o n d i t i o n s r e q u i r e d f o r d i s s o c i a t i o n o f t h e p r o t e i n A-p e p t i d o g l y c a n complex must i r r e v e r s i b l y d e n a t u r e t h e p r o t e i n . R e a s s o c i a t i o n o f monomeric p r o t e i n B and t h e f o r m a t i o n o f p r o t e i n Bp A p o r t i o n o f p r o t e i n B r e s i s t a n t to. d i g e s t i o n by p r o n a s e r e m a i n s i n t h e membrane f o l l o w i n g t r e a t m e n t w i t h p r o n a s e . 147 Whereas, p r o t e i n B was r e a d i l y e x t r a c t e d f r o m t h e membrane w i t h SDS a t 37°C, h i g h e r t e m p e r a t u r e s ( a b o u t 100°C) were r e q u i r e d t o s o l u b i l i z e p r o t e i n Bp. P r o t e i n Bp was i s o l a t e d and p u r i f i e d by g e l f i l t r a t i o n i n t h e p r e s e n c e o f SDS ( P i g s . 4 7 , 4 8 ) . I t s amino a c i d c o m p o s i t i o n ( T a b l e I I I ) was n o t e n r i c h e d i n h y d r o p h o b i c amino a c i d s when compared t o p r o t e i n B i n s p i t e o f t h e i n c r e a s e d d i f f i c u l t y i n s o l u b i l i z i n g i t f r o m t h e membrane. T h u s , i t s i n t e r a c t i o n w i t h t h e o t h e r components o f t h e membrane may not be p r i m a r i l y h y d r o p h o b i c , a l t h o u g h a s m a l l h y d r o p h o b i c s e q u e n c e c a n n o t be e x c l u d e d . P r o n a s e t r e a t -ment must remove a p o r t i o n o f t h e a m i n o - t e r m i n a l s e q u e n c e o f p r o t e i n B s i n c e p r o t e i n Bp c o n t a i n e d an a m i n o - t e r m i n a l v a l i n e r e s i d u e ( F i g . 16) whereas t h e a m i n o - t e r m i n a l amino g r o u p o f t h e p o l y p e p t i d e c h a i n o f p r o t e i n B was not a v a i l a b l e f o r r e a c t i o n ( P i g . 16). F u r t h e r e v i d e n c e on t h e n a t u r e o f t h e i n t e r a c t i o n s o f p r o t e i n B i n the.".membrane was o b t a i n e d f r o m r e c o n s t i t u t i o n s t u d i e s . The c r i t e r i o n we u s e d t o show t h a t p r o t e i n B had been r e a s s o c i a t e d t o a s i m i l a r s t a t e t o t h a t i n t h e n a t i v e mem-b r a n e was t h e f o r m a t i o n o f p r o t e i n Bp upon d i g e s t i o n w i t h p r o n a s e . P r o t e i n B was f r e e d o f d e t e c t a b l e p h o s p h o l i p i d and l i p o p o l y s a c c h a r i d e f o r t h e s e e x p e r i m e n t s . D i g e s t i o n o f p r o t e i n B i n t h e p r e s e n c e o f SDS p r o d u c e d numerous s m a l l f r a g m e n t s w h i c h m i g r a t e d n e a r t h e dye f r o n t on S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . When t h e d e t e r g e n t was removed by d i a l y s i s f o r 24 h a g a i n s t a 10 mM T r i s - H C l b u f f e r , pH 7 - 5 , c o n t a i n i n g 20 mM M g C l 2 , a p r e c i p i t a t e o f r e -147a F i g . 47. P u r i f i c a t i o n of protein Bp by gel f i l t r a t i o n i n the presence of SDS. Pronase-treated c e l l wall was extracted twice with SDS, f i r s t at 37°C for 1 h, then at 10-oc for 15 min. Peptidoglycan was removed by c e n t r i -fugation at 120,000 x g for 1 h. The second extract was dialyzed against d i s t i l l e d water, freeze-dried, and then dissolved to 2 mg protein/ml i n 10 ml of column buffer, contain-ing 0.1$ 2-mercaptoethanol. After heating at 100°C for 5 min, the sample was applied to a column of Sephadex G100 (5 x 40. cm) connected i n series to a column of Sephadex 6B of the same dimensions. Proteins were eluted with 0.1 M sodium phosphate buffer, pH 7.2, contain-ing 1% SDS. Fractions (10 ml) were co l l e c t e d , the absorbance measured at 280 nm and the protein content determined by SDS-gel e l e c t r o -phoresis. Fractions (88-92, p r o f i l e 1) containing protein Bp were pooled and reapplied to the same column system. Fractions (89-93, p r o f i l e 2) containing protein Bp were pooled, dialyzed and freeze-dried. 148 T — r F r a c t i o n N u m b e r 149 B p J P i g . 48. S D S - p o l y a c r y l a m i d e g e l s c a n o f p u r i f i e d p r o t e i n Bp r u n i n s y s t e m 1. The g e l (10% a c r y l a m i d e ) was s t a i n e d w i t h C o o m a s s i e B l u e and s c a n n e d a t 550 nm. 150 associated protein B was formed. Treatment of t h i s material with pronase gave a fragment with a molecular weight of about 20,000 (Pig. 49, scan 3) which appeared to be s i m i l a r to protein Bp prepared by digestion of the native membrane. I f the MgCl 2 was replaced by 20 mM EDTA i n the d i a l y s i s buffer, pronase digestion of the precipitated protein B yielded multiple digestion fragments (Fig. 49, scan 2 ) . These results suggest that i n the presence of divalent cations protein B w i l l re-associate to a state similar to that i n the native membrane. The conditions for reassembly were further examined (Table V). The. amount of protein Bp produced by pronase digestion of protein B reassociated i n the presence of MgCl 2 was taken as 100$. No intact protein B remained under the d i -gestion conditions used (Fig. 49, scan 3)- D i a l y s i s of protein B i n SDS against buffers containing manganous or calcium ions produced yie l d s of protein Bp similar to that formed with magnesium ions. I f the sample of protein B was frozen, l y o p h i l i z e d , or converted to form B* by heating ( 4 ) , p r i o r to attempted reassembly, no pronase-resistant digestion fragments were produced. This supports the previous suggestion that protein B as isolated from the membrane with SDS retains some native conformation which i s lost upon heating. The importance of protein-protein interactions i n the protection of a portion of the protein B molecule from diges-t i o n by pronase was supported by the following observation. Addition of p u r i f i e d lipopolysaccharide or soy bean phospho-l i p i d s i n up to a 10-fold and 50-fold excess by weight over 151 B 1 c m f - » F i g . 49. Production of p r o t e i n Bp by pronase d i g e s t i o n of r e a s s o c i a t e d p r o t e i n B. Coomassie Blue-stained SDS-polyacrylamide gels were scanned at 550 nm. 1, p u r i f i e d p r o t e i n B; 2, e f f e c t of pronase (1 mg/25 mg p r o t e i n B) on p r o t e i n B which had been d i a l y z e d f o r 24 h against 10 mM T r i s - H C l b u f f e r , pH 7-5, c o n t a i n i n g 20 mM EDTA; 3, e f f e c t of pronase on p r o t e i n B which had been d i a l y z e d against 10 mM T r i s - H C l b u f f e r , pH 7-5, c o n t a i n i n g 20 mM MgCl 2. 152 T a b l e V C o n d i t i o n s f o r r e a s s o c i a t i o n o f p r o t e i n B T r e a t m e n t % Bp number o f p r o n a s e -r e s i s t a n t p e aks No d i a l y s i s D i a l y s i s w i t h M g 2 + Mn2+ Ca2+ EDTA 20 100 97 158 31 m u l t i p l e s i n g l e s i n g l e s i n g l e m u l t i p l e P r o t e i n B: F r o z e n L y o p h i l i z e d h e a t e d 0 0 0 none none none L i p o p o l y s a c c h a r i d e added P h o s p h o l i p i d added 100 83 s i n g l e s i n g l e P r o t e i n B (1 ml) i n 1% SDS was d i a l y z e d a g a i n s t 1 1 o f 10 mM T r i s HC1, pH 7.5, c o n t a i n i n g e i t h e r 20 mM M g C l 2 , M n C l 2 , C u C l 2 o r EDTA a t 220C f o r 24 h. A f t e r d i a l y s i s , t h e p r e p a r a t i o n was t r e a t e d w i t h p r o n a s e a t an e n z y m e : p r o t e i n r a t i o o f 1:25 f o r 2 h a t 37°C. The amount o f p r o t e i n Bp p r o d u c e d u n d e r t h e s e c o n d i t i o n s a f t e r d i a l y s i s a g a i n s t T r i s b u f f e r c o n t a i n i n g 20 mM M g C l 2 was t a k e n as \Q.0%. In some e x p e r i m e n t s p r o t e i n B was f r o z e n , l y o p h i l i z e d , o r h e a t e d a t 100°C f o r 5 m i n , o r l i p o p o l y s a c c h a r i d e o r p h o s p h o l i p i d was added p r i o r t o d i a l y s i s ( s e e t e x t ) . 153 p r o t e i n B, r e s p e c t i v e l y , p r i o r t o t h e d i a l y s i s s t e p d i d n o t i n c r e a s e t h e y i e l d o f t h e p r o n a s e r e s i s t a n t , f r a g m e n t . C r o s s l i n k i n g o f p r o t e i n A - p e p t i d o g l y c a n complexes The e v i d e n c e p r e s e n t e d above s u g g e s t s t h a t e x t e n s i v e p r o t e i n - p r o t e i n i n t e r a c t i o n s o c c u r i n t h e o u t e r membrane. The r e l a t i o n s h i p s o f t h e o u t e r membrane p r o t e i n s t o one a n o t h e r was examined by t h e use o f c r o s s l i n k i n g a g e n t s and two-d i m e n s i o n a l S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . P r o t e i n A-j_- and p r o t e i n A 2 ~ p e p t i d o g l y c a n complexes p r e -p a r e d f r o m g l u c o s e - ^ a n d c a s e i n amino a c i d - g r o w n c e l l s , r e s p e c -t i v e l y , were t r e a t e d w i t h i n c r e a s i n g amounts o f t h e c r o s s l i n k e r DSP. As shown i n F i g . 50, b o t h p r o t e i n s Ai_ and A 2 c r o s s l i n k e d t o f o r m d i m e r , t r i m e r , and h i g h e r o l i g o m e r s . The t r i m e r was a l w a y s p r o d u c e d i n e x c e s s o v e r t h e t e t r a m e r w h i c h s u g g e s t s t h a t p r o t e i n A m ight be o r g a n i z e d as t r i m e r s i n t h e membrane. A l t h o u g h p r o t e i n s Aj_ and A 2 were no t w e l l r e s o l v e d by t h e g e l s y s t e m t h e c o n t e n t o f Ai_ and A2 i n t h e two p r e p a r a t i o n s was c o n f i r m e d by SDS e l e c t r o p h o r e s i s a t an a l k a l i n e pH (79). C r o s s l i n k i n g o f p r o t e i n A t o o l i g o m e r s i n p r o t e i n A-p e p t i d o g l y c a n complexes was a l s o o b s e r v e d w i t h g l u t a r a l d e h y d e ( F i g . 51). S i n c e t h e p r o t e i n A - p e p t i d o g l y c a n complexes a l s o c o n t a i n l i p o p r o t e i n ( p r o t e i n F) c o v a l e n t l y bound t o t h e p e p t i d o g l y c a n (44), i t was p o s s i b l e t h a t p r o t e i n A c o u l d be c r o s s l i n k e d t o t h e p e p t i d o g l y c a n t h r o u g h t h i s p r o t e i n . T h i s p o s s i b i l i t y was examined as d e s c r i b e d i n F i g . 52. The p r o t e i n A - p e p t i d o g l y c a n 15'I 3 4 5 ( A ) 3 A • 6. 7 8 9 JO F i g . 50. C r o s s l i n k i n g of p r o t e i n A-peptidoglycan complexes with DSP. Samples (100A* 1; 0.1 mg p r o t e i n ) of p r o t e i n A-peptidoglycan complexes prepared from glucose-grown (samples 2-5) and casein amino acid-grown c e l l s (samples 6-9) were t r e a t e d with 0/*g (samples 2 and 6 ) , 40/*g (samples 3,7), 200 A*g (samples 4 ,8) , and 600 A* g (samples of 5,9) of DSP. The samples were s o l u b i l i z e d with 9 volumes of SDS-electro-phoresis sample b u f f e r and then resolved on 9% polyacrylamide g e l s l a b s . Samples 1 and 10 contain as molecular weight markers bovine serum albumin (66,000), ovalbumin (46,000), and lysozyme (14,400). 1 5 5 1 2 3 4 5 6 7 8 9 |0 Pig. 51. Crosslinking of protein A-peptidoglycan complexes with glutaraldehyde. Samples (100/*1; 0.1 mg protein) of protein A-peptidoglycan complexes prepared from glucose-grown (samples 2-5) and casein amino acid-grown c e l l s (samples 6-9) were treated with 0% CA) (samples 2 and 6), 0.05% (samples 3 and 7), 0.1% (samples 4 and 8) and 0.5% (samples 5 and 9). Pin a l concentration of glutaraldehyde i n 0.2 M triethanolamine buffer, pH 8.5. The samples were s o l u b i l i z e d with 9 volumes of SDS-electrophoresis buffer and then resolved on 9% polyacrylamide gel slabs. Samples 1 and 10 contain as molecular weight markers bovine serum albumin (66,000), ovalbumin (46,000) and lysozyme (14,400). 155a Pig. 52. Lack of crosslinking of protein A to the peptidoglycan layer by DSP treatment of protein A-peptidoglycan complexes. The protein A-peptidoglycan complex, prepared from glucose-grown c e l l s was suspended to 0.3 mg protein/ml i n 10 ml of 0.2 M triethanolamine buffer, pH 8.5. DSP (5 mg) was added from a stock solution (30 mg/ml i n dimethyl sulfoxide). After 1 h at 22°C, 2.5 ml of 1 M Tris-HCl buffer, pH 8.5, was added and the complex was recovered by centrifugation at 100,000 x g for 1 h at 15°C The crosslinked complex was extracted with 1% SDS at 100°C for 15 min. The peptidoglycan was recovered by centrifugation and reextracted by 1% SDS, 1% 2-mercaptoethanol at 100°C for 15 min. The peptidoglycan was removed by centrifugation. A control experiment without added DSP was performed i n p a r a l l e l . Scan 1, SDS-polyacryla-mide gel of protein A extracted by SDS from control peptidoglycan complexes. Scan 2, SDS-polyacrylamide gel of crosslinked protein extracted from DSP-treated peptidoglycan complexes. Scan 3, SDS-polyacrylamide gel of second SDS extract of DSP-treated peptidoglycan complexes. A l l samples were made 10% i n gl y c e r o l , heated at 100°C for 5 min and then resolved i n 5$ polyacrylamide gels run with a phosphate buffer system 1 at neutral pH. Gels were--stained with Coomassie Blue and scanned at 550 nm. 157 complex was f i r s t reacted with DSP and then extracted with 1% SDS. Uncrosslinked protein A i s extracted by SDS at 100°C (scan 1) as were i t s crosslinked oligomers (scan 2). The peptidoglycan, and any proteins linked to i t , was reextracted as before but i n the presence of 1% 2-mercaptoethanol to cleave the crosslinking agent. No further protein A was released (scan 3) indicating that i t had not been crosslinked to the bound li p o p r o t e i n or d i r e c t l y to the peptidoglycan. Crosslinking of outer membrane C e l l wall, composed of outer membrane and peptidoglycan, and i s o l a t e d outer membrane preparations were treated with di f f e r e n t levels of DSP (Pig. 53). Proteins A and D 2 (molec-ular weight 18,000) were less r e a d i l y crosslinked than proteins B and F, the free form of the li p o p r o t e i n . The a b i l i t y of the proteins to be crosslinked i s determined by the degree of exposure of suitably disposed amino groups. Thus, protein B, which was most readily crosslinked, was also most reactive with the amino group reagent fluorescamine and was readily digested by pronase. In contrast, protein A, which was less readily crosslinked i n outer membrane preparations, reacted more slowly with fluorescamine and was resist a n t to digestion by pronase. The effect of DSP crosslinking of pretreating the outer membrane with pronase was studied. Pronase cleaves protein B to a fragment, protein Bp, which remains embedded i n the membrane, but does not digest protein A. In the pronase-158 Ammm B— 4 5 6 7 8 9 10 fr M ' • a a m If K 4Mk — -F i g . 53- C r o s s l i n k i n g of c e l l w a l l and outer membrane with DSP. Samples ( 1 0 0 A * 1 ; 0 . 1 mg p r o t e i n ) o f c e l l w a l l (samples 2-5) and outer membrane (samples 6-9) prepared from g l u c o s e -grown c e l l s were t r e a t e d with 0A*-g (samples 2 and 6), 20A*.g (samples 3 and 7), 40/*g (samples 4 and 8) and 60 A*g (samples 5 and 9) of DSP. The samples were s o l u b i l i z e d w i t h two volumes of SDS e l e c t r o p h o r e s i s sample b u f f e r and r e s o l v e d on 7.5$ p o l y a c r y l a m i d e g e l s l a b s . Samples 1 and 1 0 contained as molecular weight markers bovine serum albumin ( 6 6 , 0 0 0 ) , ovalbumin ( 4 6 , 0 0 0 ) , and lysozyme (14,400). 159 treated membrane protein A was crosslinked to the dimer and trimer. In contrast to intact protein B, protein Bp was not readily crosslinked (Fig. 54). Thus, the functional groups responsible for the crosslinking of protein B must be situated on the exposed portion of this protein. This supports the view that the a c c e s s i b i l i t y of the reactive groups on the protein are important i n determining i t s ease of reaction with the crosslinker. The formation of the dimer and trimer of protein A occurred when r e l a t i v e l y high levels of DSP (2 mg/mg protein) were reacted with the outer membrane. This was confirmed by two-dimensional SDS-polyacrylamide gel electrophoresis i n which the crosslinked products separated i n the f i r s t dimension were cleaved by 2-mercaptoethanol p r i o r to entering the separating gel of the second dimension. The cleaved products could then be identified, from t h e i r c h a r a c t e r i s t i c rate of migration (Fig. 55). The formation of the dimer and trimer of protein A by crosslinking with both the outer membrane and the protein A-peptidoglycan complex suggests that protein A i s arranged In the same way i n both preparations. At the higher l e v e l of crosslinker, protein B gave a high molecular weight complex that penetrated the k% stacking gel but did not enter the 7-5$ polyacrylamide gel of the f i r s t dimension. Crosslinking of protein A to protein B was never observed. rcan Interaction of proteins B and F with peptidoglyc When the c e l l wall (outer membrane-peptidoglycan) pre-160 ( A ) 3 ( A ) 2 B P P i g . 5 4 . C r o s s l i n k i n g of pronase-treated outer membrane w i t h DSP. Outer membrane was t r e a t e d with pronase at an enzyme: p r o t e i n r a t i o of 1 : 2 5 f o r 2 h at 37°C. The outer membrane was recovered by c e n t r i f u g a t i o n at 1 2 0 , 0 0 0 x g f o r 1 h and resuspended to about 1 mg of protein/ml i n 0 . 2 M tr i e t h a n o l a m i n e b u f f e r , pH 8 . 5 . Samples ( 1 0 0 / * 1 ; 0 . 1 mg p r o t e i n ) of digested membrane were t r e a t e d with O ^ i g (sample 1 ) , 2 0 0 y*.g (sample 2 ) , and 6 0 0 , M g (sample 3) of DSP. The samples were s o l u b i l i z e d w i t h nine volumes of SDS e l e c t r o p h o r e s i s sample b u f f e r and then r e -solved on 9% polyacrylamide g e l s l a b s . Sample 4 contained as molecular weight markers bovine serum albumin ( 6 6 , 0 0 0 ) , ovalbumin ( 4 6 , 0 0 0 ) , and lysozyme ( 1 4,400). 161 m i A B ' F i g . 55. Two-dimensional g e l of products from c r o s s l i n k e d outer membrane p r o t e i n s . A sample (100/*1; 0.1 mg p r o t e i n ) of outer membrane was tr e a t e d i n the 200 / A g of DSP then s o l u b i l i z e d w i t h two volumes of SDS e l e c t r o p h o r e s i s sample b u f f e r , and r e s o l v e d on a f i r s t dimension 1.5% polyacrylamide g e l . The c r o s s l i n k e d products were cleaved i n the second dimension by 2-mercaptoethanol and re s o l v e d on a 12% p o l y -acrylamide g e l . The p o s i t i o n s of monomer (A), dimer ( A ) 2 , and t r i m e r (A)3, of p r o t e i n A, and the high molecular weight oligomer of p r o t e i n B, ( B ) x , are i n d i c a t e d . A stained first dimension g e l i s placed along the top of the g e l s l a b . 162 p a r a t l o n was t r e a t e d w i t h DSP u n d e r I d e n t i c a l c o n d i t i o n s t o t h o s e u s e d i n t h e p r e v i o u s e x p e r i m e n t w i t h t h e i s o l a t e d o u t e r membrane, a s i m i l a r t w o - d i m e n s i o n a l g e l p a t t e r n was o b t a i n e d ( F i g . 56). However, i t was c o n s i s t e n t l y o b s e r v e d t h a t a much l a r g e r c r o s s l i n k e d complex o f p r o t e i n B was fo r m e d w h i c h c o u l d n o t e n t e r t h e k% s t a c k i n g g e l i n t h e f i r s t d i m e n s i o n . I n o r d e r t o d e t e r m i n e i f t h i s was due t o t h e c r o s s l i n k i n g o f p r o t e i n B t o t h e p e p t i d o g l y c a n , e i t h e r d i r e c t l y o r t h r o u g h a n o t h e r p r o t e i n , t h e f o l l o w i n g e x p e r i m e n t was p e r f o r m e d . The c e l l w a l l p r e p a r a t i o n was c r o s s l i n k e d w i t h DSP and t h e n e x t r a c -t e d w i t h a b u f f e r c o n t a i n i n g 1% sodium d o d e c y l s u l f a t e and 10$ g l y c e r o l i n 62.5 mM T r i s - H C l , pH 6.8, a t 100° f o r 3 min. The i n s o l u b l e p e p t i d o g l y c a n and a t t a c h e d p r o t e i n s were r e e x t r a c t e d w i t h t h i s b u f f e r b u t c o n t a i n i n g 1% 2 - m e r c a p t o e t h a n o l . A c o n t r o l e x p e r i m e n t was c a r r i e d out i n w h i c h t h e c r o s s l i n k e r was o m i t t e d . As a f u r t h e r c o n t r o l , t h e i s o l a t e d o u t e r membrane was t a k e n t h r o u g h t h e above p r o c e d u r e . The r e s u l t s o f t h i s e x p e r i m e n t a r e shown i n F i g . 57. I n t h e a b s e n c e o f DSP t h e f i r s t e x t r a c t o f t h e c e l l w a l l c o n t a i n e d a l l o f t h e o u t e r mem-b r a n e p r o t e i n (sample 1). The f i r s t e x t r a c t o f t h e c r o s s l i n k e d c e l l w a l l showed t h e p r e s e n c e o f t h e d i m e r and t r i m e r o f p r o t e i n A (sample 2) w h i c h c o u l d be c l e a v e d by t h e a d d i t i o n o f 2-m e r c a p t o e t h a n o l (sample 3). The s e c o n d e x t r a c t o f t h e non-c r o s s l i n k e d sample c o n t a i n e d v i r t u a l l y no p r o t e i n s (sample 4). However, t h e s e c o n d e x t r a c t i o n o f t h e c r o s s l i n k e d c e l l w a l l w i t h t h e 2 - m e r c a p t o e t h a n o l - c o n t a i n i n g b u f f e r r e l e a s e d p r o t e i n s B and F f r o m t h e p e p t i d o g l y c a n (sample 5). I n c o n t r a s t t o c e l l 163 (B) x (A)3(A). t I F i g . 56. Two-dimensional gel of products from crosslinked c e l l wall. A sample of c e l l wall, consisting of outer membrane and peptidoglycan was treated as described i n F i g . 52. The positions of monomer (A), dimer ( A ) 2 , and trimer (A)g, of protein A, and the high molecular weight oligomer of protein B, (B) are indicated. A stained f i r s t dimensional gel i s placed along the top of the gel slab. 164 L _2_ _3_ 4 5 _6 J L 8 9 10 P i g . 57. C r o s s l i n k i n g of prot e i n s B and F to the peptidoglycan l a y e r . Preparations (1 ml; 1 mg p r o t e i n ) of c e l l w a l l (samples 1-5) and outer membrane (samples 6-10) were t r e a t e d w i t h 2 mg of DSP f o r 30 sec. A f t e r a d d i t i o n of excess T r i s -HCl, pH 8.5, two volumes of SDS e l e c t r o p h o r e s i s sample b u f f e r without 2-mercaptoethanol were added and the p r o t e i n s were s o l u b i l i z e d by heating at 100°C f o r 3 min. The supernatant ( f i r s t e x t r a c t ) was removed a f t e r c e n t r i f u g a t i o n of the mixture at 120,000 x g f o r 1 h at 15°C and the p e l l e t was r e - e x t r a c t e d with three volumes of sodium dodecyl s u l f a t e e l e c t r o p h o r e s i s sample b u f f e r c o n t a i n i n g 1% 2-mercaptoethanol (second e x t r a c t ) . The peptidoglycan was removed by c e n t r i f u g a t i o n at 120,000 x g f o r 1 h at 15 C. Cont r o l preparations (1 ml) untreated w i t h DSP were c a r r i e d through the same procedure. Samples 1 and 6, f i r s t e x t r a c t of c o n t r o l p r e p a r a t i o n ; samples 2 and 7, f i r s t e x t r a c t of DSP-treated p r e p a r a t i o n ; samples 3 and 8, same as samples 2 and 7 but t r e a t e d w i t h 2-mercaptoethanol p r i o r to e l e c t r o p h o r e s i s ; samples 4 and 9, second e x t r a c t of c o n t r o l p r e p a r a t i o n ; samples 5 and 10, second e x t r a c t of DSP-treated p r e p a r a t i o n . 165 w a l l , a l l the c r o s s l i n k e d products of the I s o l a t e d outer membrane was r e a d i l y s o l u b i l i z e d i n the f i r s t e x t r a c t (sam-ples 6-8) as the second e x t r a c t d i d not contain any p r o t e i n s (samples 9 ,10). The r e s u l t s i n d i c a t e that p r o t e i n s B and F can be c r o s s l i n k e d to the peptidoglycan. This might occur by l i n k -ing to a f r e e amino group of the peptidoglycan i t s e l f or by l i n k i n g to the bound l i p o p r o t e i n . However, the l i n k i n g of p r o t e i n B or p r o t e i n F to the bound l i p o p r o t e i n or peptido-glycan could be i n d i r e c t through the other p r o t e i n . In t h i s case a c r o s s l i n k e d product con t a i n i n g both p r o t e i n s B and F should be formed. F i g . 58 shows the e f f e c t of t r e a t i n g a c e l l w a l l p r e p a r a t i o n w i t h DSP at a r e l a t i v e l y low l e v e l (0.4; mg DSP/mg p r o t e i n ) . Besides a dimer of p r o t e i n A and one of p r o t e i n B, a c r o s s l i n k e d product w i t h a molecular weight of about 40,000 and c o n t a i n i n g one molecule each of p r o t e i n s B and F was detected. This product gave two spots of both p r o t e i n s B and F on the g e l f o l l o w i n g cleavage with 2-mercaptoethanol. This i s probably due to the presence of i n -t e r n a l c r o s s l i n k i n g such that one or both of the p r o t e i n s r e t a i n s a more compact conformation on denaturation w i t h SDS which would a f f e c t i t s r a t e of m i g r a t i o n on e l e c t r o p h o r e s i s . A small amount of i n t e r n a l l y c r o s s l i n k e d p r o t e i n A, which comigrated with p r o t e i n B i n the f i r s t dimension, can a l s o be seen i n F i g . 58. The presence of a dimer of p r o t e i n F was detected when c r o s s l i n k e d m a t e r i a l s i m i l a r to that used i n the previous 1 6 6 ( A ) 2 ( B ) 2 B - F o ; b F i g . 58. Two dimensional g e l of products from c r o s s l i n k e d c e l l w a l l . A sample (100 A»1; 0.1 mg p r o t e i n ) of c e l l w a l l was t r e a t e d w i t h 40 A*g of DSP, s o l u b i l i z e d w i t h two volumes of SDS e l e c t r o p h o r e s i s sample b u f f e r , and r e s o l v e d on a 7-5$ polyacrylamide g e l . The c r o s s l i n k e d products were cleaved i n the second dimension with 2-mercaptoethanol and r e s o l v e d on a 12$ polyacrylamide g e l . The p o s i t i o n s of the c r o s s l i n k e d products c o n t a i n i n g both p r o t e i n s B and F (B-F a, molecular weight, 42,200; and B-F D, molecular weight, 40,300), and the dimers of p r o t e i n A ((A) 2, molecular weight, 80,000) and p r o t e i n B ((B)2, molecular weight, 70,500) are i n d i c a t e d . A stained f i r s t dimension g e l i s placed along the top of the g e l s l a b . 167 experiment was run on two-dimensional gels containing higher concentrations of polyacrylamide (Pig. 5 9 ) . These re s u l t s indicate that at least some of the molecules of protein B and protein F i n the outer membrane must be i n close proximity to one another, and that there may be groups of molecules of protein F as suggested by Inouye (133). The.se re s u l t s do not prove that either protein B or F i s linked through the other to the peptidoglycan layer but they are not Inconsistent with t h i s p o s s i b i l i t y . The results of pr o t e o l y t i c digestion, covalent l a b e l l i n g and c r o s s l i n k i n g have been integrated to give a model for the organization of the proteins of the outer membrane of E. c o l i . This model Is presented i n the Discussion section. 168 F i g . 59. Two dimensional g e l of products from c r o s s l i n k e d c e l l w a l l . A c r o s s l i n k e d preparation of c e l l w a l l s i m i l a r to that described i n F i g . 6 was separated on a f i r s t dimension 12% polyacrylamide g e l to resolve low molecular weight c r o s s -l i n k e d products. The c r o s s l i n k e d p r o t e i n s were cleaved i n the second dimension wi t h 2-mercaptoethanol and r e s o l v e d i n a 15% polyacrylamide g e l . The p o s i t i o n of the c r o s s l i n k e d products of p r o t e i n s B and F (B-F) and the dimer of p r o t e i n F, ((F)2) 9-re i n d i c a t e d . A. stained f i r s t dimension g e l i s placed along the top of the g e l s l a b . 169 DISCUSSION P r o p e r t i e s of the outer membrane p r o t e i n s P r o t e i n B P r o t e i n B i s r e l e a s e d from the outer membrane by detergent treatment and may t h e r e f o r e be c l a s s i f i e d as an i n t e g r a l membrane p r o t e i n (156). T h i s p r o t e i n has an amino a c i d compo-s i t i o n that appears to be c h a r a c t e r i s t i c of membrane p r o t e i n s . Namely, a low content of c y s t e i n e , an excess of a c i d i c amino a c i d s over b a s i c , and a moderate content of hydrophobic amino a c i d s (204). The amino a c i d composition of p r o t e i n B presen-t e d i n t h i s t h e s i s agrees with t h a t determined by Henning's group f o r p r o t e i n I I * from E. c o l i B/r (87). T h i s suggests that p r o t e i n B and Henning's p r o t e i n I I * are the same p r o t e i n (Table I ) . Schnaitman has shown th a t p r o t e i n 3 from E. c o l i 0 111 Is composed of two d i f f e r e n t p o l y p e p t i d e s , both of which are he a t - m o d i f i a b l e (88). The s i n g l e band on SDS-gels found before and a f t e r h e a t i n g , the k i n e t i c s of conversion o f p r o t e i n B to form B*, the cyanogen bromide cleavage p a t t e r n , the amino t e r m i n a l a n a l y s i s , the p r o d u c t i o n of s i n g l e p r o t e a s e - r e s i s t a n t fragments by pronase, t r y p s i n and chymotrypsin treatment o f the outer membrane, a l l suggest t h a t p r o t e i n B from E. c o l i NRC 482 i s a s i n g l e p o l y p e p t i d e . The same c o n c l u s i o n was reached by Henning f o r the h e a t - m o d i f i a b l e p r o t e i n from E. c o l l B/r. The d i f f e r e n c e i n Schnaitman./s r e s u l t s may be 170 b e c a u s e t h e c e l l s he u s e d were grown on s u c c i n a t e r a t h e r t h a n g l u c o s e o r b e c a u s e o f d i f f e r e n c e s i n t h e s t r a i n u s e d . The c y a n o g e n b r o m i d e c l e a v a g e p a t t e r n o b t a i n e d f o r p r o t e i n B on S D S - g e l s d i f f e r s f r o m t h a t r e p o r t e d by o t h e r w o r k e r s . H e n n i n g ' s g r o u p f o u n d t h a t p r o t e i n I I * c o n t a i n s f i v e m e t h i o -n i n e r e s i d u e s and c y a n o g e n b r o m i d e t r e a t m e n t r e s u l t e d I n t h e p r o d u c t i o n o f f i v e m a j o r f r a g m e n t s w i t h m o l e c u l a r w e i g h t s o f 14 ,000, 11,000, 9,000, 2,000, 1,000 as d e t e r m i n e d by S D S - g e l e l e c t r o p h o r e s i s (87). S c h n a i t m a n (88) s e p a r a t e d p r o t e i n 3 by i o n - e x c h a n g e c h r o m a t o g r a p h y i n t o two p o l y p e p t i d e s , 3a and 3b t h a t d i f f e r e d i n t h e i r c y a n o g e n b r o m i d e c l e a v a g e p a t t e r n s . P r o t e i n 3a gave two m a j o r p e p t i d e s (M.W. 22,000 and 12,000) and a l a r g e number o f s m a l l e r p e p t i d e s on S D S - g e l s a f t e r c y a n o g e n b r o m i d e c l e a v a g e . The S D S - g e l p a t t e r n f o r t h e c y a -nogen b r o m i d e c l e a v a g e p r o d u c t s o f p r o t e i n 3b showed two b r o a d bands w i t h d i f f e r e n t m o l e c u l a r w e i g h t s f r o m t h a t d e t e r m i n e d f o r t h e f r a g m e n t s d e r i v e d f r o m p r o t e i n 3a. The r e l a t i o n s h i p o f t h e two m a j o r p e p t i d e s (M.W. 18,000 and 15,000) o b t a i n e d by c y a n o g e n b r o m i d e t r e a t m e n t o f p r o t e i n B t o t h e s e r e s u l t s i s n o t c l e a r . The d i f f e r e n c e may be a c c o u n t e d f o r by t h e use o f d i f f e r e n t g e l s y s t e m s o r d i f f e r e n c e s due t o s t r a i n o r g r o w t h c o n d i t i o n s . No a m i n o - t e r m i n a l d e r i v a t i v e was d e t e c t e d f o r p r o t e i n B. The n a t u r e o f t h e b l o c k i n g g r o u p was n o t d e t e r m i n e d , however, i t may be c a r b o h y d r a t e (88) o r p e r h a p s a p y r r o l i d i n e c a r -b o x y l i c r e s i d u e . H e n n i n g ' s g r o u p r e p o r t e d t h a t p r o t e i n I I * has an a m i n o - t e r m i n a l a l a n i n e (87). However, no f u r t h e r de-171 r i v a t i v e s were r e l e a s e d by s u b s e q u e n t Edman d e g r a d a t i o n s . The h e a t - m o d i f i a b l e p r o t e i n c h a r a c t e r i z e d i n t h i s t h e s i s may d i f f e r f r o m t h e p r o t e i n c h a r a c t e r i z e d by H e n n i n g ' s g r o u p by t h e a m i n o ' - t e r m i n a l amino a c i d . P r o t e i n B (M.W. 28,500) was c o n v e r t e d t o a h i g h e r m o l e -c u l a r w e i g h t f o r m B* (M.W. 33,400) upon h e a t i n g . The t e m p e r a t u r e - d e p e n d e n t c o n v e r s i o n o f p r o t e i n B t o B* a c c o u n t s f o r t h e m u l t i p l e p r o t e i n bands o b s e r v e d by a number o f a u t h o r s (81,82,177)• T h e s e w o r k e r s had h e a t e d membrane e x t r a c t s a t 70°C f o r 20 min. T h i s r e s u l t s i n t h e c o n v e r s i o n o f about 25% o f p r o t e i n B t o f o r m B*. Peak 7 o f In o u y e and Yee (82), peak C o f S c h n a i t m a n (8l) and b a n d D o f Koplow and G o l d f i n e (177) r e p r e s e n t u n c o n v e r t e d p r o t e i n B, w h i l e peak 6 o f Inouye and Yee, peak B o f S c h n a i t m a n and band C o f Koplow and G o l d f i n e c o r r e s p o n d t o f o r m B* ( T a b l e I ) . P r o t e i n 3 o f S c h n a i t m a n (88) and I I * o f H e n n i n g show s i m i l a r h e a t - m o d i f i -a b l e c h a r a c t e r i s t i c s t o p r o t e i n B and may be t h e same p r o t e i n . S c h n a i t m a n (8 1)-suggested t h a t t h e c o n v e r s i o n o f p r o t e i n t o B* was due' t o u n f o l d i n g o f t h e p r o t e i n c a u s i n g an i n c r e a s e d l e v e l o f SDS b i n d i n g and c o n v e r s i o n t o t h e r i g i d r o d c o n f o r m a -t i o n p r o p o s e d by R e y n o l d s and T a n f o r d (152) f o r S D S - p r o t e i n . c o m p l e x e s . I t was c o n f i r m e d t h a t p r o t e i n B u n f o l d s i r r e v e r s i b l y upon h e a t i n g , however no f u r t h e r b i n d i n g o f SDS o c c u r r e d . T h i s s u g g e s t s t h a t p r o t e i n B as i s o l a t e d f r o m t h e membrane con t a i n s some n a t i v e s t r u c t u r e w h i c h I s l o s t upon h e a t i n g . T h i s i s c o n f i r m e d by t h e o b s e r v a t i o n t h a t p r o t e i n B, b u t n o t f o r m B* i s a b l e t o r e a s s o c i a t e i n t o an a r r a n g e m e n t s i m i l a r t o 172 that found i n the n a t i v e membrane. The true molecular weight of p r o t e i n B i s unknown. Henning and coworkers have determined from amino a c i d a n a l y s i s and by summation of the molecular weights of the peptides formed on cleavage with cyanogen bromide, that the molecular weight of p r o t e i n B i s 27,000, c l o s e r to the molecular weight of the unmodified form (87). Molecular weight determinations by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s are subject to e r r o r (204) as are determinations based on minimal chemical molecular weights by amino a c i d a n a l y s i s . The true molecular weight of p r o t e i n B may be c l o s e r to the modified form since heating would cause the p r o t e i n to a t t a i n a more extended s t r u c t u r e with l o s s of a l l n a t i v e s t r u c t u r e . Conversion to a r i g i d rod conformation would make comparisons of p r o t e i n B t o standard p r o t e i n s i n SDS more meaningful. Further s t u d i e s on t h i s pro-t e i n are r e q u i r e d before i t s molecular weight can be e s t a b l i -shed w i t h c e r t a i n t y . P r o t e i n A P r o t e i n A i s f i r m l y attached to the peptidoglycan and-.as such i s r e s i s t a n t to p r o t e o l y t i c d i g e s t i o n . L i k e p r o t e i n B, i t i s only moderately hydrophobic, has a low content of cyteine and contains more a c i d i c residues than b a s i c . The amino a c i d composition of p r o t e i n A agrees with that determined by Fiosenbusch (8.0). f o r the matrix p r o t e i n and by Henning's group f o r p r o t e i n 1 suggesting that they are the same p r o t e i n (Table I ) . The matrix p r o t e i n contains four methionine r e s i d u e s , 173 however I t i s not r e a d i l y cleaved by cyanogen bromide. This r e s u l t s i n the production of peptides, the molecular weight of which, when summed, g r e a t l y exceed that of the i n t a c t p r o t e i n . The incomplete cleavage makes comparisons of SDS-gel patterns of separated fragments,.from d i f f e r e n t l a b o r a t o r i e s d i f f i c u l t . E. c o l i NRC 482 contains two d i f f e r e n t matrix p r o t e i n s , A-i_ and A 2 that can be resolved by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s at an a l k a l i n e pH (system 2). Both are as-soci a t e d with the peptidoglycan, have s i m i l a r amino a c i d compositions, and give s i m i l a r spectrum of peptides on SDS-gels a f t e r cyanogen bromide cleavage, suggesting that they may be two forms of the same polypeptide. P r o t e i n s 1 and 2 of Schnaitman are l i k e l y equivalent to pr o t e i n s A-^  and A 2 (Table I ) . This i s supported by the f i n d i n g that l i k e Schnaitman's p r o t e i n 2, p r o t e i n A 2 i s under c a t a b o l i t e r epres-s i o n . No p r o t e i n equivalent to p r o t e i n A 2 i s found i n most E. c o l i s t r a i n s i n c l u d i n g K 12 (91,140), JE 1011 (179) and B/r (91,149). E. c o l i B/r does however contain two forms of the matrix p r o t e i n , l a and l b that d i f f e r i n only one region of the polypeptide chain (142). The r e l a t i o n s h i p of p r o t e i n s l a and l b t o p r o t e i n A.^  i s not c l e a r , however i t i s l i k e l y that one of the forms of p r o t e i n I i s the same as p r o t e i n A]_. I t i s apparent that the matrix p r o t e i n v a r i e s with s t r a i n and growth c o n d i t i o n s . The r e l a t i o n s h i p of these v a r i a t i o n s to the f u n c t i o n of the matrix p r o t e i n as a passive pore through the outer membrane remains to be. e l u c i d a t e d . 174 Organization of proteins i n the outer membrane The experiments described i n the second part of thi s thesis have been primarily directed towards understanding the arrangement of the proteins i n the outer membrane of E. c o l i and t h e i r relationship to some of the other components of thi s membrane. Attempts to use intact c e l l s i n these studies met with limited success. Whereas pronase rea d i l y digested certain proteins of the outer membrane i n isola t e d c e l l envelope or outer membrane preparations, the major proteins i n intact c e l l s were unaffected by thi s treatment. Removal of a fr a c -t i o n of the lipopolysacchardie by EDTA treatment did not result i n the loss of any major outer membrane proteins from intact c e l l s and these proteins remained in s e n s i t i v e to diges-t i o n . Thus, there does not appear to be an intimate relationship between this pool of lipopolysaccharide and the major outer membrane proteins. However, the p o s s i b i l i t y that the non-releasable portion of the lipopolysaccharide might be protecting the proteins from digestion was examined using a heptose-deficient mutant which the 0-antigen and core regions of the lipopolysaccharide were absent. There was no change i n the pattern of digestion i n t h i s mutant. Moreover, i n spite of the lower levels of proteins A and B found i n mutants of t h i s type (86,177,179) there was no change i n the behaviour of the other outer membrane proteins. This suggests that these proteins i n the mutant must be arranged i n a similar way to those i n the parent s t r a i n , and that the resistance of 175 t h e p r o t e i n s i n i n t a c t c e l l s was n o t due t o t h e l i p o p o l y -s a c c h a r i d e . The p o s s i b i l i t y t h a t r e s i s t a n c e was due t o t h e u n a v a i l a b i l i t y o f c e r t a i n g r o u p s a t t h e s u r f a c e o f t h e membrane i s s u p p o r t e d by t h e f i n d i n g t h a t d a n s y l c h l o r i d e was u n a b l e t o l a b e l c e l l s u r f a c e p r o t e i n s i n b o t h r o u g h and smooth s t r a i n s o f S a l m o n e l l a t y p h i m u r l u m (238). However, t y r o s i n e r e s i d u e s a r e e x p o s e d a t t h e s u r f a c e o f t h e membrane i n b o t h t h i s o r g a n i s m and i n Pseudomonas f a c 1 1 i s s i n c e l a c t o p e r o x i d a s e -c a t a l y z e d i o d i n a t i o n o f p r o t e i n s o f m o l e c u l a r w e i g h t s 16,000 and 14 ,000, r e s p e c t i v e l y , o c c u r r e d i'n i n t a c t c e l l s o f t h e s e o r g a n i s m s (58,59). A l s o , t h e a b i l i t y t o l a b e l s u r f a c e com-p o n e n t s o f i n t a c t c e l l s o f J E and NS-1 s u g g e s t s t h a t r e a c t i v e g r o u p s a r e a v a i l a b l e on t h e s u r f a c e o f t h e c e l l . The u s e o f c e l l e n v e l o p e , c e l l w a l l ( o u t e r membrane-p e p t i d o g l y c a n ) and i s o l a t e d o u t e r membrane p r e p a r a t i o n s gave a c l e a r e r u n d e r s t a n d i n g o f t h e a r r a n g e m e n t o f t h e o u t e r mem-b r a n e p r o t e i n s . The s i m i l a r i t y i n r e s p o n s e o f t h e p r o t e i n s t o p r o t e o l y t i c d i g e s t i o n , l a b e l l i n g and c r o s s l i n k i n g i n t h e d i f f e r e n t p r e p a r a t i o n s a r g u e s a g a i n s t a m a s s i v e a r t i f a c t u a l r e a r r a n g e m e n t o f t h e o u t e r membrane p r o t e i n s i n s u b c e l l u l a r ' p r e p a r a t i o n s . R e c e n t o b s e r v a t i o n s , i n c l u d i n g t h o s e p r e s e n t e d i n t h i s t h e s i s have p e r m i t t e d an u p d a t i n g o f t h e models o f t h e o r -g a n i z a t i o n o f o u t e r membrane components ( F i g s . 5 a - i ) . The model p r e s e n t e d i n Fig .6Q summarizes t h e r e s u l t s o f p r o -t e o l y t i c d i g e s t i o n , , c o v a l e n t l a b e l l i n g and c r o s s l i n k i n g s t u d -i e s . T h i s model i s s c h e m a t i c o n l y and does n o t r u l e out 176 alternative arrangements of the proteins. Lipopolysaccharide i s dis t r i b u t e d on the outside of the bil a y e r with the polar carbohydrate chains extending into the medium (45). The EDTA-releasable f r a c t i o n of the lipopoly-saccharide i s s t a b i l i z e d by divalent cations such as magnesium and calcium (38,61) and probably does not interact primarily with protein. The phospholipids are dist r i b u t e d mainly i n the inner layer of the bilayer or are covered by proteins when in the outer layer as proposed recently by Nikaido (107,282). Proteins C, D]_ and E are probably exposed at the surface of the membrane since they are readily extracted with sodium dodecyl sulfate and are digested by pronase. Protein , which i s resistant to digestion and i s not readily crosslinked, must be protected by i t s arrangement i n the membrane. The bound lipoprotein extends from the peptidoglycan into the outer membrane (44). I f the free and bound form of the lipop r o t e i n form a channel (Pigs. 5 g and i ) as proposed by Inouye (130) then they w i l l interact with the hydrophobic regions of the membrane. The existence of groups of protein P molecules i s supported by the detection of a crosslinked dimer of protein F. The apparent lack of reaction of the lipoprotein with antibodies at the surface of intact c e l l s (123) does not necessarily exclude the penetration of the outer membrane by the lipo p r o t e i n complex. Its external sur-face might be protected from int e r a c t i o n with the antibody by lipopolysaccharide or other proteins. This i s supported by the finding that protein F, the free form of the li p o p r o t e i n , 177 l i k e protein B can be digested at the surface of is o l a t e d outer membrane preparations but not i n intact c e l l s . A l t e r n a t i v e l y , single lipoprotein molecules might span the distance from the peptidoglycan to the outer membranes i l l u s t r a t e d i n most models (Figs. 5c,d,e, and f ) . However, i t appears from sequence analysis that the function of the lipop r o t e i n i s to serve as an interface between a hydrophobic and a hydrophilic zone. Channel models would f i t t h i s concept of l i p o p r o t e i n function. A lip o p r o t e i n complex proposed by Inouye would not be located i n the periplasmic space (Fig. 5h) since the hydrophobic amino acid residues would have to be i n contact with the water of the periplasmic space. The i n a b i l i t y of proteases to digest proteins A.]_ and A 2 . may not be due to the absence of these proteins at the surface of the membrane since they probably act as receptors for certain bacteriophages (91,98,105)• Rosenbusch (80) has shown that molecules of protein A are arranged i n a regular array on the peptidoglycan i n isola t e d protein A-peptidoglycan complexes. Protein A was not digested by pronase i n these preparations, but i t was readily digested when s o l u b i l i z e d . This observation was confirmed both with complexes containing primarily protein A]_ and with complexes of protein A 2. Thus, the pronase resistance of these proteins i s probably a function of the folding of the protein molecule or of the in t e r a c t i o n between i t and other molecules of protein A. Moreover, prot-eins A-j_ and A 2 must be s i m i l a r l y disposed i n the outer 178 membrane. T h i s I s s u p p o r t e d by. t h e i r s i m i l a r amino a c i d c o m p o s i t i o n s . P r o t e i n A a l s o can f o r m c h a n n e l s t h r o u g h l i p o p o l y s a c c h a r i d e - p h o s p h o l i p i d v e s i c l e s (13*0 . T hese p r o p e r t i e s s u g g e s t t h a t p r o t e i n A spans t h e o u t e r membrane i n c o n f l i c t w i t h I n o u y e ' s model ( F i g . 5 i ) . R o s e n b u s c h (80) u s i n g e l e c t r o n m i c r o s c o p y has s u g g e s t e d t h a t p r o t e i n A f orms a l a t t i c e s t r u c t u r e w i t h h e x a g o n a l symmetry on t h e o u t e r f a c e o f t h e p e p t i d o g l y c a n . The r e s u l t s i n t h i s t h e s i s a l s o i n d i c a t e t h a t m o l e c u l e s o f p r o t e i n A must i n t e r a c t w i t h one a n o t h e r s i n c e o l i g o m e r s o f t h i s p r o t e i n were i s o l a t e d a l t h o u g h t h e number o f s u b u n i t s i n t h e o l i g o m e r c o u l d n o t be d e t e r m i n e d . P r o t e i n A can be c r o s s l i n k e d t o t h e d i m e r and t r i m e r b u t no l i n k i n g t o o t h e r p r o t e i n has been d e t e c t e d . T h u s , p r o t e i n A i s p r o b a b l y r e s t r i c t e d t o p a t c h e s i n t h e membrane and t h e c h a n n e l m i g h t be f o r m e d by t h e a s s o c i a t i o n o f t h r e e m o l e c u l e s o f t h i s p r o t e i n . T h i s would be c o n s i s t e n t w i t h t h e morpho-l o g i c a l r e s u l t s o f R o s e n b u s c h (80) who s u g g e s t e d t h a t 1 t o 3 m o l e c u l e s o f p r o t e i n A f o r m a u n i t , and w i t h t h e i s o l a t i o n o f o l i g o m e r s o f t h i s p r o t e i n . S u r p r i z i n g l y , no c r o s s l i n k i n g o f p r o t e i n A t o t h e p e p t i d o g l y c a n was d e t e c t e d . P r o t e i n B a p p e a r s t o be e x p o s e d a t t h e s u r f a c e o f t h e membrane s i n c e a bout 25$ o f t h e m o l e c u l e c o u l d be removed by p r o n a s e d i g e s t i o n t o l e a v e a f r a g m e n t o f 20,000 m o l e c u l a r w e i g h t embedded i n t h e membrane. H e n n i n g and c o w o r k e r s (131, 136) have f o u n d t h a t t r y p s i n d i g e s t i o n o f p r o t e i n B i n g h o s t s gave a f r a g m e n t w i t h a m o l e c u l a r w e i g h t o f 25,000. A s i m i l a r 179 fragment was produced by digestion with chymotrypsin. The exposure of protein B at the surface was confirmed by covalent l a b e l l i n g with fluorescamine. Protein B was more re a d i l y l a b e l l e d than protein A i n agreement with t h e i r apparent degree of exposure at the surface of the membrane determined with pro t e o l y t i c digestion. The exposed portion of protein B i s responsible for the ease with which t h i s protein i s crosslinked to i t s e l f to form high molecular weight aggregates. The presence of protein-protein interactions between molecules of protein B i s supported by the divalent cation-dependent reassociation of t h i s protein which can occur i n the absence of phospholipid and lipopolysac-charide. However, the portion of the native structure which i s retained when protein B i s s o l u b i l i z e d with sodium dodecyl sulfate at 37°C i s necessary to ensure the:reassociation of protein B into structures, which on the basis of cleavage with pronase to y i e l d protein Bp, may resemble the arrangement of protein B i n the native outer membrane. Protein Bp has an amino acid composition which i s not markedly more hydrophobic than protein B. This would be consistent with the primary inte r a c t i o n of protein B with components of the membrane being other than hydrophobic. Although most of the interactions which we have found are between molecules of the same protein, as also seems to be the case with the erythrocyte membrane (296), an association of protein B with protein F was detected. These proteins can be crosslinked to the peptidoglycan possibly through the 180 l i p o p r o t e i n w h i c h i s c o v a l e n t l y bound t o t h e p e p t i d o g l y c a n . The a s s o c i a t i o n o f p r o t e i n s B and P w i t h t h e bound l i p o p r o t e i n w ould r e s t r i c t t h e i r m o b i l i t y i n t h e membrane. The non-c o v a l e n t i n t e r a c t i o n o f p r o t e i n A w i t h t h e p e p t i d o g l y c a n m ight a l s o have t h e same e f f e c t . T h u s , t h e l a t e r a l m o b i l i t y o f t h e o u t e r membrane p r o t e i n s i s l i k e l y t o be r e s t r i c t e d . T h i s c o u l d r e s u l t i n t h e e x i s t e n c e o f s e p a r a t e r e g i o n s o f p r o t e i n I n t h e membrane. The a b s e n c e o f d e t e c t a b l e i n t e r a c t i o n s between p r o t e i n A and B wou l d s u p p o r t t h i s h y p o t h e s i s . T h u s , as H e n n i n g and c o w o r k e r s have s u g g e s t e d (13), t h e S i n g e r -N i c h o l s o n model o f membrane s t r u c t u r e (240) may n o t a p p l y t o t h e o u t e r membrane o f E. c o l i . L P S P i g . 6 0 . S c h e m a t i c model f o r t h e arrangement o f p r o t e i n s i n t h e o u t e r membrane o f E. c o l i . D i v a l e n t c a t i o n s ( • ) , p h o s p h o l i p i d ( P L ) , l i p o p o l y s a c c h a r i d e ( L P S ) , p e p t i d o g l y c a n (PG) and p r o t e i n s A t o P a r e i n d i c a t e d . 182 BIBLIOGRAPHY 1 . G l a u e r t , A . M . and T h o r n l e y , M . J . ( 1 9 6 9 ) Annu. Rev. M i c r o b i o l . 23, 1 5 9 - 1 9 8 . 2 . Osborn,M.J. (1969) Annu. Rev. Biochem. 38, 5 0 1 - 5 3 8 . 3. R e a v e l e y , D . A . and Burge,R.E. ( 1 9 7 2 ) Adv. i n M i c r o b . P h y s i o l . 7, 1-81. 4. Braun,V. and Hantke,K. ( 1 9 7 4 ) Annu. Rev. Biochem. 43, 8 9 - 1 2 1 . 5. C o s t e r t o n , J . W . , Ingram,J.M. and Cheng,K.J. ( 1 9 7 4 ) B a c t e r i o l . Rev. 3 8 , 8 7 - 1 1 0 . 6. Osborn,M.J., R i c k , P . D . , Lehmann,V., R u p p r e c h t ,E. and Singh,M. ( 1 9 7 4 ) Ann. N.Y. A c a d . S c i . 2 3 5 , 5 2 - 6 5 . 7. Bayer,M.E. ( 1 9 7 4 ) Ann. N.Y. A c a d . S c i . 2 3 5 , 6 - 2 8 . 8. Bayer,M.E. ( 1 9 7 5 ) i n "Membrane B i o g e n e s i s , M i t o c h o n d r i a , C h l o r o p l a s t , B a c t e r i a " ( T z a g o l o f f , A . , e d . ) , pp. 3 9 3 - 4 2 7 , Plenum P r e s s , New Y o r k . 9. Murray,R.G.E. ,. S t e e d , P . and E l s o n , H . E . ( 1 9 6 5 ) Can. J . M i c r o b i o l . , 1 1 , 5 4 7 - 5 6 0 . 1 0 . D e P e t r i s , S . ( 1 9 6 7 ) J . U l t r a s t r u c t , Res. 1 9 , 4 5 - 8 3 -1 1 . White,D.A., L e n n a r z , W . J . and Schnaitman,C.A. ( 1 9 7 2 ) J . B a c t e r i o l . 1 0 9 , 6 8 6 - 6 9 0 . 1 2 . Schnaitman,C.A. ( 1 9 7 0 ) J . B a c t e r i o l . 1 0 4 , 8 8 2 - 8 8 9 . 1 3 . Schnaitman,C.A. ( 1 9 7 0 ) J . B a c t e r i o l . 104, 890-901. 14. Osborn,M.J., G a n d e r , J . E . , P a r i s i , E . and C a r s o n , J . ( 1 9 7 2 ) J . B i o l . Chem. 2 4 7 , 3 9 6 2 - 3 9 7 2 . 1 5 . M a r t i n , E . L . and MacLeod,R.A. ( 1 9 7 1 ) J . B a c t e r i o l . 1 0 5 , I I 6 O-II67. 1 6 . Nanninga,N. ( 1 9 7 0 ) J . B a c t e r i o l . 1 0 1 , 2 9 7 - 3 0 3 . 1 7 . Bayer,M.E. and Remsen,C.C. ( 1 9 7 0 ) J . B a c t e r i o l . 1 0 1 , 3 0 4 - 3 1 3 . 1 8 . T o u r t e l l o t t e , M . E . and Z u p n i k , J . S . ( 1 9 7 3 ) S c i e n c e 1 7 9 , 84 - 8 6 . 183 19. T o u r t e l l o t t e , M . E . (1972) I n "Membrane M o l e c u l a r B i o l o g y " ( F o x , C . F . and K e i t h , A . D . , e d s . ) , pp. 439-470, S i n a u e r A s s o c i a t e s I n c . , S t a m f o r d , Conn. 20. R o b e r t s o n , J . D . (1964) i n " C e l l u l a r Membranes i n D e v e l o p m e n t " (M.Locke, e d . ) , pp. l - 8 l , Academic P r e s s , New Y o r k . 21. Gellman,N.S. and Lakoyanova,M.A. (1967) " R e s p i r a t i o n and P h o s p h o r y l a t i o n o f B a c t e r i a " Plenum P r e s s , New Y o r k . 22. Lemberg,R. and B a r r e t t , J . (1973) i n " C y t o c h r o m e s " , pp. 217-326, A c a d e m i c P r e s s , -New Y o r k . 23- Simoni,R.D. (1972) i n "Membrane M o l e c u l a r B i o l o g y " ( F o x , C ( v p l , and K e i t h , A . D . , e d s . ) , S i n a u e r A s s o c i a t e s I n c . , S t a m f o r d , Conn. 24. H a r o l d , P . M . (1972) B a c t e r i o l , Rev. 36, 172-230. 25. Kaback,H.R. and Hong,J. (1973) CRC C r i t i c a l Review i n M i c r o b i o l . 3, 333-376. 26. Boos,W. (1974) Annu. Rev. Biochem. 43, 123-146. 27. B e l l , R . M . , Mavis,R.D., Osboirn,M.J. and V a g e l o s , P . R . (1971) B i o c h i m . B i o p h y s . A c t a 249, 628-635. 28. White,D.A., A l b r i g h t , F . R . , L ennarz,W.J. and Schnaitman,C.A. (1971) B i o c h i m . B i o p h y s . A c t a 249, 636-642. 29. C r o n a n , J . E . and V a g e l o s , P . R . (1972) B i o c h i m . B i o p h y s . A c t a 265, 25-60. 30. Lennarz,W.J. (1972) A c c o u n t s o f Chem. Res. 5, 361-367-31. R o g e r s , H . J . and P e r k i n s , H . R . (1968) " C e l l W a l l s and Membranes" E. and F.N. Spon L t d . , London. 32. R o g e r s , H . J . (1970) B a c t e r i o l . Rev. 34, 194-204. 33- Ghuysen,J.M. and Shockman,G.D. (1973) i n " B a c t e r i o l Membranes and W a l l s " ( L e i v e , L . , e d . ) , pp. 37-130, M a r c e l D e k k e r , New Y o r k . 34. Osborn,M.J. (1971) i n " S t r u c t u r e and F u n c t i o n o f B i o l o g i c a l Membranes" ( R o t h f I e l d , L . , e d . ) , pp. 343-400, Academic P r e s s , New Y o r k . 35. Osborn,M.J., G a n d e r , J . E . , and P a r i s ! , E . (1972) J . B i o l . Chem'. 247, 3973-3986. 184 36. R o t h f i e l d , L . and Romeo,D. (1971) i n " S t r u c t u r e and F u n c t i o n o f B i o l o g i c a l Membranes" ( R o t h f i e l d , L . , e d . ) , pp. 251-284, Academic P r e s s , New Y o r k . 37. R o t h f i e l d , L . and Romeo,D. (1971) B a c t e r i o l . Rev. 35, 14-38. 38. N i k a i d o , H . (1973) i n " B a c t e r i o l Membranes and W a l l s " ( L e i v e , L . , e d . ) , pp. 131-208, M a r c e l D e k k e r , New Y o r k . 39- S t r o m i n g e r , J . L . and T i p p e r , D . J . (1974) i n "Lysozyme" (Osserman,E.F., C a n f i e l d , R , E . and B e y c h o k , S . , e d s . ) , pp. 169-184, A c a d e m i c P r e s s , New Y o r k . 40. Braun,V., G n i r k e , H . , H e n n i n g , U . and Rehn,K. (1973) J. B a c t e r i o l . 114, 1264-1270. 41. F o r s b e r g , C . W . , C o s t e r t o n , J . W . and MacLeod,R.A. (1970) J . B a c t e r i o l . 104, 1338-1353. 42. Weidel,W. and P e l z e r , H . (1964) Advan. E n z y m o l . 26, 193-232. 43. F o r s b e r g , C . W. , Rayman,M.K., C o s t e r t o n ,J . VJ. and MacLeod,R.A. (1972) J. B a c t e r i o l . 109, 895-905. 44. Braun,V. (1975) B i o c h i m . B i o p h y s . A c t a 415, 335-377-45. Shands,J.W. (1968) J. B a c t e r i o l . 90, 266-270. 46. Weidel,W. (1958) Annu. Rev. M i c r o b i o l . 12, 27-48. 47. F o r g e , A . , C o s t e r t o n , J . W . and K e r r , K . A . (1973) J. B a c t e r i o l , 113, 445-457. 48. DeVoe,I.W., C o s t e r t o n , J . W . and MacLeod,R.A. (1971) J-B a c t e r i o l . 106, 659-671. 49. G i l l e l a n d , H . E . , S t i n n e t , J . D . , R o t h , I . L . and Eagon,R.G. (1973) J. B a c t e r i o l 113, 417-432. 50. M u h l r a d t , P . F . , M e n z e l , J . , G o l e c k i , J . R . and Sp e t h , V . (1974) E u r . J. Biochem. 43, 533-539-51. L e v y , S . B . and L e i v e , L . (1968) P r o c . N a t l . A c a d . S c i . U.S.A. 61, 1435-1439. 52. L u d e r w i t z , 0 . , W e s t p h a l , 0 . , Staub,A.M. and N i k a i d o , H . (1971) i n " M i c r o b i a l T o x i n s " (Weinbaum,G., K a d i s , S . and A j l , S . J . e d s . ) , V o l . 4, pp. 145-233, Academic P r e s s , New Y o r k . 185 53. A s h w e l l , G . and H i c k m a n , J . (1971) i n " M i c r o b i a l T o x i n s " (Weinbaum,G., K a d i s , S . and A j l , S . J . e d s . ) , V o l . 4, pp. 235-266, Academic P r e s s , New Y o r k . 54. Osborn,M.J. and R o t h f i e l d , L . I . (197D i n " M i c r o b i a l T o x i n s " . (Weinbaum,G., K a d i s , S . and A j l , S . J . e d s . ) , V o l . 4, pp. 331-350, A c a d e m i c P r e s s , New Y o r k . 55. Robbins,P.W. and W r i g h t , A . (1971) i n " M i c r o b i a l T o x i n s " (Weinbaum,G., K a d i s , S . and A j l , S . J . e d s . ) , V o l . 4 , pp. 351-368, A c a d e m i c P r e s s , New Y o r k . 56. S t o c k e r , B . A . D . and Makela,P.H. (1971) i n " M i c r o b i a l T o x i n s " (Weinbaum,G., K a d i s , S . and A j l , S . J . e d s . ) , V o l . 4, pp. 369-438, Academic P r e s s , New Y o r k . 57- Shands,J.W. (1971) i n " M i c r o b i a l T o x i n s " (Weinbaum,G., K a d i s , S . and A j l , S . J . e d s . ) , V o l . 4, pp. 127-144, A c a d e m i c P r e s s , New Y o r k . 5 8 . F r e e r , J . H . and S a l t o n , M . J . R . (1971) i n " M i c r o b i a l T o x i n s " (Weinbaum,G., K a d i s , S . and A j l , S . J . e d s . ) , V o l . 4 , pp. 67-126, A c a d e m i c P r e s s , New Y o r k . 59. P a p a h a d j a p o u l o s , D . and M i l l e r , N . (1967) B i o c h i m . B i o p h y s . A c t a 135, 624-638. 60. Haest,C.W.M., d e G i e r , J . and vanDeenan,L.L.M. (1969) Chem. Phys. L i p i d s 3, 413-417. 61. L e i v e , L . (1974) Ann. N.Y. A c a d . S c i . 235, 109-129-62. C o s t e r t o n , J . W . and Cheng,K.J. (1975) J . A n t i m i c r o b i a l Chemotherapy 1, 363-377-63. R o a n t r e e , R . J . , Kuo,T., MacPhee,D.G. and S t o c k e r , B . A . 0 . (1969) Amer. Soc. M i c r o b i o l . , B a c t e r i o l . P r o c . p. 79, A b s t M76. 64. Tamaki,S., S a t o , T . and M a t s u h a s h i , M . (197D J - B a c t e r i o l 105, 968-975-65- W i l k i n s o n , R . G . , Gemski,P. and S t o c k e r , B . A . D . (1972) J . Gen. M i c r o b i o l . 70, 527-554. 66. Schnaitman,C.A. (197D J - B a c t e r i o l . 108, 545-552. 67. Schnaitman,C.A. (1971) J . B a c t e r i o l . 108, 553-563. 68. M u l r a d t , P . F . and G u l e c k i , J . R . (1975) E u r . J . Biochem. 51, 343-352. 186 69. F i l i p , C , F l e t c h e r , G . , Wulff,J.L. and Earhart,C.F. (1973) J . B a c t e r i o l , 115, 717-722. 70. J o s e l e a u - P e t i t , D . and Kepes,A. (1975) Biochim. Biophys. Acta 406, 36-49. 71. B i r d s e l l , D . C . and Cota-Robles,E.H. (1967) J . B a c t e r i o l . 93, 427-437. 72. De Pamphilis,M.L. (197D J • B a c t e r i o l . 105, 1184-1199-73- Miuru,T. and Mizushima,S. (1968) Biochim. Biophys. Acta 150, 159-161. 74. Miuru,T. and Mizushima,S. (1969) Biochim. Biophys. Acta 193, 268-276. 75. Osborn,M.J. and Munson,R. (1974) i n "Methods i n Enzymol." ( F l e i s c h e r , S . and Packer,L., eds.), V o l . 31, pp. 6 4 2 -653, Academic Press, New York. 76. Yamato,I., Anraku,Y. and Hirosawa,K. (1975) J- Biochem. 77, 705-718. 77- Mizushima,S. and Yamada,H. (1975) Biochim. Biophys. Acta 375, 44-53-78. Wolf-Watz,H., Normark,S. and Bloom,G.D. (1973) J -B a c t e r i o l . 115, 1191-1197-79- Bragg,P.D. and Hou,C. (1972) Biochim. Biophys. Acta 274, 4 7 8 - 488 . 80. Rosenbusch,J.P. (1974) J . B i o l . Chem. 2 4 9 , 8019-8029. 81. Schnaitman,C.A. (1973) Arch. Biochem. Biophys. 157, 541-552. 82. Inouye,M. and Yee,M.L. (1973) J- B a c t e r i o l . 113, 304-312. 83. Ames,G.F.L. (1974) J . B i o l . Chem. 2 4 9 , 6 3 4 - 644 . 8 4 . Holland,L.B. and Tuckett,S. (1972) J . Supramol. S t r u c t . 1, 77-97. 85. Uemura,S. and Mizushima,S. (1975) Biochim. Biophys. Acta 413, 163-176. 86. Ames,G.F.L., Spudich,E.N. and Nikaido,H. (1974) J . B a c t e r i o l . 117, 406-4l6. 87. Garten,W., Hindennach,I. and Henning,U. (1975) Eur. J. Biochem. 59, 215-221. 187 88. S c h n a i t m a n , C . A . (1974) J . B a c t e r i o l . 118, 442-453. 89. Schnaitman,C.A. (1970) J . B a c t e r i o l . 104, 1404-1405. 90. Weber,K. and Osborn,M (1969) J . B i o l . Chem. 244, 4406-4412. 91. S c h n a i t m a n , C . A . (1974) J . B a c t e r i o l . 118, 454-464. 92. Laemmli,U.K. (1970) N a t u r e 227, 680-685. 93. K a l t s c h m i d t , E . and Wittmann,H.G. (1970) A n a l . Biochem. 36, 401-412. 94. 0 ' F a r r e l l , P . H . (1974) J . B i o l . Chem. 250, 4007-4021. 95. Johnson,W.C., S i l h a v y , T . J . and Boos,W. (1975) A p p l . M i c r o -b i o l . 29, 405-413. 96. R u s s e l l , R . R . B . (1976) Can. J . M i c r o b i o l . 22, 83-91. 97. Ames,G.F.L. and N i k a i d o , K . (1976) B i o c h e m i s t r y 15, 616-623. 98. H e n n i n g , U . and H a l l e r , I . (1975) FEBS L e t t . 55, 161-164. 99- R o b i n s o n , A . and Tempest,D.W. (1973) J • Gen. M i c r o b i o l . 78, 361-370. 100. M c i n t o s h , M . A . and E a r h a r t , C . F . (1976) Biochem. B i o p h y s . Res. Commun. 70, 315-322. 101. Hantke,K. and Braun,V. (1975) FEBS L e t t . 59, 277-281. 102. Luckey,M., Wayne,R. and N e i l a n d s , J . B . (1975) B i o c h e m . B i o p h y s . Res. Commun. 64, 687-693. 103. Hancock,R.E.W. and Reeves,P. (1976) J . B a c t e r i o l . 127, 98-108. 104. V e r k l e i j , A . J . , L u t e n b e r g , E . J . J . and V e r v e r g a e r t , P . H . J . T . (1976) B i o c h i m . B i o p h y s . A c t a 426, 581-586. 105. S k u r r a y , R . A . , Hancock,R.E.W. and R e e v e s , P . (1974) J . B a c t e r i o l . 119, 726-735-106. M o l d o w , C , R o b e r t s o n , J . and R o t h f i e l d , L . (1972) J . Memb. B i o l . 10, 137-152. 107- N i k a i d o , H . (1976) B i o c h i m . B i o p h y s . A c t a 433, 118-132. 108. Wu,H.C. (1972) B i o c h i m . B i o p h y s . A c t a 290, 274-289-109- L e i v e , L . (1965) Biochem. B i o p h y s . Res. Commun. 21, 290-296. 188 110. Bragg,P.D. and Hou,C. (1971) FEBS L e t t . 15, 142-144. 111. Duckworth,D.H., B e v e r s , E . M . V e r k l e l j , A . J . , OpDenKamp, J.A.F. and VanDeenan,L'.L.M. (1974) A r c h . Biochem. B i o p h y s . 165, 379-387. 112. C r o n a n , J . E . (1968) J . B a c t e r i o l . 95, 2054-2061. 113. Marr,A.G. and I n g r a h a m , J . L . (1962) J . B a c t e r i o l . 84, 1260-1267. 114. L u g t e n b e r g , H . , M e l j e r , J . , P e t e r s , R . , VanderHoek,P. and V a n A l p h e n , L . (1975) FEBS L e t t . 58, 254-258. 115. M l z u s h i m a , S . (1976) P r o c . F e d . E u r . Biochem. Soc. p. 273, A b s t . 336. 116. S c h n a i t m a n , C . A . , Smith,D. and d e S a l a s , M . F . (1975) J . V i r o l . 15, 1121-1130. 117. Braun,V. and Rehn,K. (1969) E u r . J . Biochem. 10, 426-438. 118. Braun,V. and Bosch,V. (1972) P r o c . N a t l . A c a d . S c i . U.S.A. 69, 970-974. 119- Braun,V. and Bosch,V. (1972) E u r . J . Biochem. 28, 51-69. 120. Inouye,M. (1975) I n "Membrane B i o g e n e s i s , M i t o c h o n d r i a , C h l o r o p l a s t s , B a c t e r i a " ( T z a g o l o f f , A . , e d . ) , pp. 351-391. Plenum P r e s s , New Y o r k . 121. Braun,V. and W o l f f , H . (1970) E u r . J . Biochem. 14, 387-391. 122. Hantke,K. and Braun,V. (1973) E u r . J . Biochem. 34, 284-296. 123. Braun,V., Bosch,V., Klumpp,E.R., N e f f , I . , Mayer,H. and S c h l e c h t , S . (1976) E u r . J . Biochem. 62, 555-566. 124. Braun,V. (1973) J . I n f e c t . D i s . 128 ( S u p p l e m e n t ) , 9-15-125. Inouye,M. (1971) J • B i o l . Chem. 246, 4834-4838. 126. Inouye,M., Shaw,J. and Shen,C. (1972) J . B i o l . Chem. 247, 8154-8159. 127. H i r a s h i m a , A . , Wu,H.C., V e n t a k e s w a r a n , P . S . and Inouye,M. (1973) J . B i o l . Chem. 248, 5654-5659. 128. Bosch,V. and Braun,V. (1973) FEBS L e t t , 34, 307-310. 129. Braun,V., Hantke,K. and Hennlng,U. (1975) FEBS L e t t . 60, 2.6-28. 189 130. H a l e g o u a , S . , H i r a s h i m a , A . and Inouye,M. (1974) J . B a c t e r i o l . 120, 1204-1208. 131. Henning,U., Hohn,B. and S o n n t a g , I . (1973) E u r . J . Biochem. 39, 27-36. 132. Braun,V. Rehn,K. and W o l f f , H . (1970) B i o c h e m i s t r y , 9, 5041-5049, 133. Inouye,M. (1974) P r o c . N a t l . A c a d . S c i . U.S.A. 71, 2396-2400. 134. Nakae,T. (1976) J . B i o l . Chem. 251, 2176-2178. 135- Inouye,M. and Yee,M.L. (1972) J . B a c t e r i o l . 112, 585-592. 136. Henning,U., Rehn,K. and Hohn,B. (1973) P r o c . N a t l . A c a d . S c i . U.S.A. 70, 2033-2036. 137. H a l l e r , I . , Hohn,B. and Henning , U . (1975) B i o c h e m i s t r y 14, 478-484. 138. Nakamura,K., O s t r o v s k y , D . N . , Miyazawa,T. and M i z u s h i m a , S . (1974) B i o c h i m . B i o p h y s . A c t a 332, 329-335. 139. M i z u s h i m a , S . (1974) Biochem. B i o p h y s . Res. Commun. 6 l , 1221-1226. 140. H i n d e n n a c h , I . and Henning , U . (1975) E u r . J . Biochem. 59, 207-213. 141. Garten,W., H i n d e n n a c h , I . and Henning,U. (1975) E u r . J . Biochem. 60, 303-307. 142. S c h m i t g e s , C . J . and Henning , U . (1976) E u r . J . Biochem. 63, 47-52. 143. Bragg,P.D. (1965) B i o c h i m . B i o p h y s . A c t a 96, 263-271. 144. Weber,K. and K u t e r , D . J . (1971) J . B i o l . Chem. 246, 4504-4509. 145. L e n a r d , J . (1971) Biochem. B i o p h y s . Res. Commun. 45, 662-668. 146. Weber,K. and Osborn,M. (1975) i n "The P r o t e i n s " T h i r d E d i t i o n ( N e u r a t h , H . and H i l l , R . L . , e d s . ) , V o l . 1, pp. 179-223, Academic P r e s s , New Y o r k . 147. Schroeder,W.A., S h e l t o n , J . B . and S h e l t o n , J . R . (1969) A r c h . Biochem. B i o p h y s . 130, 551-556. 148. Schnaitman,C.A. (1973) A r c h . Biochem. B i o p h y s . 157, 553-560. 190 1 4 9 . G a r t e n ,W . and H e n n l n g , U . (1974) E u r . J . Biochem. 47, 343-352. 150. K i d r o n l , G . and Welnbaum,G. (1975) B i o c h i m . B i o p h y s . A c t a 399, 4 2 8 - 446 . 151. K i d r o n i , G . and Weinbaum,G. (1975) B i o c h i m . B i o p h y s . A c t a 399, 447-459. 152. R e y n o l d s , J . A . and T a n f o r d , C . (1970) J . B i o l . Chem. 2 4 5 , 5161-5165. 153. Henning,U., H i n d e n n a c h , I . and H a l l e r , I . (1976) FEBS L e t t . 61, 4 6 - 4 8 . 154. C h a i , T . and F o u l d s , J . (1974) J . M o i . B i o l . 85, 465-474. 155. D a v i e s , P . L . and Bragg,P.D. (1972) B i o c h i m . B i o p h y s . A c t a 266, 273-284. 156. S i n g e r , S . J . (1971) " S t r u c t u r e and F u n c t i o n o f B i o l o g i c a l Membranes" ( R o t h f i e l d , L . I . , e d . ) , pp. 145 -222, Academic P r e s s , New Y o r k . 157. Nomura,M. (1967) Annu. Rev. M i c r o b i o l . 21, 257-284. 158. Wu,M.C. and H e a t h , E . C . (1973) P r o c . N a t l . A c a d . S c i . U.S.A. 159. O v e r a t h , P . , B r e n n e r , M . , G u l i k - K r z y w i c k i , T . , S c h e h t e r , E . and L e t e l l i e r , L . (1975) B i o c h i m . B i o p h y s . A c t a 389, 358-569. 160. Kamio,Y. and N i k a i d o , H . (1976) B i o c h e m i s t r y 15, 2561-2570. 161. W e s t p h a l , 0 . and Jann,K. (1965) i n "Methods i n C a r b o h y d r a t e Chem." ( W h i s t l e r , R . L . , e d . ) , V o l . 5, pp. 83-91 Academic P r e s s , New Y o r k . 162. Brown,M.R.W. (1975) i n " R e s i s t a n c e o f Pseudomonas  a e r u g i n o s a " (Brown,M.R.W., e d . ) , pp. 71-99, W i l e y , New Y o r k . 163. K a t e s , M . (1972) i n " L a b o r a t o r y T e c h n i q u e s i n B i o c h e m i s t r y and M o l e c u l a r B i o l o g y " (Work,T.S. and Work,E., e d s . ) , V o l . 3, pp. 267-610. A m e r i c a n E l s e v i e r P u b l i s h i n g Co. I n c . , New Y o r k . 164. H e p p e l , L . A . (1971) i n " S t r u c t u r e and F u n c t i o n o f B i o l o g i c a l Membranes" ( R o t h f i e l d , L . I . , e d . ) , Academic P r e s s , New Y o r k , 2 2 3 -247 . 165. MacGregor,D.R. and E l l i k e r , P . R . (1958) Can. J . M i c r o b i o l . 4,. 499-503. 166. Repaske,R. (1958) B i o c h i m . B i o p h y s . A c t a 30, 225-232. 191 167. Zachari.us ,R.M. , Z e l l , T . E . , M o r r i s o n , J .H. and Woodlock, J . J . (1969) A n a l . Biochem. 30, 148-152. 168. L e i v e , L . S h o v l i n , V . K . and Mergenhagen,S.E. (1968) J . B i o l . Chem. 24_3 6384-6391. 169. H a y a s h i , K . (1975) A n a l . Biochem. 67, 503-506. 170. Payne,J.W. and G i l v a r y , C . ( 1 9 6 8 ) J . B i o l . Chem. 243, 6291-6294. 171. Lowry,O.H., R o s e b r o u g h , N . J . , P a r r , A . L . and R a n d a l l , R . J . (1951) J . B i o l . Chem. 193, 265-275. 172. Nakae,T. (1975) Biochem. B i o p h y s . Res. Commun. 64, 1224-1230. 173. N i k a i d o , H . and Nakae,T. (1973) J . I n f e c t . D i s . 128, 530-533. 174. L i n d s a y , S . S . , W h e e l e r , B . , S a n d e r s o n , K . E . and C o s t e r t o n , J . W . (1973) Can. J . M i c r o b i o l . 19, 335-343-175. Hirs,C.H.W. (1967) i n "Methods i n E n z y m o l " (Hirs,C.H.W., e d . ) , V o l . 11, pp. 197-199, Academic P r e s s , New Y o r k . 176. Habeeb,A.F.S.A. (1972) i n "Methods i n E n z y m o l . " 25, (Hirs,C.H.W. and T i m a s h e f f , S . N . , e d s . ) , V o l . 25, pp. 457-464, Academic P r e s s , New Y o r k . 177. K o p l o w , J . and G o l d f i n e , H . (1974) J . B a c t e r i o l . 117, 527-543. 178. P a r t o n , R . (1975) J . Gen. M i c r o b i o l . 39., 113-123. 179. S i n g h , A . P . and R e i t h m e i e r , R . A . P . (1975) J . Gen. A p p l . M i c r o b i o l . 21, 109-118. 180. R a n d a l l , L . L . (1975) J - B a c t e r i o l . 123., 41-46. 181. I r w i n , R . T . , C h a t l e r j e e , A . K . , S a n d e r s o n , K . E . and C o s t e r t o n , J.W. (1975) J . B a c t e r i o l . 124, 930-941. 182. S m i t , J . , Kamio,Y. and N i k a i d o , H . (1975) J . B a c t e r i o l . .124, 942-958. 183. Bayer,M.E., K o p l o w , J . and G o l d f i n e , H . (1975) P r o c . N a t l . A c a d . S c i . U.S.A. 7.2., 5145-5149. 184. H e n n i n g , U . (1975) Annu. Rev. M i c r o b i o l . 29, 45-60. 185. Henning,U. and Schwarz,U. (1973) i n " B a c t e r i o l Membranes and W a l l s " ( L e i v e , L . , e d . ) , pp. 413-438, M a r c e l D e k k e r , New Y o r k . 192 186. H a l l e r , I . and Henning,U. (1974) P r o c . N a t l . A c a d . S c i . U.S.A. 71, 2018-2021. 187. S c h w i z e r , M . , S o n n t a g , I . and H e n n i n g , U . (1975) J . M o i . B i o l . 93, 11-21. 188. Brown,A.D. (1969) B a c t e r i o l . Rev. 28, 296-329. 189. Murray,R.G.E. (1963) Can. J . M i c r o b i o l . 9, 381-392. 190. B u c k m i r e , F . L . A . and Murray,R.G.E. (1970) Can. J . M i c r o b i o l . 16, 1011-1022. 191. Henning,U., Rehn,K., Braun,V., Hohn,B. and Schwarz,U. (1972) E u r . J . Biochem. 26, 570-586. 192. Normark,S. (1969) J • B a c t e r i o l . 98, 1274-1277. 193. Normark,S. and Wolf-Watz,H. (1974) Ann. M i c r o b i o l . ( I n s t . P a s t e u r ) 125B, 211-226. 194. W e s t l i n g - H a g g s t r o m , B . and Normark,S. (1975) J . B a c t e r i o l . 123, 75-82. 195. R o d o l a k i s , A . , Thomas,P. and S t a r k a , J . (1973) J . Gen. M i c r o b i o l . 75, 409-416 (1973). 196. S t a r k a , J . , D i S a v i n o , D . , M i c h e l , G . , R o d o l a k i s , A . and Thomas, P. (1974) Ann. M i c r o b i o l . ( I n s t . P a s t e u r ) 125B, 227-232. 197. Hakenbeck,R., Goudell,E.W. and Schwarz,U. (1974) FEBS L e t t . 40, 261-264. 198. Hartmann,R., B o c k - H e n n i g , S . B . and Schwarz,U. (1974) E u r . J . Biochem. 4 l , 203-208. 199. L a z d u n s k i , C . and S h a p i r o , B . M . (1972) J . B a c t e r i o l . I l l , 495-498. 200. L a z d u n s k i , C . and S h a p i r o , B . M . (1972) J . B a c t e r i o l . I l l , 499-509. 201. Matsuzawa,H., Hayakawa,K., S a t o , T . and I m a h o r i , K . (1973) J . B a c t e r i o l . 115, 436-442. 202. M a r t i n , H . H . , Lehmann,R., H e r z o g , U . and K a u l , U . (1974) Ann. N.Y. A c a d . S c i . _ 235, 283-293-203. Weinbaum,G. and Okuda,S. (1968) J . B i o l . Chem. 243, 4358-4363. 204. S t e c k , T . L . and F o x , C . F . (1972) i n "Membrane M o l e c u l a r B i o l o g y " ( F o x , C . F . and K e i t h , A . , e d s . ) , pp. 27-75, S i n a u e r A s s o c i a t e s I n c . , S t a n f o r d , C o n n . , U.S.A. 1 9 3 205. Davson,H. and D a n e l l i , J . F . (1952) "The P e r m e a b i l i t y o f N a t u r a l Membranes" 2nd E d i t i o n , Cambridge U n i v . P r e s s , London and New Y o r k . 2 0 6 . Blumberg,P. and S t r o m i n g e r , J . L . (1974) B a c t e r i o l . Rev. 38, 291-235. 207. Weidel,W., F r a n k , H . and M a r t i n , H . H . (I960) J.Gen. M i c r o b i o l . 22, 1 5 8 -166 . 208. M a r t i n , H . H . (1963) J • T h e o r . B i o l . 5 , 1 - 3 4 . 209. Inouye,M. and G u t h r i e , J . P . (1969) P r o c . N a t l . A c a d . S c i . U.S.A. 6 4 , 957-961. 210. Inouye,M. and Par d e e , A . B . (1970) J . B i o l . Chem. 2 4 5 , 5813-5819. 211. James,R. (1975) J . B a c t e r i o l . 1 2 4 , 918-929. 212. G u d a s , L . J . , James,R. and Pardee,A.B. (1976) J . B i o l . Chem. 251, 3470-3479. 213. Churchward,G.G. and H o l l a n d , I . B . (1976) FEBS L e t t 62, 347-350. 2 1 4 . F r o s t , G . E . and R o s e n b e r g , H . (1973) B i o c h i m . B i o p h y s . A c t a 3 3 0 , 90-101. 215. Langman,L., Young,I.G., F r o s t , G . E . , R o s e n b e r g , H . and G i b s o n , F . (1972) J . B a c t e r i o l . 112, 1 1 4 2 - 1 1 4 9 . 216. P o l l a c k , J . R . , Ames,B.N.-and N e i l a n d s , J . B . (1970) 1 0 4 , 635-639. 217. Luckey,M., P o l l a c k , J . R . , Wayne,R., Ames,B.N. and N e i l a n d s , J.B. (1972) J . B a c t e r i o l . I l l , 731-738. 218. Hantke,K. and Braun,V. (1975) FEBS L e t t . 4 9 , 301-305. 219. T a y l o r , A . L . and T r o t t e r , C D . (1972) B a c t e r i o l . Rev. 36', 505-524 . 220. Braun,V. and W o l f f , H . (1973) FEBBS L e t t . 3 4 , 7 7 - 8 0 . 221. Braun,V., S c h a l l e r , K . and W o l f f , H . (1973) B i o c h i m . B i o p h y s . A c t a 323, ' 8 7 - 9 7 . 222. Braun,V., Bosch,V., Hantke,K. and S c h a l l e r , K . (1974) Ann. N.Y. A c a d . S c i . 235, 66 - 8 2 . 223. F r e d e r i c q , P . and Smarda,J. (1974) Ann. M i c r o b i o l . ( I n s t . P a s t e u r ) 118, 7 6 7 - 7 7 4 . 194 224. Wayne,R. and Neilands, J . B. (1975) J . B a c t e r i o l . 121, 497-503. 225. Guterman,S.K. (1973) J • B a c t e r i o l . 114, 1217-1224. 226. Guterman,S.K. and Dunn,L. (1973) J . B a c t e r i o l . 114, 1225-1230. 227. White,J.C., DiGirolama,P.M., Fu,M.L., Preston,V.A. and Bradbeer,C. (1973) J • B i o l . Chem. 248, 3478-3986. 228. DiMasi,D.R., White,J.C., Schnaitman,C.A. and Bradbeer, C. (1973) J . B a c t e r i o l . 115, 506-513-229. Bradbeer,C, Woodrow,M.L. and K h a l i f a h , L . I . (1976) J . B a c t e r i o l . 125, 1032-1039. 230. Sabet,S.F. and Schnaitman,C.A. (1973) J • B i o l . Chem. 248, 1797-1806. 231- Randall-Hazelbauer,L.L. and Schwartz,M. (1973) J- B a c t e r i o l . 116, 1436-1446. 232. Szmelcman,S. and Hofnung,M. (1975) J . B a c t e r i o l . 124, 112-118. 233- Hazelbauer,G.L. (1975) J- B a c t e r i o l . 124, 119-126. 234. Wallach,D.F.H. (1972) Biochim. Biophys Acta 265, 61-83-235- Carraway,K.L. (1975) Biochim. Biophys. Acta 415, 379-410. 236. Kabir,S. (1975) Can. J . M i c r o b i o l . 21, 1132-1136. 237- Gow,J.A., Parton,R. and Wardlaw,A.C. (1976) Proc. Can. Fed. B i o l . Soc. 19, p. 5, Abst. 19-238. Schindler,P.R.G. and Teuber,M. (1975) Arch. M i c r o b i o l . 102, 29-33-239. Capaldi,R.A. and Green,D.E. (1972) FEBS L e t t . 25, 205-209. 240. Singer,S.J. and Nicholson,G.L. (1972) Science 175, 720-731-241. Hirashima,A., Childs,G. and Inouye,M. (1973) J . Moi. B i o l . 79, 373-389. 242. Zusman,D.R. (1973) Arch. Biochem. Biophys. 159, 336-341. 243. Lee,N. and Inouye,M. (1974) FEBS L e t t . 39, 167-170. 244. Voynow,P. and Kurland,C.G. (1971) Biochem. 10, 517-529. 195 245. Weber,H.J. (1972) M o l e c . Gen. G e n e t . 119, 233-248. 246. R a n d a l l , L . L . and H a r d y , S . J . S . (1975) M o i . Gen. Ge n e t . 137, 151-160. 247. Cancedda,R. and S c h l e s i n g e r , M . J . (1974) J . B a c t e r i o l . 117, 290-301. 248. H i r a s h i m a , A . and Inouye,M. (1973) N a t u r e . 242, 405-407. 249. H i r a s h i m a , A . , Wang,S. and Inouye,M. (1974) P r o c . N a t l . A c a d . S c i . U.S.A. 71, 4l49-4l53. 250. Wang,S., Marcu,K.B. and Inouye,M. (1976) Biochem. B i o p h y s . Res. Commun. 68, 1194-1200. 251. Braun,V. and Bosch,V. (1973) FEBS L e t t . 34, 302-306. 252. James,R. and G u d a s , L . J . (1976) J . B a c t e r i o l . 125, 374-375-253- Kepes,A. and A u t i s s i e r , F . (1972) B i o c h i m . B i o p h y s . A c t a 265, 443-469-254. F i e d l e r , F . and G l a s e r , L . (1973) B i o c h i m . B i o p h y s . A c t a 300, 467-485-255- S l a t e r , M . and S c h a e c h t e r , M . (1974) B a c t e r i o l . Rev. 38, 199-221. 256. M c l n t y r e , T . M . and B e l l , R . M . (1975) J . B i o l . Chem. 250, 9053-9059-257. R a n d a l l , L . L . (1975) J - B a c t e r i o l . 122, 347-351-258. R a z i n , S . (1972) B i o c h i m . B i o p h y s . A c t a 265, 241-296. 259- K u s h n e r , D . J . (1969) B a c t e r i o l . Rev. 33, 302-345. 260. H e l e n i u s , A . and Simons,K. (1975) B i o c h i m . B i o p h y s . A c t a 415, 29-79-261. R a z i n , S . , M o r o w i t z , H . J . and T e r r y , T . M . (1965) P r o c . N a t l . A c a d . S c i . U.S.A. 54, 219-262. R a c k e r , E . and S t o e c k e n i n s , W . (1974) J . B i o l . Chem. 249, 662-663-263. Kagawa,Y. and R a c k e r , E . (197D J - B i o l . Chem. 246, 5477-5487. 264. S e k i z a w a , J . and F u k a i , S . (1973) B i o c h i m . B i o p h y s . A c t a 307, 104-117. 265. Nakamura,K. and M i z u s h i m a , S . (1975) B i o c h i m . B i o p h y s . A c t a , 413, 371-393-196 266. Rittenhouse,H.G., McFadden,B.A., Shumway,L.K. and H e p t i n s t a l l , J . (1973) J . B a c t e r i o l . 115, 330-340. 267. Gruss,P., Gmeiner,J. and Martin,H.H. (1975) Eur. J . Biochem. 57, 411-414. 268. Bennett,R.L. and R o t h f i e l d , L . I . (1976) J . B a c t e r i o l . 127, 498- 504. 269. Weiner,A.M., P i a t t , T . , and Weber,K. (1972) J . B i o l . Chem. 247, 3242-3251. 270. L i n , J . J . C . and Wu,H.C.P. (1976) J . B a c t e r i o l . 125, 892-904. 271. Inouye,S., Takeishi,K., Lee,N., DeMartini,M., Hirashima, A. and Inouye,M. (1976) J . B a c t e r i o l . 127, 555-563-272. DeMartini,M., Inouye,S. and Inouye,M (1976) J . B a c t e r i o l . 127, 564-571-273. Montgomery,R. (1957) Arch. Biochem. Biophys. 67, 378-386. 274. Weissbach,A. and Hurwitz,J. (1958) J . B i o l . Chem. 234, 705-709. 275. Osborn,M.J. (1963) Proc. N a t l . Acad. S c i . U.S.A. 50, 499- 506. 276. Bacchus,A.N. and Javor,G.T. (1975) A n t i m i c r o b i o l Agents and Chemotherapy 8, 387-389-277. Konisky,J., Soucck,S., P r i c k , K . , Davies,J.K. and Hammond,C. (1976) J . B a c t e r i o l . 127, 249-257. 278. Pugsley,A.P. and Reeves,P. (1976) J . B a c t e r i o l . 127, 218-228 . 279. Wu,H.C. and Li n , J . J . C . (1976) J . B a c t e r i o l . 126, 147-156. 280. Ames,B.N. (1966) i n "Methods i n Enzymol." (Neufeld,E.F. and Ginsburg,V., eds.), V o l . 8, pp. 115-118, Academic Press, New York. 281. Gross,E. (1967) "Methods In Enzymol." (Hirs,C.H.W., ed.), V o l , 11, pp. 238-255, Academic Press, New York. 282. Nakae,T. and Nikaido,H. (1975) J . B i o l . Chem. 250, 7359-7365. 283. Konisky,J. and Liu,C.T. (1974) J . B i o l . Chem. 2 4 9 , 835-840.. 284. Kwok-KwongLi,J. and Fox,C.F. (1975) J . of U l t r a s t r u c t , Res. 52, 120-133 (1975). 197 285- S c a n d e l l a , C . J . and K o r n b e r g , A . (1971) B i o c h e m i s t r y . 10, 4447-4456. 286. V a n H o l d e , K . E . (1971) P h y s i c a l B i o c h e m i s t r y , P r e n t i c e -H a l l , I n c . , Englewood C l i f f s , New J e r s e y . 287. T a n f o r d , C , N o z a k i , V . , R e y n o l d s , J . A . and Makino,S. (1974) B i o c h e m i s t r y 13, 2369-2376. 288. Fish,W.W., R e y n o l d s , J . A . and T a n f o r d , C . (1970) J . B i o l . Chem. 245, 5166-5168. 289. A c k e r s , G . K . (1967) J . B i o l . Chem. 242, 3237-3238. 290. R e y n o l d s , J . A . and T a n f o r d , C . (1970) P r o c . N a t l . A c a d . S c i . U.S.A. 66, 1002-1007. 291. R o b i n s o n , N . C . and T a n f o r d , C . (1975) B i o c h e m i s t r y 14, 369-378. 292. C l a r k e , S . (1975) J . B i o l . Chem. 280, 5459-5469. 293. T a k a g i , T . , T s u j i i , K . and Shirahama,K. (1975) J . Biochem. 77, 939-947. 294. N e l s o n , C . A . (197D J . B i o l . Chem. 246, 3895-3901. 295. H o l z w o r t h , G . (1972) i n "Membrane M o l e c u l a r B i o l o g y " ( F o x , C P . and K e i t h , A . D . , e d s . ) , pp. 228-286, S i n a u e r A s s o c i a t e s I n c . , S t a m f o r d , C o n n . 296. Wang,K. and R i c h a r d s , F . M . (1974) J . B i o l . Chem. 249, 8005-8018. 297. Wang,K. and R i c h a r d s , F . M . (1975) J . B i o l . Chem. 250, 6622-6626. 298. S t e c k , T . L . (1972) J . M o i . B i o l . 66, 295-305. 299. U d e n f r i e n d , S . , S t e i n , S . , B o h l e n , P . , Dairman,W., L e i m g r u b e r , W. and W e i g e l e , M . (1972) S c i e n c e 178, 871-872. 300. Hawkes,S.P., Meehan,T.D. and B i s s e l l , M . S . (1976) Biochem. B i o p h y s . Res. Commun. 68, 1226-1233. 198 A p p e n d i x C a l c u l a t i o n o f i n t r i n s i c v i s c o s i t y (286) The r e l a t i v e v i s c o s i t y ( n r ) o f a s o l u t i o n i s t h e r a t i o o f s o l u t i o n t o s o l v e n t v i s c o s i t y : n s = 1 + * $ no" wherev i s a shape f a c t o r and / i s t h e f r a c t i o n o f a s o l u t i o n volume o c c u p i e d by s o l u t e . The shape f a c t o r i s a f u n c t i o n o f t h e a x i a l r a t i o o f t h e s o l u t e p a r t i c l e . The s p e c i f i c v i s c o s i t y ( n s p ) i s a measure o f t h e f r a c t i o n a l change i n v i s c o s i t y p r o d u c e d by a d d i n g s o l u t e : n s p = o $ t h e r e f o r e n s p = n - 1 s i n c e $ = Cv where C i s grams o f s o l u t e p e r m i l l i l e t e r o f s o l u t i o n and v i s t h e p a r t i a l s p e c i f i c volume o f t h e s o l u t e , t h e n n S p = v and • n S p = v C The l i m i t o f n&g/C as C o i s t h e i n t r i n s i c v i s c o s i t y n . 199 E x p e r i m e n t a l l y , t h e r e l a t i v e v i s c o s i t y ( n r ) i s d e t e r m i n e d a t a number o f s o l u t e c o n c e n t r a t i o n s as • n s = t s / s n o t 0 / o where a r e t h e d e n s i t i e s o f t h e s o l u t i o n and s o l v e n t r e s p e c t i v e l y . The i n t r i n s i c v i s c o s i t y i s d e t e r m i n e d by p l o t t i n g n S p / C as a f u n c t i o n o f C and e x t r a p u l a t e d t o i n f i n i t e d i l u t i o n (C = o ) . C a l c u l a t i o n o f t h e d i m e n s i o n s o f a p r o t e i n - S D S complex (152,286) The i n t r i n s i c v i s c o s i t y i s r e l a t e d t o t h e h y d r o d y n a m i c volume o f a p a r t i c l e by: n = ^ ( v 2 + & i V]_ + i>2 ^2°) where i s t h e simha shape f a c t o r , V2 i s t h e p a r t i a l s p e c i f i c volume o f t h e p r o t e i n , S> ^  i s t h e g o f H 2 0 p e r g o f p r o t e i n , v-^ 0 i s t h e s p e c i f i c volume o f w a t e r , S 2 i s t h e g o f SDS p e r g o f p r o t e i n and v 2 ° i s t h e s p e c i f i c volume o f SDS. The f o l l o w i n g v a l u e s were t a k e n f r o m R e y n o l d s and T a n f o r d (152): v 2 = 0.725 c c / g i-L =0.9 a s s u m i n g 1.4 g o f SDS p e r g V]_° = 1 c c / g i 2 = 1.4 g o f SDS p e r g o f p r o t e i n v 2 ° =0.886 c c / g 200 A s s u m i n g a p r o l a t e e l l i p s o i d , t h e a x i a l r a t i o (a/b) was d e t e r m i n e d f r o m t h e simha r e l a t i o n s h i p (286) between t h e shape f a c t o r and a/b. The a b s o l u t e v a l u e s f o r t h e r a d i i o f t h e p a r t i c l e was c a l c u l a t e d f r o m t h e known p a r t i c l e volume ( i i TT ab^) where 3 (1 a / b ) b 3 = M ( v 2 + ^ 1 v 1 ° + ^2 ? 2°) 3 N t h e r e f o r e C a l c u l a t i o n o f S t o k e s r a d i u s o f a p r o t e i n - S D S complex (152, 288,289) A c k e r s (289) has shown t h a t t h e e l u t i o n o f a s o l u t e m oving t h r o u g h a p o r o u s g e l c h r o m a t o g r a p h i c column depends on t h e S t o k e s r a d i u s ( R s ) o f t h e s o l u t e p a r t i c l e : R s = A + B e r f c - 1 K d where A and B a r e c o n s t a n t s o f t h e p a r t i c u l a r g e l and t h e d i s t r i b u t i o n c o e f f i c i e n t : V t " V o where Ve i s t h e e l u t i o n volume o f t h e s o l u t e , V^-, t h e t o t a l volume o f t h e column and V 0 , t h e v o i d volume o f t h e column. E r f c ~ ^ K ( j i s t h e i n v e r s e o f t h e complement o f t h e s t a n d a r d e r r o r f u n c t i o n . 201 The f r a c t i o n o f t h e i n t e r i o r volume o f t h e g e l a v a i l a b l e f o r d i s t r i b u t i o n o f t h e s o l u t e ( K d ) i s d e f i n e d as t h e a r e a u n d e r a random d i s t r i b u t i o n p r o b a b i l i t y c u r v e f o r p a r t i c l e s t h e same s i z e as t h e s o l u t e and g r e a t e r . M a t h e m a t i c a l l y , t h i s i s t h e complement o f t h e s t a n d a r d e r r o r f u n c t i o n . C a l c u l a t i o n o f p a r t i a l s p e c i f i c volume o f a p r o t e i n - S D S complex (292) The s e d i m e n t a t i o n c o e f f i c i e n t o f a p a r t i c l e a t t h e p o s i t i o n r a V g i s d e f i n e d as ST,M ( r a v g ) = ( r - r 0 ) / f 2 r ~ where Sij^jy; i s t h e c o e f f i c i e n t a t a g i v e n t e m p e r a t u r e i n a g i v e n medium; r a V g = ( r Q + r)/2 where r 0 i s t h e d i s t a n c e o f t h e a p p l i e d sample f r o m t h e c e n t e r o f r o t a t i o n (4.96 cm) and r i s t h e d i s t a n c e o f t h e sample a t t i m e + and w i s t h e a n g u l a r v e l o c i t y o f t h e r o t o r ( w 2 t = 17.4 x 10" s e c - 1 ) . The S 2 o * w v a l u e s were c a l c u l a t e d f r o m : S20,w = ST,M . ( l - v f ? n , w ) n20,w d - v / T , M where v i s t h e p a r t i a l s p e c i f i c volume o f t h e p r o t e i n - S D S complex. n T M and y T M a r e t h e v i s c o s i t y and d e n s i t y r e s -p e c t i v e l y a t r a y g w h i l e n 2 o j W and/> 20,w a r e t n e v i s c o s i t y and d e n s i t y o f w a t e r a t 20°C. The v i s c o s i t i e s and d e n s i t i e s o f s t a n d a r d s u c r o s e s o l u t i o n s i n H2O and D2O were measured by 202 v i s c o m e t r y and be w e i g h i n g known volumes r e s p e c t i v e l y . The % (w/w) s u c r o s e was d e t e r m i n e d f r o m t h e r e f r a c t i v e i n d e x and t h e v i s c o s i t y and d e n s i t y a t r a v g was e s t i m a t e d f r o m s t a n d a r d p l o t s o f v i s c o s i t y and d e n s i t y as a f u n c t i o n o f % (w/w) s u c r o s e r e s p e c t i v e l y . The p a r t i a l s p e c i f i c volume (v) was c a l c u l a t e d f r o m S H n H _ i v = n p  fV S H n H _ / H SD n 0 where s u b s c r i p t s H and D r e f e r t o v a l u e s measured i n H2O and D2O, s i s t h e d e t e r m i n e d s e d i m e n t a t i o n c o e f f i c i e n t i n a s o l u t i o n o f d e n s i t y , f , and n, t h e v i s c o s i t y d e t e r m i n e d a t t h e h a l f - d i s t a n c e o f t r a v e l , r a v g > C a l c u l a t i o n o f t h e m o l e c u l a r w e i g h t o f a p r o t e i n - S D S complex (287) The m o l e c u l a r w e i g h t o f a p r o t e i n - S D S complex M = M p (1 + S d) where M p i s t h e m o l e c u l a r w e i g h t o f t h e p o l y p e p t i d e and £ 3 i s t h e g o f SDS bound p e r g o f p r o t e i n . The f o l l o w i n g e q u a t i o n can be a p p l i e d t o p r o t e i n - S D S c o m p l e x e s : S = M (1 - vf) 6 "rr n NR S 203 where M, v , R s, and s a r e t h e m o l e c u l a r w e i g h t , p a r t i a l s p e c i f i c v o l u m e . S t o k e s r a d i u s , and s e d i m e n t a t i o n c o e f f i c i e n t r e s p e c t i v e l y , o f t h e p r o t e i n - S D S complex. The S t o k e s r a d i u s was d e t e r m i n e d by g e l f i l t r a t i o n i n t h e p r e s e n c e o f SDS. The p a r t i a l s p e c i f i c volumes o f p r o t e i n - S D S complexes were d e t e r m i n e d by s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i n H 20 and D 2 0 - c o n t a i n i n g g r a d i e n t s . P u b l i c a t i o n s 1. Reithmeier, R.A.F. and Bragg, P.D. (19 74) " P u r i f i c a t i o n and C h a r a c t e r i z a t i o n o f a Heat - m o d i f i a b l e P r o t e i n from the Outer Membrane of E s c h e r i c h i a c o l i " , Proc. Can. Fed. B i o l . Soc. 17, 112 ( A b s t r a c t 445). 2. Reithmeier, R.A.F. and Bragg, P.D. (1974) " P u r i f i c a t i o n and C h a r a c t e r i z a t i o n of a Heat - m o d i f i a b l e P r o t e i n from the Outer Membrane of E s c h e r i c h i a c o l i " , FEBS L e t t . 41, 195-198. 3. Singh, A.P. and Reithmeier, R.A.F. (1975) "Leakage of P e r i -p lasmic Enzymes from C e l l s of H e p t o s e - d e f i c i e n t Mutants of E s c h e r i c h i a c o l i , A s s o c i a t e d w i t h A l t e r a t i o n s i n the P r o t e i n Component of the Outer Membrane", J . Gen. Appl. M i c r o b i o l . 21, 109-118. 4. Reithmeier, R.A.F. and Bragg, P.D. (1975) " O r g a n i z a t i o n of P r o t e i n s i n the Outer Membrane of E. c o l i " , Proc. Can. Fed. B i o l . Soc. 18, 153 (A b s t r a c t 609). 5. Reithmeier, R.A.F. and Bragg, P.D. (1976) " S p e c i f i c Reassoc-i a t i o n of a D e t e r g e n t - s o l u b i l i z e d P r o t e i n from the Outer Membrane of E s c h e r i c h i a c o l i " , Proc. Can. Fed. B i o l . Soc. 19, 5 ( A b s t r a c t 18). 6. Reithmeier, R.A.F. and Bragg, P.D. (1977) "Molecular Char-a c t e r i z a t i o n o f a H e a t - m o d i f i a b l e P r o t e i n from the Outer Membrane of E s c h e r i c h i a c o l i " , Arch. Biochem. B i o p h y s . ( i n p r e s s ) . 7. Reithmeier, R.A.F. and Bragg, P.D. (1977) " O r g a n i z a t i o n o f P r o t e i n s i n the Outer Membrane of E s c h e r i c h i a c o l i , I. P r o t -e o l y t i c D i g e s t i o n and L a b e l l i n g S t u d i e s " , Biochim. Biophys. Acta (submitted f o r p u b l i c a t i o n ) . 8. Reithmeier, R.A.F. and Bragg, P.D... (1977) " O r g a n i z a t i o n of P r o t e i n s i n the Outer Membrane of E s c h e r i c h i a c o l i , I I . Cross-l i n k i n g S t u d i e s " , Biochim. Biophys. Acta (submitted f o r pub-l i c a t i o n ) . 9. Reithmeier, R.A.F., Singh, A.P. and Bragg, P.D. (1977) " A l t e r a t i o n s i n the P r o t e i n Components of the Outer Membrane of Gamma-radiation R e s i s t a n t Mutants", M i c r o b i o s L e t t , (sub-m i t t e d f o r p u b l i c a t i o n ) . 

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