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Structure-function studies of the Pseudomonas aeruginosa porin Oprp Sukhan, Anand 1996

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STRUCTURE-FUNCTION STUDIES OF T H E PSEUDOMONAS AERUGINOSA PORTN OPRP by ANAND SUKHAN B.Sc, University of Manitoba, 1985 M.Sc., University of Manitoba, 1989 A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A December 1996 © Anand Sukhan 1 9 9 6 f  In  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be granted her  for  It  is  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  r  ii  ABSTRACT  The gene encoding the Pseudomonas aeruginosa phosphate-specific porin OprP was subjected to both linker and epitope insertion mutagenesis. Nine of the 13 linker mutants expressed protein at levels comparable to the wild type gene and were shown to be properly exposed at the cell surface. Four of the linker mutants expressed protein at reduced levels which were not detectable at the cell surface. A foreign epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was cloned into the linker sites of 12 of the 13 mutants. Seven of the resultant epitope insertion mutants expressed surface-exposed protein. Two of these mutants presented the foreign epitope at surface-accessible regions as assessed using a malarial epitope-specific monoclonal antibody. The data from these experiments were used to create a topological model of the OprP monomer which was tested and supported by further mutagenic procedures. In order to determine the function that individual lysine residues serve in the transport of anions through the channels of this protein, the first nine amino-terminal lysine residues of OprP were substituted with glutamates. The mutant proteins were purified and the channels they formed were characterized by reconstituting the purified porins in planar lipid bilayer 74  121  membranes. In comparison to the wild-type protein, the Lys , Lys  , and Lys  126  mutants all 74  displayed reduced levels of conductance at KC1 concentrations below 1 M and the Lys  and  121  Lys  mutants no longer exhibited a saturation of conductance at high anion concentrations. In  addition, the ability of phosphate ions to inhibit the conductance of CI 121  channels formed by the Lys  -  ions through the  mutant was greatly reduced, while their ability to inhibit the CI  iii  74  conductance of the Lys 121  74  Lys , Lys  mutant was reduced by approximately 2-fold. To clarify the roles that  126  , and Lys  play in regulating the channel characteristics of OprP, these amino acids  were replaced with either glycine or glutamine residues. Analysis of these mutants revealed that 126  74  both Lys  and Lys  may serve to funnel anions toward the binding site, but only the presence  121  of Lys  is required tor the formation of the inorganic phosphate-specific binding site of OprP.  iv  T A B L E OF CONTENTS  ii  ABSTRACT T A B L E OF CONTENTS  iv  LIST OF FIGURES  ix  LIST OF TABLES  xi  ACKNOWLEDGEMENTS  xii  INTRODUCTION  1  Pseudomonas aeruginosa  1  The cell wall of gram negative bacteria  4  The inner membrane  4  The periplasmic space  5  The outer membrane  6  Porins of gram negative bacteria: Functional aspects  7  Porins of gram negative bacteria: Structural aspects  11  Phosphate transport across the outer membrane of gram negative bacteria  16  Aims of this thesis  18  MATERIALS AND METHODS  19  Bacterial strains and plasmids  19  Media and growth conditions  19  Chemicals  22  General recombinant D N A techniques  23  V 1. Enzymes and plasmid manipulation  23  2. Agarose gel electrophoresis  23  3. Synthetic oligonucleotides  24  4. D N A sequencing  24  General protein and immunological techniques  25  1. SDS-polyacrylamide gel electrophoresis  25  2. Western immunoblotting  25  3. Antibodies  26  4. Indirect immunofluorescence  27  Construction of plasmid pAS27  28  Linker insertion mutagenesis  31  Malarial epitope insertion  31  Site-directed lysine mutagenesis  32  Isolation of outer membrane fractions  34  Detergent solubilization of OprP  34  F P L C purification of OprP  35  Purification of OprP from SDS-polyacrylamide gels  36  Planar lipid bilayer experiments  37  RESULTS  Single-channel conductance  37  Anion selectivity  37  Phosphate ion specificity  38 39  vi  A. Structural analysis of OprP 1. Linker insertion mutagenesis 1.1 Introduction  39 '.  '.  1.2 Expression of OprP in an E. coli background  39 39  1.3 Linker insertion mutagenesis of OprP at a unique Eco RV restriction enzyme site  40  1.4 Semi-random linker mutagenesis of OprP  40  1.5 Expression of the linker insertion mutant forms of OprP in E. coli  42  1.6 Surface localization of the OprP linker insertion mutants  45  1.7 Functional analysis of selected linker insertion mutants.  46  1.8 Summary  49  2. Epitope insertion mutagenesis of OprP  52  2.1 Introduction  52  2.2 Insertion of the malarial epitope into the linker sites of OprP mutants  52  2.3 Expression of the malarial epitope insertion mutant proteins  53  2.4 Surface localization of the epitope insertion mutant proteins  57  2.5 Surface localization of the inserted malarial epitopes  59  2.6 Summary  61  3. Modeling of the OprP monomer  63  3.1 Introduction  63  3.2 Structural implications of linker insertion mutagenesis  63  3.3 Structural implications of epitope insertion mutagenesis  65  vii  3.4 Testing of the proposed OprP topological model i) Deletion mutagenesis of the proposed fifth surface-exposed loop  66  3.5 Testing of the proposed OprP topological model ii) Epitope insertion mutagenesis of the proposed seventh surface-exposed loop  69  3.6 Summary  69  B. Functional analysis of O p r P 4. Site-directed mutagenesis of specific lysine residues of OprP  72  4 ! Introduction  72  4.2 Site-directed mutagenesis of lysine residues  73  4.3 Expression of amino-terminal lysine substitution mutant proteins  74  4.4 Single-channel conductance of amino-terminal lysine substitution mutant proteins  74  4.5 Anion selectivity of amino-terminal lysine substitution mutant proteins...  79  4.6 Phosphate specificity of amino-terminal lysine substitution mutants  81  4.7 Channel characteristics of L y s ^ G l y , Lys->Gln and L y s ' ' - > G l u 74  121  126  mutant proteins  85  4.8 Expression and membrane localization of carboxy-terminal lysine substitution mutant proteins  85  4.9 Summary  87  DISCUSSION  90  Topological model of OprP  91  The role of specific lysine residues in defining the channel characteristics of OprP  99  Summary REFERENCES  ix  LIST OF FIGURES  Figure 1. Procedure used in the creation of plasmid pAS27  29  Figure 2. Restriction map of plasmid pAS27  30  Figure 3. Western immunoblot of outer membranes of E. coli CE1248 containing OprP  41  Figure 4. Western immunoblot of outer membranes of E. coli CE1248 containing OprP linker insertion plasmids pPLl-pPL13  44  Figure 5. Indirect immunofluorescence of E. coli CE1248 containing plasmid pAS27 or plasmid pTZ19U  47  Figure 6. Western immunoblot of E. coli CE1248 containing OprP-encoding plasmids with the malarial epitope inserted at sites PL1, PL2 and PL4 to PL13  54  Figure 7. Western immunoblot of E. coli CE1248 containing OprP-encoding plasmids with the malarial epitope inserted at sites PL1, PL2 and PL4 to PL13  55  Figure 8. Indirect immunofluorescence oiE. coli strain CE1248 containing the OprP-encoding plasmid with the malarial epitope inserted at site PL10  60  Figure 9. Topological model of the monomelic structure of OprP based on linker and epitope insertion  64  Figure 10. Whole cell Western immunoblot of E. coli CE1248 expressing the OprP mutants deleted for sections of the proposed fifth surface-exposed loop  68  Figure 11. Indirect immunofluorescence of E. coli CE1248 containing the OprP-encoding plasmid with the malarial epitope inserted in the proposed seventh surface-exposed loop  70  Figure 12. Topological model of the monomeric structure of OprP  71  Figure 13. Whole cell lysate Western immunoblot of outer membranes of E. coli CE1248 expressing the OprP substitution mutant proteins  75  Figure 14. Single-channel events of wild-type OprP and Lys  121  —>Glu mutant porin  77  Figure 15. KC1 concentration-conductance relationship of selected OprP Lys—>Glu mutants  78  Figure 16. Phosphate-induced inhibition of chloride conductance  83  Figure 17. Phosphate-induced inhibition of chloride conductance  84  Figure 18. Whole ceil Western immunoblot of E. coli CE1248 expressing the OprP substitution mutant proteins Lys —>Glu and Lys —> Glu  88  Figure 19. Topological model of the monomeric structure of OprP  89  Figure 20. Topological model of the monomeric structure of OprP  106  Figure 21. Schematic diagram of the p-barrel structure of the E. coli porin OmpF  117  Figure 22. Conceptual model for the structure of OprP  118  Figure 23. Hydropathy plots for OprP  119  361  385  xi  LIST OF TABLES  Table I. Strains and plasmids used in this study  20-21  Table IL Codon changes of OprP substitution mutants  33  Table III. Insertion sites of OprP linker mutants  43  Table IV. Expression and surface exposure of OprP linker mutant proteins  48  Table V. Channel activity of selected OprP linker mutant proteins  50  Table VI. Expression and surface exposure of OprP malarial epitope insertion mutant proteins  58  Table VII. Single-channel conductance of OprP Lys—>Glu mutant proteins  76  Table VIII. Anion selectivity of OprP Lys—>Glu substitution mutant proteins  80  Table LX. Phosphate inhibition of chloride conductance through the OprP Lys—>Glu substitution mutant proteins  82  Table X. Channel characteristics of Lys->Gly and Lys->Gln mutant proteins  86  xii  ACKNOWLEDGEMENTS I w o u l d like thank my research supervisor Robert E. W. Hancock for taking me into his lab and giving me the benefit of his experience. I w o u l d also like to thank all the members of the Hancock lab, both past and present, who have help me along the way. The assistance of the following members of the Hancock lab was indispensible to the completion of this thesis; Manjeet Bains, Susan W. Farmer, Eileen G. Rawling, Richard J. Siehnel and Rebecca S.-Y. Wong. In addition; I w o u l d also like to thank the  members of my advisory committee as well as various members of the  Department of Microbiology and Immunology for their helpful advice. The financial support of the Medical Research Council of Canada is gratefully acknowledged. Finally, I w o u l d like to thank my parents whose unfailing support throughout the past five and a half years made this experience far less painful than it otherwise might have been.  1  INTRODUCTION  Pseudomonas aeruginosa  The gram negative bacterium Pseudomonas aeruginosa is a member of a large group of rod-shaped aerobic microorganisms (Palleroni, 1986). The cell w a l l of this organism is typical of that of most gram negative bacteria, being composed of two lipid bilayers separated by a thin periplasmic space. The external surface of the cell wall is covered w i t h a number of pili and also contains a single polarly located flagellum. This latter structure has been shown to provide the cells w i t h a means of locomotion. Members of the Pseudomonas genus are often identified by the characteristic pigments w h i c h they produce. In the case of P. aeruginosa, cultures of the organism release the fluorescent compound pyoverdine into the growth media. This molecule, along w i t h the nonfluorescent compound pyochelin, acts as an iron chelating scavenger (Cox, 1980; Cox and A d a m s , 1985). The growth requirements of P. aeruginosa are quite minimal. This allows the organism to occupy a wide variety of different environments; some of w h i c h w o u l d be rather inhospitable towards  other  more fastidious bacteria. Various species of  Pseudomonas have been isolated from samples of soil and lake water (Palleroni, 1986), and there is evidence that they may inhabit seawater as well (Tanoue et a l , 1995). Members of this genus of bacteria have also been found to inhabit the areas surrounding the roots of certain plants (Scher et a l , 1988). While the prevalence of this bacterium i n the environment should make it the cause of frequent human disease, a lack of efficient defenses against the immune system means that the organism is rarely able to establish a long term infection. However, i n the case of immunocompromised hosts (eg. A I D S patients or individuals undergoing chemotherapy), the organism has been shown to be capable of causing severe problems (Schimpff et a l , 1970).  2  The pathogenicity of P. aeruginosa has been demonstrated i n infections of patients suffering from burns of the skin (Pruitt and McManus, 1984)". The injured tissues of these patients lack many of the common mechanisms of defense. This deficiency allows bacteria, w h i c h w o u l d normally be quickly eliminated from these surfaces,  the  opportunity to establish a foothold on the body of the prospective host. Once an infection has been established, P. aeruginosa is able to elaborate a number of intriguing mechanisms w h i c h aid i n the spread of the bacteria throughout the body. Several different proteases are secreted by P. aeruginosa w h i c h serve to destroy the surrounding host tissues and allow the bacteria to invade further into the body (Morihara and Tsuzuki, 1977). Other enzymes act to release various molecules from the host environment w h i c h are essential for the long term survival of the microorganism (Vasil, 1986). In addition to these proteins, the previously mentioned iron chelators pyoverdine and pyochelin are also released into the surrounding environment. These molecules b i n d to available iron(III) ions w h i c h are normally extremely insoluble. The iron-chelator complex is bound by the bacterium via a surface-exposed receptor and is then brought into the cell cytoplasm by the actions of various membrane associated proteins (Meyer et al., 1990). The internalized molecule is stripped of its iron load and shunted back out into the external media where the process is repeated. This mechanism is an efficient means by w h i c h P. aeruginosa is able to obtain sufficient quantities of iron(III) w h i c h w o u l d normally be bound up by various host chelating proteins (Sriyosachati and Cox, 1986). Another category of patients who must endure the effects of P. aeruginosa infections are those who suffer from the genetically acquired disease cystic fibrosis (CF). Symptoms of this disease include an inability to adequately clear fluids from the lungs. Individuals suffering this genetic affliction have been found to produce a mutated form of a chloride channel w h i c h is found i n the plasma membrane of epithelial cells w h i c h line the lungs (Welsh and Liedke, 1986). The lack of a normal chloride channel results i n  3  the unusually high levels of fluid accumulation which are characteristic of the disease. The large volumes of fluid present i n the lungs of these patients not only make breathing extremely laborious but they also produce a detrimental effect on the immunological competency of the surrounding tissues. This deficiency i n local immunity allows bacteria the chance to settle i n for a prolonged occupation. The ability of P. aeruginosa to produce a viscous surface layer is thought to enhance its ability to infect the lungs of C F patients. This mucoid layer, w h i c h is secreted by the bacteria into the extracellular environment, is composed of a polymer of mannuronic and glucuronic acid residues (Pedersen et al., 1989). The viscosity of this substance, along w i t h its high concentration of negative electrical charge, serves to inhibit the deposition of antibodies on the surface of the invading bacteria. This surface layer not only obstructs the phagocytosis of this microorganism by indigenous leukocytes (Bayer et al., 1991), but it may also serve to increase the ability of the bacteria to b i n d to the epithelial cell surface of the lungs (Marcus and Baker, 1985). This mucoid layer therefore qualifies as a virulence factor which is elaborated by P. aeruginosa when the organism inhabits the lungs of C F patients. While P. aeruginosa is not a common source of human infections, clinical studies have shown that once an individual has become colonized by this bacterium, the eradication of such an infection is often very difficult. This is due to a high degree of intrinsic resistance to antibiotics which is common among the various pseudomonads (Bryan,. 1979). These high levels of resistance are thought to be due to the unique features of this bacterium's outer membrane. The membrane, w h i c h is extremely impermeable i n P. aeruginosa, functions as an initial screen by w h i c h the organism is able  to  discriminate  between  various  molecules  present  i n the  extracellular  environment. The l o w permeability of the P. aeruginosa outer membrane limits the extent to w h i c h various molecules (such as antibiotics) are capable of entering the cell.  4  The cell w a l l of gram negative bacteria  The cell walls of all gram negative bacteria are composed of three separate domains; the inner membrane, the periplasmic space and the outer membrane. Each of these structures serve a unique role i n maintaining an equilibrium between  the  extracellular environment and the internal milieu of the cell. They each also play a role in the uptake of nutrients by the cell. In addition to these three components, many bacteria produce an extracellular capsule layer. This surface  structure has  been  implicated i n providing the cells w i t h a means of inhibiting the phagocytic capacity of neutrophils and macrophages (Horwitz and Silverstein, 1980) i n a manner similar to that of the P. aeruginosa mucoid layer.  The inner membrane The inner membranes of most gram negative bacteria are composed of a small variety of different phospholipids (Nikaido and Vaara, 1987). These molecules are arranged i n a symmetrical bilayer membrane w i t h the polar head groups oriented towards either the cytoplasm of the cell or the aqueous matrix of the periplasmic space. The hydrophobic tails of the phospholipids are faced towards each other and associate by means of strong, nonpolar interactions. The arrangement of the lipids i n such a manner  provides a formidable barrier to the non-specific transit of hydrophilic  molecules across the membrane.  Passage of such molecules is limited by  the  hydrophobic core of the membrane. However, this structure provides little impediment to the transport of even moderately hydrophobic compounds. The l i p i d molecules are interspersed w i t h a number of different proteins which serve various functions. Some of these proteins act to generate energy w h i c h is needed for the active transport of nutrients into the cell (Dassa and Hofnung, 1985) while others function as receptors that bind certain substrates w h i c h find their way into the periplasmic space (Kristjansson  5  and Hollcher, 1979). The bound molecules are then brought through the membrane b y the action of these receptors i n concert with-those of other inner membrane associated proteins. Other inner membrane proteins act as pumps w h i c h serve to expel noxious compounds from the cell interior (Li et al., 1995); still other proteins carry out the synthesis of various components of the cell wall (Tuomanen, 1986).  The periplasmic space The area separating the inner and outer membranes of gram negative bacteria is referred to as the periplasmic space. The name of this region is deceptive, since it is composed not of empty space, but of a viscous, gelatinous substance. This material, w h i c h has been termed peptidoglycan, was shown to be comprised of linear strands of two different amino sugars (N-acetylglucosamine and N-acetylmuramic acid). Short peptides (normally four amino acid residues long) w h i c h serve to cross-link the individual strands via peptide bonds are bound to the N-acetylmuramic acid residues. The peptidoglycan layer acts as a stabilizing force i n the cell wall. The covalent or strong non-covalent attachment of this layer to proteins located i n the outer membrane has been observed i n a number of different bacteria (Nikaido and Vaara, 1987). In addition to the structural role played by the constituents of the periplasmic space, this area of the cell also serves to determine the types of molecules w h i c h are able to enter the cell cytoplasm as well as the rates at which these compounds cross the membranes. Several substrate-binding proteins have been shown to reside i n this region of the cell wall. The specificity of these proteins is usually towards molecules w h i c h are essential to the growth and survival of the cell. By maintaining i n the periplasm a low concentration of free substrate, these proteins ensure the continuous flow of those molecules through the outer membrane. Both the maltose-binding protein of E. coli and the phosphate-binding protein of P. aeruginosa have been shown to be responsible for keeping the free periplasmic concentrations of their respective substrates far below that  6  of the external environment (Kellerman and Ferenci, 1982; Poole and Hancock, 1984). Transport of appropriately charged molecules into the periplasmic space is also thought to be enhanced by the presence of an electrical potential (the Donnan potential) w h i c h is maintained across the outer membrane by a number of periplasmically-localized fixed charges (Stock et a l , 1977).  The outer membrane Like the inner membrane, the outer membrane of gram negative bacteria is composed of a number of phospholipid molecules. In addition, this outer layer also contains lipopolysaccharide (LPS) which is only found i n the cell walls of bacteria (Nikaido and Vaara, 1987). This molecule is comprised of the lipid A core and a hydrophilic polysaccharide tail. The lipid A component is anchored i n the membrane while the hydrophilic tail extends away from the surface. The arrangement of the outer membrane is such that the LPS molecules form the external face of the membrane while the phospholipids line the inner face. The individual LPS molecules have been shown to be both covalently and non-covalently attached to certain outer membrane proteins (Hancock and Karunaratne, 1990). They have also been found to form inter-molecular interactions via the mutual attraction of certain sites on these molecules to various divalent cations (eg. M g ^ ) . The presence of these ions has been shown to be important to the maintenance of the stability of the outer membrane (Hancock et al., 1994). Like most biological membranes, the outer membranes of gram negative bacteria contain a number of proteins which serve various functions. One of the major protein constituents of the outer membrane of E. coli is O m p A . While this protein has been shown to play a role i n maintaining the structural stability of the outer membranes of this bacteria, more recent studies have led to the proposal that O m p A may also function as a channel (Sugawara and Nikaido, 1992). Another protein which serves to stabilize the structure of the E. coli outer membrane is the Braun lipoprotein. This protein is  7  localized on the inner face of the outer membrane and has been shown to be covalently bound to the peptidoglycan layer of the periplasmic space (Braun and Sieglin, 1970). Since the presence of the hydrophilic L P S side chains on the external surface provides an effective means by which to limit the uptake of hydrophobic compounds and the nonpolar nature of the inner lipid core does not allow the passage of hydrophilic molecules, the outer membrane provides an excellent barrier to nonspecific transport through the cell surface. The impermeability of this bilayer necessitates the inclusion of certain proteins i n the outer membrane w h i c h are capable of facilitating the uptake of various nutrients from the environment. The permeability differences displayed b y various gram negative bacteria have been shown to be at least partially dependent on differences i n the number and the types of these proteins w h i c h are expressed i n the outer membrane of these organisms.  Porins of gram negative bacteria: Functional aspects  The presence of porins i n the outer membranes of gram negative bacteria serves to provide these organisms w i t h a means by which to obtain various growth factors from the extracellular environment. These proteins form channels w h i c h span the membrane and allow the passage of molecules from the external m e d i u m into the periplasmic space (Hancock, 1987a). Functional analyses of these channels have been accomplished  using a number  of different  techniques.  These  include  uptake  experiments using whole cells expressing the porin of interest (Nikaido, 1986) as well as model membrane studies of purified proteins (Hancock, 1987b). The latter category of methods provide a finer level of biophysical examination of the channels as they allow better control of the environment to which the porins are exposed. However, both types of experimental systems have been used to gain valuable information o n the function of this class of bacterial outer membrane proteins.  8  The majority of porins examined to date have been shown to form non-specific channels. This means that they allow the passive diffusion of molecules through the outer membrane provided that the size of the molecule is below the exclusion limit of the channel. Porins found i n E. coli which have been shown to display these traits include the well characterized proteins O m p F and O m p C . Some of the earliest studies of the mechanisms of porin function have been performed on these proteins (Nikaido and Rosenberg, 1983). Functional analyses have shown that the O m p F and O m p C channels share many common features, including an estimated diameter of slightly more than 1 n m and an exclusion limit of approximately 600 daltons. The channels of these porins have also been shown to exhibit a slight (OmpF) to moderate (OmpC) selectivity towards the transport of cations over anions. However, these two porins differ i n their single-channel conductances as measured using artificial membrane systems. W h e n reconstituted into bilayers, purified O m p F exhibits an average singlechannel conductance of 2.1 nS i n I M KC1. Using these same methods the single-channel conductance of the O m p C porin was shown to be 1.5 nS (Nikaido and Rosenberg, 1983). These apparent differences i n function have been determined to be due to differences i n the electrochemical nature of the amino acid residues w h i c h are located within the channels of these two proteins. Proteins analogous to O m p F and O m p C have been identified i n the outer membranes of other gram negative bacteria (Lee and Schnaitman, 1980). Unlike the constitutively expressed O m p F and O m p C porins, PhoE is only expressed i n the outer membrane when cells experience phosphate deprivation (Overbeeke and Lugtenberg, 1980, Poole and Hancock, 1986). The production of this porin under these conditions is related to its selectivity for the transport of anions. The negatively-charged phosphate ions are able to more easily enter the cell via these anionselective channels. However, the ability of PhoE to transport many anions as efficiently as it does phosphate ions has placed this protein i n the category of the general diffusion  9  porins (Benz et al., 1984). Despite these differences i n i o n selectivity, PhoE channels have been shown to possess conductance levels and exclusion limits similar to those of O m p F and O m p C (Benz and Hancock, 1982). This is consistent w i t h the fact that the amino acid sequence of PhoE is quite similar to the sequences of these other two porins (Jeanteur et a l , 1991). Another E. coli porin w h i c h is expressed i n response to particular growth conditions is the maltose-specific porin LamB. Expression of this protein i n the outer membrane is induced upon growth of the organism i n l o w concentrations of carbon sources other than maltose (Schwartz, 1987). Studies w i t h both whole cells and isolated protein have shown that this porin forms channels that exhibit a distinct preference for the transport of maltose and maltodextran sugars (Wandersman et al., 1979; Benz et al., 1986). This specificity has been shown to be due to the presence of a substrate-binding site. Mutants w h i c h have been isolated by selecting for growth on large maltodextran molecules have been found to express LamB proteins w h i c h contain deletions i n specific regions of the protein sequence (Misra and Benson, 1988). These regions are thought to be involved i n maintaining the substrate-specific nature of this particular porin. The related enteric bacterium Salmonella enterocolitica has been shown to express a LamB protein w h i c h is very homologous to the E. coli protein (Schulein and Benz, 1990). Several proteins w h i c h have been shown to exhibit porin-like behaviour have been identified i n the outer membrane of P. aeruginosa. These include the nonspecific channel forming protein O p r F (Benz and Hancock, 1981) as well as the specific porins O p r D (Trias and Nikaido, 1990a), OprB (Hancock and Carey, 1980) and O p r P (Hancock et al, 1982). While O p r F has been shown to be the most common constitutively expressed porin found i n P. aeruginosa, both the low level of channel activity of this protein combined w i t h the l o w diffusion rate of solutes through the few active channels appear to be responsible for the relative impermeability displayed by the  outer  membrane of this bacteria. The exclusion limit of the channels formed by O p r F has been  10  experimentally determined to be well above that of the E. coli general diffusion porins (in vitro estimates place it at over 3000 daltons, whereas in vivo data suggests a channel diameter of at least 1.56 nm). However, despite this apparently larger channel size, the rate of diffusion of solutes through the channels of O p r F was about two orders of magnitude lower than the rates afforded by the E. coli porins O m p F and O m p C . This apparent contradiction has been theorized to be due to the fact that only a small proportion of the OprF porins form the large channels while the majority of the proteins form  channels w i t h considerably smaller exclusion limits. Planar lipid bilayer  experiments w i t h O p r F have shown that addition of this protein to bilayers results i n the incorporation of channels which can be divided into two categories according to measured conductance. The vast majority of channels exhibited conductances of 0.34 nS while a small minority of larger channels w i t h conductances of approximately 5 nS were also observed (Benz and Hancock, 1981; Nikaido et al., 1991). The homologous E. coli outer membrane protein O m p A has recently been shown to form channels w h i c h share some of the characteristics displayed by the O p r F channels (Sugawara and Nikaido, 1992; 1994). Since the differences i n function displayed by these two proteins from that of the classical general diffusion porins w o u l d dictate that their threedimensional structure must differ somewhat from that of the other channel forming proteins, the topological arrangements of O m p A and OprF w i l l not be discussed. Both O p r D and OprP represent specific porins w h i c h are expressed i n the outer membrane of P. aeruginosa under specific environmental conditions. The wild-type expression of O p r D is low and the conditions that result i n enhanced expression have not been well defined. The substrate specificity of the channels formed by this porin mean that the transport of essential molecules across the outer membrane is not solely dependent on passive diffusion. The proposed binding site of O p r D is thought to be specific for the structure of basic amino acids (Trias and Nikaido, 1990a). However, it appears that the structural similarity of the antibiotic imipenem to that of these amino  11  acids allows this molecule to also gain entry into the cells via the channels of this porin (Trias and Nikaido, 1990b). Strains which express O p r D have been shown to be significantly more sensitive to the presence of imipenem than O p r D deficient strains. The  growth of P. aeruginosa under phosphate-starvation conditions (< 0.2 m M )  induces the expression of the phosphate-specific porin OprP (Hancock et al., 1982). The channels formed by this protein demonstrate a strong selectivity for the transport of anions over cations (Benz et al., 1983). This feature is also exhibited b y the analogous phosphate-starvation inducible E. coli porin PhoE (Benz et al., 1984). However, unlike the E. coli porin, OprP has been shown to possess a binding site w h i c h makes the channels specific for phosphate ions. The presence of this binding site means that the channels of O p r P transport phosphate ions at approximately a 100-fold higher rate than they do other similarly sized anions (Hancock and Benz, 1986). G i v e n the l o w general outer membrane permeability of P. aeruginosa, this specificity is thought to be essential to this porin's role i n the acquisition of phosphate ions when the extracellular concentrations are extremely low. The sequence of the recently cloned P. aeruginosa poly-phosphate specific porin O p r O was shown to be quite similar to that of O p r P (76% identity, 16% conserved substitutions) (Siehnel et a l , 1992). The differences i n substratespecificity displayed by these two proteins appear to be due to minor variations i n the amino acid sequences.  Porins of gram negative bacteria; Structural aspects  A number of different techniques have been employed to determine the structure of bacterial porins. These include both genetic and biophysical approaches. The data gathered from these various studies have shown that these proteins share a number of common structural features, including a high percentage of (3-sheet content. The differences i n function exhibited by the various porin channels appears to be due to  12  specific regions of their amino acid sequences, and i n fact, only a small number of individual residues have been implicated i n defining the unique channel characteristics of these different proteins. Insertion and deletion mutagenesis of the genes encoding various different porins has shown that only certain sections of the expressed proteins were permissive for these types of alterations (Boulain et al., 1986; Bosch and Tommassen, 1987; Roque and McGroarty, 1990). Data previously obtained from circular dichroism analysis indicated that these proteins contain a large number of (3-strands. Thus, it seemed likely that the sites w h i c h were permissive for the insertion or deletion of small peptides were located i n loop regions which connected the individual p-strands. The insertion of a foreign epitope into these permissive sites allowed for the identification of those loop regions which were exposed at the surface of whole cells (Charbit et al., 1986; Agterberg et al. 1987). Although a number of topological models have been based on data generated by the previously described methods, the most accurate procedure available for the assessment of the three-dimensional configuration of a given protein is that of X-ray crystallography. While this technique has been used to determine the structures of several ct-helical proteins, it has only been used sparingly for the analysis of porin structures. This has been due to the difficulties encountered i n crystallizing these proteins. Recent successes i n growing suitable crystals has allowed this technique to be used to create structural models for a small number of porins. These models have been compared to those generated by genetic analyses to strengthen the  structural  predictions for these proteins. The first porin structure to be solved by X-ray diffraction analysis was a porin of Rhodobacter capsulatus. The model which was based on this analysis showed this protein as being arranged i n a complex of three identical monomers (Weiss et al., 1990). Chemical cross-linking studies had previously provided evidence that these proteins  13  formed oligomeric structures i n the outer membrane (Angus and Hancock, 1988). The individual subunits were arranged around a central axis and were tilted at a slight angle (approximately 16°) toward that axis. Each monomer assumed the conformation of a p-barrel which was composed of 16 strands. The barrel enclosed a central waterfilled channel. The amino acid sequences of the p-strands tended to alternate between hydrophobic and hydrophilic residues. This w o u l d seem appropriate if one side of the P-sheet were to face the interior of the channel while the other side faced the lipid core of the membrane. This is i n contrast to the sequences of the membrane spanning segments of ct-helical membrane proteins which contain predominately hydrophobic residues (Taylor, et al., 1994). The P-strands of the porin monomer structures were shown to be connected by loop regions. The loops exposed to the periplasmic space were found to be relatively shorter than those exposed to the cell surface. These larger surface-exposed loops appear to play a much more important role i n defining the channel characteristics of the protein than the periplasmically-exposed loops. Six of the eight surface-exposed loops (loops 1, 4, 5, 6, 7 and 8 ) were shown to form an umbrella-like structure over part of the opening to the central channel. This umbrella appears to limit the size of the molecules that are capable of passing through the channels of this protein. The third surfaceexposed loop was shown to be positioned i n a way which allowed it to exert a substantial influence over the passage of molecules through the channel. This region of the protein was seen to fold back into the channel and create an eyelet structure approximately half-way d o w n the channel. This eyelet is believed to constrict the internal diameter of the pore and further limit the diffusion of solutes. The crystal structures of the highly homologous E. coli general diffusion porins O m p F and PhoE were found to be remarkably similar to that of the R. capsulatus porin (Appendix-Figure 21) (Cowan et a l , 1992). This is somewhat surprising, given that the amino acid sequence of this protein does not show a great deal of homology w i t h that  14  of the other three porins. Each of the E. coli proteins were also found to exist i n the outer membrane as a trimer of three identical subunits. Like the structure of the R.. capsulatus porin, the models of the E. coli porins showed the third surface-exposed loop as being folded back into the channel and the second loop as interlocking w i t h the neighbouring monomer. The locations of the loop regions i n the crystal models of these porins coincided w i t h the placement of these regions i n topological models based on mutagenic procedures (van der Ley et al., 1987; Benson et al., 1988). The sections of these proteins w h i c h were proposed as composing the loop regions were shown to be the same sections that were permissive for insertion and deletion mutagenesis. In addition to verifying the location of the loops, specific alterations i n the amino acid sequences of these proteins have shown that certain residues located i n and around these loops serve to define their individual channel properties (Misra and Benson 1988). Combinatorial mutagenesis of O m p F , O m p C , and PhoE have shown that regions located i n the amino-terminal half of these proteins are involved i n determining the specific channel characteristics of these porins (Benz et al., 1989). Genetic manipulations w h i c h resulted i n the combination of different portions of these proteins helped to identify several discrete regions implicated as serving to define both the exclusion limits and the i o n selectivities of the channels. These experiments have shown that the third surface-exposed loop contains a number of residues w h i c h play a role i n the functioning of the channels. Mutagenic studies have shown that the region between residues 108 and 133 i n O m p F are responsible for the cation selective nature of the channels (Benson et al., 1988). Other experiments have shown that L y s  125  i n PhoE serves to govern the i o n  selectivity of the channels of this protein (Bauer et a l , 1989). A l l of these residues are contained i n the third surface-exposed loops of the proposed topological models. Thus, the results of these experiments helped to strengthen the proposal of an interior location for this particular surface-exposed loop region.  15  Since the structure of the maltose-specific LamB porin was found to contain a large amount of (3-sheet structure, it was assumed that the topology of this protein w o u l d be similar to that of the other better characterized members of the family. However, as there exists little similarity between the amino acid sequence of LamB and the other porins (Jeunteur et a l , 1991), and as this protein contains approximately 25% more residues than the other porins, it was difficult to construct a model of L a m B based on the previously solved porin structures. A recently published crystal model of this protein showed the structure to be quite similar to that of the other porins, w i t h one noted exception; the p-barrels were constructed out of 18 strands instead of 16 (Schirmer et al., 1995). Mutagenic analyses revealed that residues located i n both the second and third surface-exposed loops were important for the substrate-specificity of this p o r i n (Heine et al., 1987). In addition to these sites, certain residues arranged throughout the sequence of the protein were established as also directing the functions of the channels formed by this protein. These amino acids have been proposed to be involved i n forming a diffuse maltose binding site which serves to specifically guide the substrate through the channels and into the periplasmic space. The genes encoding the P. aeruginosa specific porins O p r D and O p r P have been cloned and expressed i n an E. coli background (Huang and Hancock, 1993; Siehnel et al., 1988). The evident homology of the O p r D sequence w i t h that of the classical porins allowed for the construction of a topological model of this protein w h i c h was based on that homology (Huang et al., 1995). Subsequent experimental testing of this model showed that O p r D does indeed appear to assume a conformation similar to that of the majority of previously characterized porins (Huang et a l , 1996). These experiments also indicated that residues contained i n the second and third surface-exposed loops of this protein might be involved i n forming the substrate binding site of the channels. Unlike the sequence of O p r D , the amino acid sequence of O p r P displays little homology to that of the other porins (Siehnel et al., 1990). In spite of this fact, O p r P  16  shares many biophysical properties w i t h these proteins, including a high proportion of (3-sheet structure (Worobec et al., 1988) and a native oligomeric configuration (Angus and Hancock, 1983). Lysine-specific chemical modifications of this protein were shown to adversely affect the single-channel conductance as well as the anion selectivity and the phosphate specificity of the OprP channels (Hancock and Benz, 1986). Similar modifications of PhoE were also shown to affect the channel properties of this porin (Darveau et al., 1984). The results of the O p r P modification experiments indicated that lysine residues contained i n the sequence of this protein were involved i n determining the various channel characteristics.  Phosphate transport across the outer membranes of gram negative bacteria  The acquisition of inorganic phosphate (phosphate ions) and phosphorylated compounds is an essential function of growing microorganisms. M a n y bacteria have been shown to possess two separate systems for the transport of phosphate ions; one w h i c h operates i n a phosphate rich environment and a second w h i c h functions under conditions of phosphate deprivation. This second group of genes is part of what is often referred to as the Pho regulon (Wanner and Latterll, 1980). The products of the E. coli Pho regulon are among the most extensively studied representatives  of this  category of bacterial proteins. M a n y other gram negative bacteria contain genes w h i c h appear to be homologous to components of this regulon (Filloux et al., 1988; Lee et al., 1989; Scholten et al., 1995). Therefore, information regarding the E. coli Pho regulon has been invaluable to gaining an understanding of the means by w h i c h other gram negative bacteria respond to conditions of phosphate deprivation. The Pho regulon of E. coli has been found to include at least 31 genes w h i c h are arranged i n eight separate operons (Wanner, 1993). The expression of these genes is induced by l o w extracellular concentrations  of phosphate.  Under conditions of  17  phosphate deprivation, the inner membrane localized PhoR sensor protein along w i t h the response regulator protein PhoB act i n concert to activate transcription of the components of the regulon. The phosphorylation of PhoB by the sensor protein occurs under conditions of phosphate deprivation. The activated PhoB protein i n turn binds to regions on the chromosomal D N A which are upstream of the individual Pho operons. These regions contain common consensus sequences and are usually referred to as Pho boxes. The Pho regulons of many enteric bacteria have also been shown to be under the control of similar upstream genetic elements. The gene products of the E. coli Pho regulon include an inner  membrane  permease (PstB), a periplasmically localized phosphate binding protein (PstS) and an outer membrane porin (PhoE). A l o n g w i t h the actions of a few other proteins, these components of the Pho regulon serve to increase the rate at which the organism is able to assimilate phosphate from the surrounding environment (Wanner, 1993). The common purpose provided by these different proteins engendered their inclusion i n a particular  subgroup  of the  Pho regulon; the  Pst transporter  system.  Proteins  homologous to members of the E. coli Pst system have been identified i n such bacteria as Shigella flexneri (Scholten et a l , 1995) and Salmonella typhimurium (Bauer et al., 1985). The Pst system of P. aeruginosa has been shown to be quite similar to that of E. coli. The genes of this system are also under the control of upstream Pho boxes and the products of these genes include a periplasmic phosphate binding protein (Poole and Hancock, 1984) and an outer membrane localized channel forming protein (OprP) (Hancock et al., 1982). Mutants which are deficient i n one or more components of this system are unable to transport inorganic phosphate when the extacellular concentration of this nutrient is l o w (Poole and Hancock, 1984). This lack of an intact high affinity phosphate transport system results i n significantly slower rates of growth for these mutants i n comparison to the w i l d type strains. The genes encoding both the periplasmic phosphate-binding protein and O p r P have been cloned and expressed i n E.  18  coli (Worobec et al., 1988; Siehnel et a l , 1988). The P. aeruginosa periplasmic phosphate binding protein was found to be extremely homologous to the E. coli PstC protein. However, the sequence of OprP displayed little homology w i t h the E. coli phosphate starvation inducible porin PhoE (Siehnel et al., 1990). Functional studies have revealed that the P. aeruginosa porin is far more selective for the transport of anions than the E. coli protein. In addition, the channels of O p r P were shown to possess a phosphatespecific binding site which was proposed to be formed b y the presence of one or more lysine residues (Benz and Hancock, 1986).  Aims of this thesis  The purpose of this thesis is to indirectly ascertain the structure of the P. aeruginosa phosphate-starvation  inducible porin O p r P and to examine h o w this  structure relates to the functions of this protein. A topological model of the structure of O p r P was created using data generated by insertion and deletion mutagenesis of the gene encoding this protein i n combination w i t h previously generated data regarding the hydrophobicity and propensity to form (3-turns of individual segments of protein sequence. Subsequently, the roles of specific residues i n the transport of phosphate ions through the channels of this porin were assessed by means of site-directed amino acid substitution mutagenesis. The results of this second set of experiments were combined w i t h those w h i c h were utilized i n the creation of the topological model i n order to gain a greater understanding of the mechanism of phosphate uptake across the P. aeruginosa outer membrane.  19  MATERIALS A N D METHODS  Bacterial strains and plasmids  Strains and plasmids used i n this study are listed i n Table I. The P. aeruginosa strain H I 03 was utilized i n the purification of w i l d type O p r P w h i c h was used for the production of polyclonal anti-OprP antiserum. The E. coli strain DH5-ocF' was used i n all procedures involved i n creating the oprP mutant plasmids. E. coli strain CE1248 was used as the host background i n all O p r P expression experiments. This strain does not produce O m p C and O m p F due to a regulatory mutation and is also deficient i n the expression of PhoE due to a gene deletion. In addition, CE1248 contains a mutation i n the phoR gene w h i c h allows for the constitutive expression of the Pho regulon. The gene encoding O p r P was originally cloned into the expression vector p M M B 6 6 H E to create plasmid pRSP-3. This latter plasmid was used as the source of the oprP gene w h i c h was subcloned into the commercial phagemid vector p T Z 1 9 U to create plasmid pAS27 (see below). Plasmid pAS27 was the parent plasmid used i n the creation of all the oprP insertion and substitution mutants. Plasmid p U C 4 K A P A used as the source of the Hinc II fragment w h i c h contained the kanamycin resistance cassette. This fragment was used i n the creation of the linker insertion mutants. Plasmids pUC18 and pUC19 were used to create the vector p U C 1 8 / 1 9 which was utilized i n the creation of plasmid pAS27.  M e d i a and growth conditions  Cells were grown overnight at 37 °C on agar plates or i n broth cultures. The P. aeruginosa strain was initially streaked on to Mueller-Hinton agar plates and grown  ON O N  15  i>1  O  d  <u  O N  C50  VH  ,<L5  O N  oo  15  O N  13  O  15 VH 15  o3 d  15  u d  03  03  •rH  •rH  o3  03  03  o  O  o o  o3  d  o3  O VH  o3 •rH  rd  fin  d  o  o  o3  03  rd  a,  -d  o  CO §3  15 VH 15  OH  o  03 OH O VH  <  o i—l  4-1  o -=! 2  VH  CO  O O  N  o o3  T3 O N N O  O  a  oo  15 O  -e-  V  PH  1  —  1  o  T-H  15  <  15  u  r>  W)  i—i  <  a  'd  o  AH  oo  o  Q  CO  (73 • V1 — H1  o o3  PQ  U  •S s  ^  H  o 15  03 15 3 D ft S  U  U I—I  VH  r>  .2 &  1  CO  SH  VH O 4-1  d  <5  d d  o  O N  i—i  Q  s  d CO  d  o  IS  15 VH  T3  O  C5H  O N  °  *a  nd  o3  > 15  or  r d  o  OH  C/3  S  a  N  VH 4-»  g  bO CO  d  P<  u to:  'I LO:  03  oo  u r D  u  O N  1—I  N H  PH  21  c/1 O  Pi 03  o  oo oo  03 03  o T3  o 1-1 pi o  a s 13 13 03  H 03  -4-> ,—<  03  H  CCi  4 3  03  CO  l-i  J3  & -a U  H  >>  03  13 a  CN  3  3  -i-> i/i  I/)  </l 4 3  </)  4 3  H  4 3  H  CO  tn 03 ft O  ft id c3  O OO 1—I  03 ft >>  U ft  O  T3  03  rj  o  "a ^  '«i  43 'ft  4 3  o i—i  N H ft T3 » — l  c/p  43 ft  i/l  a V J _ /  k>  'a  O  o j  "ft  T 3  a v  Ia cn i—i i-l OH  I  cn c/3 T3 c/5  (73  J2  03  H  cni cu CO  ft  CN CO  < ft  CU  CU CN  1—I  ft  PI  03 c/1 T3 "3 £  ft  c/i  PI o 1/1 4 2  i/i c/5  •"J 42 ^ ^3  I  •J  o  03  a  o xi u 03  5 o  t  • 1-1  -*-»  o  03  gp  ft 03  03  CN  •5H  13  03  CO  ft S  c/i  <! 43 ft ft  22  overnight at 37 °C. The cells were asceptically scraped from the plates and used to inoculate 1 litre flasks containing a defined phosphate minimal m e d i u m (phosphatesufficient conditions) (Hancock et al., 1982). These cultures were grown overnight and were then used to inoculate a 60 litre fermentor containing phosphate-deficient minimal m e d i u m (phosphate-deficient conditions). The  E. coli strains were grown overnight i n Luria-Bertani (LB) broth (lOg  tryptone, 5g yeast extract, 5g N a C l / litre) or on L B agar plates (LB broth + 2% agar). W h e n cells were harbouring plasmids, the media was supplemented w i t h 50 u g / m l ampicillin. Broth cultures were agitated on a shaker at 37 °C. W h e n strain CE1248 was used, 0.4% glucose was included to inhibit expression of LamB. In addition, 5 u M isopropyl thio-D-galactoside (IPTG) was included i n cultures used i n the indirect immunofluorescence experiments i n order to optimize expression of the plasmidencoded proteins. M e d i a components were purchased from Difco Laboratories (Detroit, MI).  Chemicals  KC1, K 2 H P O 4 and K H 2 P O 4 used i n the planar bilayer experiments were purchased from Fisher Scientific. KC1 was used unbuffered ( p H 6.0) while equal molar concentrations of K 2 H P O 4 and K H 2 P O 4 were mixed to achieve a p H of 8.0. A l l other chemicals were purchased from either Fisher Scientific, Sigma Chemicals, B D H or I C N Biomedicals Inc.  23  General recombinant D N A techniques  1. Enzymes and plasmid manipulation A l l recombinant D N A techniques were performed as described by Sambrook et al. (1989). Restriction endonucleases and D N A modifying enzymes were purchased from G i b c o / B R L , Boehringer-Mannheim, N e w England Biolabs or Pharmacia and used w i t h the accompanying buffers at the recommended concentrations. A l l restriction digests were performed at 37 °C. Ligations were performed for 2 h at room temperature or overnight at 16 °C using T4 D N A ligase. Plasmid transformation of competent cells was accomplished using the CaCf2 method. Plasmids were isolated from overnight cultures of E. coli b y the alkaline lysis method followed b y phenol/chloroform extraction and ethanol precipitation. D N A fragments were isolated from agarose gels by  either centrifuging agarose slices containing the fragments through a p l u g of  siliconized glass w o o l followed by phenol extraction and ethanol precipitation or by using a commercial D N A isolation kit (Gene Clean II, Bio 101 Inc. L a Jolla, C A ) w h i c h utilized a D N A - b i n d i n g silica matrix.  2. Agarose gel electrophoresis Analysis of restriction endonuclease digested plasmids was accomplished by electrophoresis through 1% agarose gels containing either I X TBE buffer or I X T A E buffer and 0.5 u g / m l ethidium bromide. The 10X loading buffer contained 0.25 % bromophenol blue, 0.25 % xylene cyanol FF and 30 % glycerol i n water. Gels were r u n at a constant current of 85 volts for small gels (10 cm) and 120-150 volts for large gels (25 cm). After electrophoresis the D N A bands were observed using a short-wave U V transilluminator and photographed using polaroid high-speed black and white film and a polaroid MP-4 L a n d camera fitted w i t h a lens covered by a #15 deep yellow filter. The agarose was purchased from Bio-Rad Industries.  24  3. Synthetic oligonucleotides Oligonucleotides used as primers for site-directed mutagenesis  and D N A  sequencing were synthesized on an A p p l i e d Biosystems M o d e l 392 automated D N A synthesizer (Applied Biosystems Incorporated, Foster City, C A ) . After an overnight incubation i n N H O H at 55 °C to cleave the base-protecting groups from the 5'-end of 4  the oligonucleotides, the D N A was dried by centrifugation under vacuum. The dried D N A was resuspended i n 30% N H O H and precipitated by adding two volumes of 4  isopropanol followed by centrifugation at 12,000 g for 15 min. The purified D N A was resuspended i n sterile d H 0 and quantitated by monitoring U V absorbance at 260 nm. 2  Double-stranded oligonucleotides used for the mutation of an oprP PstI site and the insertion of the malarial epitope into the oprP gene were annealed as follows. Equal quantities of each strand of D N A were combined i n sterile d H 0 , heated to 90 °C for 15 2  m i n and allowed to cool slowly to room temperature.  4. D N A sequencing Plasmid D N A was sequenced using the A B I automated fluorescent sequencing system w i t h a M o d e l 373 sequencer and P C R protocols provided by ABI. Template D N A was prepared by either using Quiawell-8 plasmid kits (Quiagen) or the polyethylene glycol precipitation method (Sambrook et a l , 1989). Primers were synthesized on an A B I D N A synthesizer. The primers and templates were combined at the recommended concentrations, and were mixed w i t h the sequencing premix w h i c h contained both fluorescent and nonfluorescent nucleotides as well as the D N A polymerase ampliTaq. The samples were then subjected to 26 rounds of a P C R cycle w h i c h consisted of a 30 second incubation at 96 °C followed by a 30 second incubation at 50 °C and a 3 minute incubation at 60 °C.  25  After amplification the D N A was purified by centrifugation through 1 m l spin columns composed of G-50 Sephadex (Pharmacia) followed b y ethanol precipitation. The  samples were then air dried, resuspended i n four u l of a 5/1 mixture of  f o r m a m i d e / E D T A ( p H 8.0) and heated at 90 °C for two minutes. The samples were immediately placed on ice and were loaded on to the gels within an hour. The gels, w h i c h contained 50% w / v urea and 4.75% acrylamide, were run at a constant power of 30 Watts for 12 hours. Automatic analysis of the sequencing data was accomplished w i t h the aid of the ABI 373 analysis software program.  General protein and immunological techniques  1. SDS-polyacrylamide gel electrophoresis Protein electrophoresis on SDS-polyacrylamide  gels was  performed  as  previously described. Separating gels contained 12% acrylamide/bis (Biorad) and stacking gels contained 2.5%. The running buffer contained 3.1g Tris, l g SDS and 14.4g glycine per litre. Whole cell lysates or isolated outer membranes were heated at 100 °C for 10 m i n prior to loading on to the gels except when nondenatured trimers were required. Undenatured proteins were not heated prior to loading. The 2X reduction mix contained 4% SDS, 20% glycerol, 125mM Tris-HCl and 4 0 m M E D T A . Electrophoresis was carried out w i t h a constant current of 120 V until the dye front reached the bottom of the gel. Both unstained and pre-stained M W t markers were purchased from Bio-Rad. Proteins were visualized by staining the gels i n a solution of 45% methanol/45% d H 0 / 2  10% acetic acid containing 0.28% Coomassie Brilliant Blue.  2. Western immunoblotting For Western immunoblotting, unstained gels were transferred to nitrocellulose membranes (Schleicher and Schuell) as previously described (Towbin et al., 1979). After  26  electrophoresis, the gels were soaked for 10 m i n i n I X blotting buffer (25mM Tris, 192mM glycine, 20% methanol). The gels were then placed on top of a piece of presoaked nitrocellulose cut to fit the gel and sandwiched between two pieces of filter paper i n a trans-blotting apparatus (Bio-Rad). The gel was then submerged i n an electrophoresis chamber filled w i t h blotting buffer. The electrotransfer of the proteins to the nitrocellulose membrane was carried out w i t h a constant current of 100 V for 1 hr. Ice packs were placed i n the chambers during the electrotransfer i n order to prevent over heating of the buffer. After blocking w i t h 3% bovine serum albumin (BSA) i n phosphate buffer saline (PBS) ( p H 7.4), the nitrocellulose membranes were incubated w i t h either a 1/1000 dilution of anti-OprP rabbit serum or a 1/5000 dilution of anti-malarial epitope monoclonal antibody i n 1% B S A / P B S for 2 hrs at room temperature o n a shaker. After washing 2X w i t h PBS, the membranes were incubated w i t h a 1/2000 dilution of alkaline phosphatase conjugated goat anti-rabbit or goat anti-mouse antibody (Bio-Rad). The membranes were washed 2X w i t h PBS and the bound antibodies were detected by incubating the membranes i n 0.1 M T r i s - H C l ( p H 9.6) containing 40 m M M g C l , 5 m M 2  BCIP and 10 m M N B T . Blots were development until the positive control exhibited a dark band the membranes were washed w i t h PBS to stop further development.  3. Antibodies Monomer specific anti-OprP rabbit serum was made during the course of a previous study (Poole and Hancock, 1986). Trimer-specific antiserum was made using F P L C purified OprP. A 3 k g  female N e w Zealand White rabbit was injected  subcutaneously w i t h 100 ug of purified OprP suspended i n 1.5 m l Freunds complete adjuvant. T w o more injections at 5 week intervals w i t h 100 ug of purified O p r P suspended i n 1.5 m l Freunds incomplete adjuvant were carried out. Three days after the  27  final injection, 20 m l of blood was drawn and the serum was assessed for reactivity w i t h O p r P by incubating dilutions w i t h nitrocellulose bound purified protein. In order to remove nonspecific antibodies, aliquots of serum were preabsorbed w i t h washed intact E. coli cells (DH-5aF' or CE1248). The pellets obtained  after  centrifuging 10 m l of an overnight culture of E. coli were resuspended i n 1 m l of the anti-OprP rabbit serum. After being incubated for 45 m i n at room temp o n an end-over end-shaker, the cells were pelleted and the serum was reabsorbed against a fresh batch of cells. The preabsorbed serum was divided into 50 ul aliquots and stored at -20 °C. The anti-malarial epitope monoclonal antibody p.f. 2A.10 was obtained from R. W i r t z (Dept. of Entomology, Walter Reed Institute of Research, Washington, D C ) and was prepared as previously described (Wong et al., 1995). Alkaline phosphatase conjugated goat anti-rabbit IgG and goat anti-mouse IgG were purchased from Bio-Rad Industries and fluorescein isothiocyanate conjugated  secondary antibodies were  purchased from Gibco BRL.  4. Indirect immunofluorescence The detection of surface exposed proteins and epitopes was accomplished using the method of Hofstra et al. (1979). Aliquots of cells (100 uls) grown overnight were pelleted, washed w i t h PBS and incubated with a 1/100 dilution of primary antibody (polyclonal anti-OprP or monoclonal anti-malarial epitope antibodies) i n P B S / 1 % B S A for 1.5 hr at room temperature. The cells were then washed 2x w i t h 1 m l of PBS and resuspended i n 100 (il  of 1% B S A / P B S containing a 1/20 dilution of fluorescein  isothiocyanate (FITC) conjugated secondary antibody mouse). After resuspended  (goat anti-rabbit or goat anti-  a 1.5 h incubation at room temperature cells were washed 2x,  i n 1% B S A / P B S and dried onto poly-lysine coated slides (Sigma).  Fluorescence was monitored using a Zeiss microscope fitted w i t h a halogen lamp and  28  filters set for emission at 525 nm. Cells were photographed using Kodak T-Max 400 black and white print film.  Construction of plasmid pAS27  The oprP gene was subcloned into pTZ19U i n two separate steps. First a vector lacking EcoRI and Psfl recognition sites was created by ligating a 900 base pair (bp) Scal/Hindlll  fragment from pUC18 to a 1.7 kilobase pair (kb) Scal/Hindlll  from pUC19 to form the vector pUC18/19. A 700 bp Hindlll/Hindlll  fragment  fragment from  pRSP-3 encoding the first 95 amino acids of OprP was gel purified and cloned into the Hindlll  site of p U C 1 8 / 1 9 (Figure 1). The resultant plasmid was then digested w i t h  EcoRI and Psfl, releasing a 27 bp fragment.  The vector was then ligated to a 27 bp  synthetic oligonucleotide which replaced the excised fragment but contained a single basepair change which destroyed the Pstl recognition site while maintaining the encoded  amino  acid  sequence.  After  verifying  the  oligonucleotide by D N A sequencing, the HindUl/HindUl  proper  orientation of  the  fragment was excised and gel  purified. A 1.2 kb Pstl/Pstl fragment from pRSP-3 encoding amino acids 35-406 of O p r P was cloned into the Pstl site of pTZ19U. This construct was then digested w i t h HindUl and ligated to the gel purified 700 bp Hindlll/Hindlll  fragment. Finally, the plasmid  was digested w i t h Pstl and ligated to an 18 bp synthetic oligonucleotide w h i c h encoded the last four amino acid residues of O p r P and contained a single basepair change w h i c h destroyed the Psfl recognition site. Proper incorporation of the oligonucleotide was again verified by D N A sequencing. This final construct was named pAS27 (Figure 2).  EcoRl HiBtnH,.PstlHindin —L-H U > — I  pRSP-3  Pstl  R  Hiiiilll digest  Hindin^lstlHMH ( own construct) Huidlll digest  ligation  Hiudlll  EcoRl , ,PstljlmdJII  * EcoRl pjfi  EcoRl*  1=1  ligation EcoRl Hindlll, * Hindlll  Hindlll digest + fragment isolation  Hi«tdin  Hindlll  Hindlll  4  ligation  Hindni  —i  Pstl  Hindlll _1  rPstl digest  Xbal  HindlllI  EcoRL. Hll * HindlTI  * Xbal  Figure 1. Procedure used in the creation of plasmid pAS27  EcoRI Kpnl BamHll  Figure 2. Restriction map of plasmid pAS27.  31  L i n k e r insertion mutagenesis  Insertion of 12 bp linkers into the oprP gene was accomplished as described previously (Wong et al., 1993). Plasmid pAS27 was partially digested b y the three frequently cutting blunt end restriction endonucleases Alul, Rsal and Thai i n the presence of varying concentrations of ethidium bromide (10-50 ng/ul). Prior to the large-scale plasmid digestion and isolation, small trial digestions were performed w i t h various concentrations of ethidium bromide i n order to establish conditions optimal for the isolation of singly cut plasmid DNA. The linker insertion mutant PL1 was created using Eco RV digested plasmid pAS27. After digestion, cut plasmid was gel purified and ligated i n equimolar ratios to a 1.3 kb Hindi fragment w h i c h was isolated from the kanamycin resistance plasmid p U C 4 K A P A . The ligation products were transformed into E. coli strain DH5-aF' and clones were selected for both ampicillin and kanamycin resistance. The resultant colonies were picked onto L B agar plates and screened for O p r P expression b y immunoblotting using  preabsorbed anti-OprP (trimer specific)  polyclonal rabbit serum. Plasmids were isolated from OprP negative clones and digested w i t h Pstl, releasing the kanamycin resistance gene and leaving a 12 bp residual linker sequence at the initial insertion site. The religated linker mutant plasmids were transformed back into DH5-aF' and characterized b y restriction enzyme digestion. Clones displaying unique digestion patterns were selected for D N A sequencing .  M a l a r i a l epitope insertion  The linker-mutant plasmids were linearized by digestion w i t h Pstl and ligated to 47 bp synthetic oligonucleotides encoding the malarial epitope at molar ratios of 1:100. The oligonucleotides were engineered so as to introduce a unique restriction enzyme recognition site (Sphl or Xbal as underlined below) when inserted into the plasmid.  32  Separate oligonucleotides were utilized for each reading frame to ensure correct translation. This resulted i n the following inserted sequences: CCG  A A C GCC A A C CCG A A C GCC A A C CCG A A C GCC GGG CAT G C A  AC C C G A A C GCC A A C C C G A A C GCC A A C C C G A A C G C A T G C A  or or  G A A C GCC A A C C C A A A C GCG A A T C C G A A T GCT C T A G A C TTG C A .  Clones were initially screened b y colony immunoblot analysis using the antimalarial epitope specific monoclonal antibody p.f. 2A.10. The plasmid D N A of selected immunopositive clones was isolated and sequenced i n order to verify the proper incorporation of the inserted epitope.  Site-directed lysine mutagenesis  The oprP substitution mutants were created using a two-step recombinant P C R method (Higuchi et al., 1988; H o et al., 1989) w i t h the oprP containing plasmid pAS27 used as the template. Mutagenic oligonucleotides (27-mers) contained mismatches w h i c h corresponded to a substitution mutation i n the encoded amino acid sequence (Table II). Reactions (100 ul total volume) contained 100 ng plasmid template (pAS27), 100 p g of primer, 200 m M each of the four dNTPs, 1-2 units Vent D N A polymerase (New  England Biolabs) and I X Vent D N A polymerase buffer. The D N A was then  subjected to 26 rounds of an amplification reaction which consisted of a 1 m i n denaturation step at 96 °C followed by a 1 min combined annealing/synthesis step at 72 °C. The amplified fragments were purified from agarose gels and the purified fragments were subcloned into appropriately digested and purified plasmid pAS27. Resultant colonies were screened for the presence of the desired construct by restriction  33  Table II. Codon changes of oprP substitution mutants. Plasmid Codon wild-type  mutant  Amino acid wild-type  mutant  pOPE13  AAG  GAG  Lys  13  Glu  13  pOPE15  AAG  GAG  Lys  15  Glu  15  pOPE25  AAG  GAG  Lys  25  Glu  25  pOPE30  AAG  GAG  Lys  30  Glu  30  pOPE74  AAG  GAG  Lys  74  Glu  74  pOPE109  AAG  GAG  Lys  109  Glu  109  pOPE121  AAG  GAG  Lys  121  Glu  121  pOPE126  AAA  GAA  Lys  126  Glu  126  pOPE181  AAG  GAG  Lys  181  Glu  181  pOPG74  AAG  GGG  Lys  74  Gly  74  pOPG121  AAG  GGG  Lys  121  Gly  121  pOPG126  AAA  GGA  Lys  126  Gly  126  pOPQ121  AAG  CAG  Lys  121  Gin  121  pOPQ126  AAA  CAA  Lys  126  Gin  126  34  enzyme mapping. Plasmids displaying the correct restriction digest maps were sequenced i n order to verify the presence of the desired mutation as well as to ensure that no errors i n the D N A were incorporated during the amplification.  Isolation of outer membrane fractions  Outer membranes were purified from both P. aeruginosa and E. coli by a method which utilized a two-step sucrose density gradient. After overnight growth, cells were pelleted by centrifigation at 3000 g for 20 min. The bacteria were resuspended i n 20 % sucrose containing 10 m M  Tris-HCl ( p H 8.0) and 50 u g / m l deoxyribonuclease and  broken by French pressing. Unbroken cells were pelleted by centrifugation and the supernatant was collected and stored on ice. Two-layered sucrose density gradients (50%/70%) were set up i n transparent, disposable centrifuge tubes  (Beckman  Instruments Inc., Palo Alto CA, U S A ) and the supernatant was layered o n top of the gradient. The tubes were then placed into a Beckman SW-41 swinging bucket rotor and spun at 100,000 g for 6-12 hours at a constant temp of 4 °C. After centrifugation, the outer membrane fraction, which migrated to the interface of the two different sucrose densities, was collected by poking a heated needle into the bottom of the tubes and draining the contents into collection vessels. The membrane fractions were diluted w i t h distilled H 2 O to reduce the sucrose concentration to below 20 % and were then pelleted by centrifugation at 200,00 g for 1 hour.  Detergent solubilization of O p r P  The nonionic detergent n-octyl-polyoxyethylene (octyl-POE) was  used  as  previously described (Garavito and Rosenbusch, 1986) to selectively solubilize O p r P contained i n isolated outer membrane fractions. Pelleted outer membranes were  35  resuspended i n 0.5 % octyl-POE containing 10 m M T r i s - H C l ( p H 8.0) and incubated at 37 °C for 1 hour. The membranes were then pelleted by a 1 hour centrifugation and the supernatant was collected and stored on ice. The pellet was resuspended i n 3% octylP O E containing 10 m M Tris-HCl ( p H 8.0) and incubated at 37 °C for 1 hour. The membranes were again pelleted by centrifugation, the supernatant was saved and the pellet was resuspended i n 3% octyl-POE/50 m M E D T A containing 10 m M T r i s - H C l ( p H 8.0) and incubated at 37 °C for 1 hour. The membranes were then centrifuged a final time, the supernatant collected and the insoluble pellet was resuspended i n distilled H2O. The three detergent solubilized fractions, along w i t h the resuspended pellet were subjected to S D S - P A G E on 12% polyacrylamide gels. After electrophoresis, the protein bands were visualized by either staining the gels w i t h a 0.28% solution of Coommassie Brilliant Blue or by electrotransferring the proteins onto nitrocellullose membranes and incubating the membranes w i t h an anti-OprP polyclonal antiserum. O p r P was found to be predominantly solubilized i n the 3% octyl-POE/50 m M E D T A supernatant fractions.  F P L C purification of O p r P  O p r P used i n the production of anti-OprP rabbit antiserum was purified from isolated P. aeruginosa outer membranes by F P L C . The octyl-POE solubilized protein solutions were placed into dialysis bags w i t h a M W cutoff of 30 k D and were then dialyzed overnight against 500 times volume 0.08% N,N-dimethyl-dodecylamide-Noxide ( L D A O ) containing 10 m M Tris-HCl (pH) and 10 m M E D T A at 4° C . This was done i n order to replace the octyl-POE w i t h L D A O which possesses a lower critical micelle concentration. The dialyzed protein was then concentrated using A m i c o n centrifuge columns. The concentrated protein sample was filtered through a syringe filter w i t h a 0.2 u m cut off and the collected filtrate was stored on ice.  36  The protein sample was loaded on to a M o n o - Q F P L C  column (Pharmacia)  w h i c h was attached to a Pharmacia F P L C system. After washing the column w i t h the buffer A (0.08% N,N-dimethyl-dodecylamide-N-oxide ( L D A O ) ; 10 m M T r i s - H C l (pH); 10 m M E D T A ) , O p r P was eluted from the column by passing a linear N a C l gradient over the column. Fractions which were shown by U V detection at 280 n m to contain large  concentrations  of  protein  were  analyzed  by  SDS-polyacrylamide gel  electrophoresis. Those fractions that were found to contain predominately O p r P were pooled and concentrated by filtration. The concentrated protein sample was reapplied to the M o n o - Q column and subjected to a second round of purification as described above. Pooled fractions of protein obtained from this second passage over the column were found to be virtually free of contaminating proteins as determined b y S D S - P A G E followed by Coomassie Blue staining of the gel.  Purification of O p r P from SDS-polyacrylamide gels  Small amounts of mutant protein required for planar lipid bilayer analysis were purified from preparative SDS-polyacylamide gels. Unheated detergent solubilized O p r P was loaded on to 12% SDS-polyacrylamide gels and electrophoresed as described above. After electrophoresis, the vertical edge of the gel was removed and stained w i t h Coommassie brilliant blue i n order to visualize OprP. The stained slice of gel was then used as a guide to localize OprP on the unstained gel, and the band was excised w i t h a razor blade. The protein was eluted from the gel slices by incubating the slices overnight i n approximately 300 ul of 10 m M T r i s - H C l ( p H 8.0) containing 0.1% SDS.  37  Planar l i p i d bilayer experiments  Analysis of the single-channel conductance, anion selectivity and  phosphate  specificity of mutant forms of OprP was accomplished using the planar l i p i d bilayer technique as previously described (Hancock and Benz, 1986). The main apparatus consisted of a Teflon chamber which contained two individual compartments. The wall separating the two  compartments contained a small hole (0.1 mm-single  channel  conductance; 0.9 mm-anion selectivity) which permitted the free passage of  bathing  solution. Prior to use the, the area surrounding the hole was  coated w i t h a small  amount of 2 % oxidized cholesterol and dried w i t h a hair dryer. The chambers were filled w i t h equal volumes of bathing solution and calomel electrodes w h i c h were connected to a voltage source and a current amplifier were placed i n either chamber. A membrane was painted over the hole by w i p i n g a small amount of oxidized cholesterol over the hole w i t h a plastic wand and the current was monitored using either a chart recorder (which was connected to a current amplifier) or a voltage meter. Once a stable base line had been established the experiment was commenced.  Single-channel conductance The bathing solutions used contained 100 mM,  1 M  or 3 M  KC1. Nanogram  quantities of purified protein were added to the bathing solution and increasing steps i n conductance were monitored w i t h a chart recorder. A t least 100 individual channel events were observed for each mutant protein examined.  A n i o n selectivity W h e n the ionic selectivity of the mutant proteins was measured the electrodes were connected directly to a current/voltage meter and the bathing solution was mM  KC1. U p o n addition of the mutant proteins a voltage of 20 mV  50  was applied and the  38  membrane current was allowed to increase approximately 100 fold before the applied voltage was turned off and the meter was switch to read voltage. A t this point 50 ul aliquots of  50 m M and 3 M KC1 were added to either compartment of the Teflon  chambers. Changes i n the zero-current membrane potential were recorded after each salt solution addition. The zero-current membrane potential of the proteins was calculated by using the Goldman-Hodgkin Katz equation.  Phosphate i o n specificity The effect that the substitution of individual amino-terminal lysine residues had on the phosphate-binding site of OprP was assessed by measuring the inhibition of conductance induced by the addition of small amounts of 1 M potassium phosphate to the bathing solution (0.1 M KC1). Equimolar concentrations of K H P 0 2  4  and K H P 0 2  4  were mixed to achieve a buffered p H of 8.0. The single-channel conductance of the individual mutant proteins i n the presence of increasing phosphate ions was compared to the channel conductance of the same proteins i n phosphate-free bathing solution. One hundred individual channel events were measured for each protein examined.  39  RESULTS  A. Structural analysis of O p r P  Chapter 1. Linker insertion mutagenesis of O p r P  1.1 Introduction Since there is not a great deal of similarity between the amino acid sequence of O p r P and the other well characterized members of the porin family, a method was needed w h i c h w o u l d differentiate between loop regions and those regions w h i c h formed the trans-membrane (3-barrel. The semi-random insertion of 12 base pair linkers into the genes of certain bacterial porins has previously been successfully used to generate topological models of these proteins. Subsequent crystallization studies have shown that this technique is able to predict the location of loop regions w i t h a reasonably high degree of reliability. While this technique does not allow for the differentiation between internally-exposed and  externally-exposed loop regions, the  inclusion of a unique restriction enzyme site within the inserted linkers provides a convenient means by which to later insert a foreign epitope which could serve as a tag. This chapter describes the creation and characterization of 13 linker insertion mutants of O p r P w h i c h were generated by both site-directed and semi-random mutagenesis.  1.2 Expression of O p r P i n an E. coli background The O p r P gene was subcloned into the phagemid vector pTZ19U as described i n the Materials and Methods section to create the O p r P expression plasmid pAS27. This construct was created i n and purified from the E. coli strain DH-5ocF'. After sequencing of the regions of the vector which were mutated so as to eliminate two Pst I sites, the purified plasmid was transformed into the porin deficient E. coli strain CE1248. Outer  40  membranes  from E. coli DH-5aF'(pAS27) and CE1248(pAS27) were purified by  centrifugation of cell envelopes through sucrose density gradients. The purified membranes were analyzed by SDS-polyacylamide gel electrophoresis and Western immunoblot analysis with anti-OprP specific polyclonal rabbit serum. The presence of O p r P was detected i n both backgrounds, but as expected, the porin deficient strain CE1248 expressed a slightly higher amount of O p r P than d i d DH-5ocF'. For this reason CE1248 was chosen as the background strain to be used i n all experiments involved i n the expression of the mutant forms of OprP (Figure 3) while the heartier DH5-otF' strain was used for the initial construction of all the mutants. Inclusion of IPTG i n the growth media was not shown to increase the expression of O p r P as determined by SDSpolyacrylamide gel electrophoresis.  1.3 Linker insertion mutagenesis of O p r P at a unique Eco RV restriction enzyme site A s an initial test of the usefulness of this technique, the kanamycin resistance cassette was ligated to Eco RV digested and gel purified pAS27. After digesting w i t h Hinc II to release the kanamycin resistance gene the religated plasmid was purified and sequenced. The 12 base pair linker was found to be properly incorporated into the Eco RV site. The reading frame of the inserted linker was such that the resulting four amino acid sequence D L Q V was inserted into OprP.  1.4 Semi-random linker insertion mutagenesis of OprP The insertion of the 12 base pair linker into multiple sites of the oprP gene was accomplished by first purifying partially digested plasmid as described i n Materials and Methods. The enzymes used i n this procedure cut at four base pair recognition sites w h i c h were scattered throughout the sequence of pAS27. Since all clones w h i c h had the kanamycin resistance cassette inserted into the coding sequence,of the oprP gene w o u l d no longer be capable of protein expression, OprP negative clones were selected for by  41  A  B  Figure 3. Western immunoblot of outer membranes of E. coli CE1248 containing OprP reacted with anti-OprP trimer specific (A) or monomer specific (B) rabbit antiserum.The sample reacted with the monomer specific antiserum was heated at 100° C for 10 min. Prestained molecular mass markers on the left were (from the top) 106, 80, 49.5, 32.5, 27.5 and 18.5 kDa.  42  immunobloting recombinants w i t h anti-OprP (trimer-specific) rabbit serum. After a Hinc II digestion which served to release the kanamycin resistance cartridge, the religated plasmids were purified and subjected to restriction enzyme digestion analysis. The  clones were grouped according to their restriction digestion patterns and  representative plasmids were sequenced. Twelve unique linker insertion mutants were isolated by this method (Table III).  1.5 Expression of the linker insertion mutant forms of OprP i n E: coli Competent E. coli CE1248 cells were transformed w i t h each of the 13 O p r P linker insertion mutant plasmids and the resulting transformants were used to inoculate 5 m l LB broth cultures which contained 50 u l / m l ampicillin to maintain the plasmid and 0.4% glucose to inhibit the expression of LamB. The cultures were grown overnight at 37 °C and cells from 100 ul aliquots were pelleted, lysed and electrophoresed o n SDSpolyacrylamide gels. After staining the gel w i t h Coomassie Blue, all 13 mutants were shown to be expressed to varying degrees (data not shown). In order to determine if these mutant proteins were properly localized to the outer membranes of the host cells, 100 m l cultures of each clone grown under the same conditions as described above were used to prepare isolated outer membrane fractions. The purified outer membranes were assessed for the presence of O p r P b y both SDSpolyacryamide gel electrophoresis and Western immunoblotting. The amounts of the mutant forms of OprP i n outer membranes was found to mirror the amounts of these proteins i n whole cells. Figure 4 depicts a Western immunoblot analysis of outer membranes containing the thirteen linker insertion mutants along w i t h the wild-type O p r P expressing plasmid pAS27. The negative control lane contained outer membranes purified from E. coli CE1248 containing the phagemid pTZ19U. Although all mutants were expressed to some degree, certain insertion sites appeared to be less capable of tolerating the presence of the inserted linker than other  43  Table in. Insertion sites of O p r P linker mutants Plasmid  Insertion site (amino acid)  Amino acids inserted  pAS27 pPLl  9  DLQV  pPL2  60  TCRS  pPL3  75  PAGP  pPL4  82  DLQV  pPL5  124  TCRS  pPL6  148  DLQV  pPL7  154  TCRS  pPL8  190  PAGP  pPL9  208  TCRS  pPLlO  226  TCRS  pPLll  287  TCRS  pPL12  309  DLQV  pPL13  333  DLQV  44  1 2 3 4 5 6 7 8 9 10 11 12 13TZ A S  —  OprP  Figure 4. Western immunoblot of outer membranes of E. coli CE1248 containing OprP linker insertion plasmids pPLl-pPL13 (lanes 1-13 respectively) and positive and negative controls plasmid pAS27 (AS) and pTZ19U (TZ). The sample were heated at 100° C for 10 min prior to loading. Prestained molecular mass markers on the left were (from the top) 106, 80,49.5, 32.5, 27.5 and 18.5 kDa.  45  sites. The O p r P insertion mutants PL3, PL8, PL12  and PL13  were all expressed at  significantly lower levels than that of the wild-type protein. A l l of the insertion mutants were shown to possess electrophoretic mobilities similar to that of wild-type OprP. Insertion of the linker into the PL2 site appeared to induce the production of a degradation product with an apparent molecular mass of approximately 30 kDa  and  two  site  smaller products of approximately 23 and 25 kDa; insertion at the PL13  appeared to induce the production of a single product w i t h a molecular mass slightly less than that of the native protein. It is possible that the presence of the linker at these sites resulted i n a slight distortion i n the folded structure of the protein w h i c h exposed a proteolytic site. However the insertion of these linkers could not have caused a profound distortion of the folding patterns since these proteins reacted w i t h the trimerspecific polyclonal serum as well as the wild-type protein, as assessed by  colony  immunoblotting (data not shown). A number of the mutant proteins appeared to possess increased trimer stability. Insertion at sites PL5, PL7, PL9, PL10 and PL11 resulted i n mutant proteins w h i c h were partially resistant to heat denaturation i n SDS. Normally, nondenatured trimers do not react w i t h monomer-specific antiserum. The fact that the high molecular weight bands of these mutants were able to bind the antibody indicated that the heat treatment affected the secondary structure of the protein without causing the dissociation of the trimers into individual monomers.  1.6 Surface localization of the OprP linker insertion mutants To  determine if the mutant forms of OprP were efficiently folded  incorporated into the outer membrane of the host cells, clones were analyzed  and by  indirect immunofluorescence for surface exposure of the mutant proteins. Cells containing the pPL plasmids, pAS27 or pTZ19U were incubated w i t h anti-OprP (trimerspecific) rabbit serum followed by  a goat anti-rabbit fluorescein isothiocyanate-  46  conjugated secondary antibody. The cells were applied to poly-L-lysine coated glass slides and viewed under a fluorescence microscope. N i n e of the 13 linker insertion mutants fluoresced at levels comparable to those exhibited by the wild-type protein (Figure 5A). These same proteins were previously shown by Western immunoblotting to be efficiently expressed and localized to the outer membrane. Cells harbouring the other four linker insertion mutant plasmids d i d not fluoresce at perceptively higher levels than that of cells carrying the negative control plasmid pTZ19U (Figure 5B; Table IV). These four clones were also previously shown to be expressed at greatly reduced levels i n the outer membrane i n comparison to the wild-type protein. Therefore the absence of detectable surface exposure displayed by these mutant forms of OprP could be due to either an inability of the proteins to be properly incorporated into the outer membrane or the fact that the binding of antibodies to cells expressing such a small amount of surface-exposed protein d i d not lead to perceptively greater fluorescence than the non-specific binding of these same antibodies to OprP-negative cells.  1.7 Functional analysis of selected linker insertion mutants The majority of the mutant proteins generated by the linker insertion method appeared to suffer few detrimental effects on either expression or surface exposure due to the presence of the inserted sequence. These findings however d i d not exclude the possibility that the channels formed by these mutants could have been affected i n some manner. To determine if the insertion of the linkers had any effect on the functional aspects of the mutant proteins, several of the insertion mutants whose expression appeared to be unaffected by the mutagenic procedure were purified from  SDS-  polyacrylamide gels as described i n the Materials and Methods section and analyzed by the planar lipid bilayer method.  47  Figure 5. Indirect immunofluorescence of E. coli strain CE1248 containing plasmid pAS27 (A) or plasmid pTZ19U (B) reacted with anti-OprP (trimer-specific) antiserum.  48  Table IV. Expression and surface exposure of OprP linker mutant proteins Plasmid  Protein expression (by SDS-PAGE)  Surface reactivity  pAS27  ++  ++  pPLl  ++  ++  pPL2  ++  ++  pPL3  +  -  pPL4  ++  ++  pPL5  ++  ++  pPL6  ++  ++  pPL7  ++  ++  pPL8  +  -  pPL9  ++  ++  pPLlO  ++  ++  pPLll  ++  ++  pPL12  +  -  pPL13  +  -  * As assessed by indirect irnmunoftuoresence using an anti-OprP specific antiserum  49  A l l of the mutant proteins tested were shown to be capable of forming stable channels when added to the salt solution bathing a l i p i d bilayer composed of oxidized cholesterol (Table V). The single-channel conductances of these proteins were not found to be significantly different from that of wild-type OprP; the variation was less than 10%. This w o u l d suggest that the sites at which these linkers had been inserted into O p r P were not critical to maintaining the three-dimensional folding pattern of the protein.  1.8 Summary The insertion of 12 base pair linkers into the gene encoding O p r P was used to differentiate between sections of this protein which were structurally flexible and sections w h i c h were relatively nonpermissive for the insertion of the linker. Four of the thirteen mutants created were shown to have been significantly affected by the mutagenesis. Their levels of expression  were drastically reduced i n the outer  membranes of the host cells and there was no evidence to show that they were capable of being properly exported to and expressed at the cell surface. The reasons for the reduction i n expression of these proteins is unknown but it is possible that the presence of the linkers at these particular sites inhibited the progressive folding kinetics of these proteins w h i c h created the opportunity for various cellular proteases to degrade the proteins before they were able to be exported to the outer membrane. Since the most structurally demanding sections of OprP should consist of those involved i n forming the (3-barrel, it w o u l d not be unreasonable to assume that these four sites may w e l l be located within sections of (3-strands. Nine of the insertion mutants were expressed at levels comparable to the w i l d type protein i n both the outer membrane and at the cell surface. The sites at w h i c h these mutations occurred must therefore not be involved with maintaining the folded structure of the mature surface localized protein. The sections of porins w h i c h have  50  Table V. Channel activity of selected OprP linker mutant proteins Plasmid  Average single-channel conductance (nS) ± S D a  pAS27  0.23 ± 0.04  pPLl  0.23 ± 0.04  pPL2  0.22 ± 0.04  pPL3  ND  pPL4  0.25 ± 0.04  pPL5  0.24 ± 0.04  pPL6  ND  pPL7  ND  pPL8  ND  pPL9  0.23 ± 0.04  pPLlO  0.23 ± 0.04  pPLll  0.24 + 0.05  pPL12  ND  pPL13  ND  a. Average of 100 single-channel events in picoSiemens ± S.D. b. N.D. = not determined  b  51  previously been shown to be most amenable to the insertion of foreign sequences are those that form the internal and external loop regions. It is possible that the reason the expression of these mutants was not noticeably affected was because the sites of these mutations were located i n loop regions of OprP.  52  Chapter 2. Epitope insertion mutagenesis of O p r P  2.1 Introduction The insertion of foreign epitopes has previously been used to determine the topological structure of a number of porins. In these studies, the surface exposure of the inserted epitopes was assessed by reacting whole cells expressing the epitope insertion mutant w i t h an epitope-specific antibody. Those clones which reacted w i t h the antibody were assumed to carry the epitope i n a surface localized loop region. A comparison of the 3-dimensional  crystal structure of proteins subjected to epitope  insertion  mutagenesis revealed that this method was able to accurately predict w h i c h sections of these proteins were exposed to the external surface. In order to gain further insight into the topological arrangement of wild-type OprP, an epitope from the circumsporozoite form of the malarial parasite Plasmodium falciparum was inserted into the unique Psf I restriction enzyme site contained w i t h i n the linker of 12 of the 13 OprP linker insertion mutants. It was hoped that this procedure  w o u l d both help to determine  which sites were flexible enough to  accommodate the insertion of a larger foreign peptide as well as aid i n the identification of specific sites which are located i n the extracellular domain of OprP.  2.2 Insertion of the malarial epitope into the linker sites of OprP mutants Oligonucleotides which encoded the repeating amino acid sequence (PNAN) . 2  3  were synthesized and purified as described i n the Materials and Methods section. The oligonucleotides were annealed and ligated to twelve of the O p r P linker insertion mutant plasmids which had previously been linearized b y digesting w i t h Psf I. Clones were screened for expression of the malarial epitope by colony immunoblotting w i t h the malarial epitope-specific monoclonal antibody p i . 2A.10. Plasmids purified from clones w h i c h were found to react w i t h the epitope-specific antibody were assessed for  53  the presence of the inserted oligonucleotide by restriction endonuclease mapping. Those plasmids which presented the correct restriction pattern were sequenced b y the di-deoxy terminator method. The results of the sequencing experiments demonstrated that the malarial epitope was correctly inserted into the Pst I site of all twelve of the selected linker insertion mutants.  2.3 Expression of the malarial epitope insertion mutant protein The effect that the insertion of the malarial epitope had o n the expression of the twelve mutant proteins was assessed by Western immunoblotting of outer membrane fractions which were purified from cells harbouring the epitope insertion mutant plasmids. The nitrocellulose-bound outer membrane proteins were reacted w i t h antiO p r P polyclonal antiserum. In order to optimize protein expression, all of the plasmids were mobilized into the porin deficient E. coli strain CE1248 w h i c h served as the background strain for all of the protein expression experiments. A s seen i n Figures 6 and 7 all of the epitope insertion mutant proteins were expressed i n the outer membrane of the host cells. However the levels of expression of these mutants were, i n most cases, reduced i n comparison to the wild-type protein. The one noticeable exception was the mutant which contains the epitope inserted at the PL10 site. The insertion of the foreign amino acid sequence at this site d i d not appear to have any effect o n the expression of the mutant protein. The three epitope insertion mutants whose linker mutant parents displayed the greatest reductions i n expression (insertions at sites PL8, PL12 and PL13) also exhibited low levels of expression. In addition, two other epitope insertion mutant proteins (sites PL6 and PL7) were expressed at noticeably lower levels than their linker insertion parents. It may be that these sites are less flexible than those of the remaining seven mutants. A l l of the epitope insertion mutant proteins demonstrated slightly reduced electrophoretic mobilities compared to wild-type OprP.  54  1 2 4 5 6 7 8 9 10 11 12 13TZ A S  OprP  Figure 6. Western immunoblot of outer membranes of E. coli CE1248 containing OprPencoding plasmids with malarial epitope inserted at sites PL1, PL2 and PL4 to PL13. Positive and negative controls were plasmids pAS27 (AS) and pTZ19U (TZ). The blots were reacted with anti-OprP (monomer-specific) antiserum. The samples were heated at 100° C for 10 min prior to loading. Prestained molecular mass markers on the left were (from the top) 106, 80,49.5, 32.5, 27.5 and 18.5 kDa.  55  1  2 4 5 6 7  8 9 10 11 12 13 T Z A S  Figure 7. Western immunoblot of outer membranes of E. coli CE1248 containing OprPencoding plasmids with malarial insertions at sites PL1, PL2 and PL4 to PL13. Positive and negative controls were plasmids pAS27 (AS) and pTZ19U (TZ). The blots were reacted with the anti-malarial epitope-specific antibody pf 2A.10. The samples were heated at 100° C for 10 min prior to loading. Prestained molecular mass markers on the left were (from the top) 106, 80, 49.5, 32.5, 27.5 and 18.5 kDa.  56  Insertion of the malarial epitope into linker site PL5 appeared to induce the production of two degradation products w i t h approximate molecular masses of 25 and 30 kDa. Surprisingly, inclusion of the epitope at insertion site PL2 d i d not induce the production of a degradation product even though the insertion of the linker alone d i d (Figure 4). The increased trimer stability displayed by some of the linker mutant proteins was reversed after insertion of the malarial epitope into the linker sites. Western immunoblotting of the outer membranes from the epitope insertion mutants w i t h the anti-malarial epitope-specific antibody was done to verify that the epitope was indeed expressed i n the mutants prior to the analysis of their surface exposure i n whole cells. If the mutant proteins d i d indeed contain the malarial epitope then the bands corresponding to OprP w o u l d bind to the anti-malarial epitope-specific antibody regardless of if the epitope was inserted i n a periplasmically-exposed or a surface-exposed region of OprP. While the degree to which the mutant proteins reacted w i t h the epitope-specific antibody was reduced i n comparison to their reactivity w i t h the OprP-specific antibodies, a careful inspection of the alkaline-phosphatase stained membrane revealed that a band which bound to the anti-malarial epitope antibody was present i n the outer membrane of every one of the epitope insertion mutants examined. The reason the anti-epitope-specific antibody failed to react w i t h the mutant proteins to the same extent as the anti-OprP-specific antibodies was likely because the anti-epitope antibody consisted of a single monoclonal antibody which bound to a single epitope contained w i t h i n the mutant proteins. The anti-OprP-specific antiserum contained a number of different antibodies which bound to a number of different epitopes distributed throughout the sequence of OprP. Therefore it w o u l d be expected that when this polyclonal antiserum was used as the primary antibody i n the Western immunoblot analysis the intensity of the reaction w o u l d be greater than when the anti-malarial epitope-specific antibody was used. The outer membranes of both clones carrying the  57  phagemid vector pTZ19U as well as those carrying plasmid pAS27 lacked a band w h i c h reacted w i t h the anti-epitope-specific antibody.  2.4 Surface localization of the epitope insertion mutant proteins To determine if the insertion of the malarial epitope into the linker site of the twelve mutant proteins had any detrimental effects on the export of the proteins to the outer membrane, whole cells expressing the mutant proteins were examined using the method of indirect immunofluorescence. The specificity of the primary antibody  was  towards wild-type OprP. The degree to which whole cells expressing the epitope insertion mutant proteins bound the antibody w o u l d reflect the surface exposure of that particular mutant. In addition, the fact that the anti-OprP antibody used i n this assay only bound to the trimer form of the protein meant that a positive reaction reflected the proper trimerization of a given mutant. A s shown i n Table V I the reactivity of the individual clones w i t h the anti-OprP antibody closely reflected the expression of these proteins i n the outer membranes as determined by Western immunoblot analysis. Cells expressing the five proteins w h i c h demonstrated significantly decreased expression levels i n isolated outer membrane preparations d i d not appear to bind the antibody at the cell surface to a greater extent than d i d those clones carrying the negative control plasmid pTZ19U. A s stated previously i n Section 1.6, the lack of detectable surface exposure could be due either to the actual lack of surface exposed protein or to the insensitivity of this assay i n discerning a difference between the specific binding of the antibodies to such a small quantity of protein and the non-specific binding of antibodies to the outer membrane proteins of the negative control. A l l the other seven proteins were shown by this method to be exported to the outer membrane at levels comparable to that of the w i l d type protein. It w o u l d therefore seem likely that if the malarial epitope was contained i n  58  Table VI. Expression and surface exposure of OprP malarial epitope insertion mutant proteins Insertion site  Protein expression (by SDS-PAGE)  Surface reactivity* Anti-OprP Anti-malarial polyclonal Ab epitope M A b  —  none  ++  ++  PL1  ++  ++  PL2  ++  :++  -  PL4  ++  ++  -  PL5  ++  ++  -  PL6  +  -  -  PL7  +  -  -  PL8  +  -  -  PL9  ++  ++  -  PL10  ++  ++  ++  PL11  ++  ++  ++  PL12  +  -  -  PL13  +  -  -  -  * As assessed by indirect immunofluoresence using either an anti-OprP specific antiserum or the malarial epitope specific MAb p.f. 2A.10.  59  a surface exposed region of any of these seven mutant proteins, the cells expressing those proteins w o u l d b i n d the anti-malarial epitope-specific antibody at the cell surface.  2.5 Surface localization of the inserted malarial epitopes To determine if any of the sites at which the malarial epitopes were inserted into O p r P faced the external surface of the cell, whole cells expressing the mutant proteins were subjected to indirect immunofluorescence w i t h the anti-malarial epitope-specific monoclonal antibody p i . 2A.10 used as the primary antibody. Clones expressing w i l d type O p r P were used as the negative control. Of the twelve clones examined by this method, only two were found to bind the anti-malarial epitope-specific antibody at levels significantly higher than the negative control (Table VI). Cells expressing the mutant proteins w i t h the epitope inserted at sites PL10 and PL11 exhibited high levels of fluorescence after incubation w i t h the antibodies (Figure 8). These same mutants were found to be capable of reacting i n whole cells w i t h antibodies directed against the native trimer form of O p r P (Table VI). It can therefore be assumed that the accessibility of these inserted epitopes to the antibody was due to the fact that they were actually located i n surface-localized sites rather than to a distortion of the native conformation of the protein w h i c h may have caused the exposure of a section of the protein which w o u l d normally be inaccessible at the cell surface. None of the other ten mutant expressing clones bound the anti-malarial epitope-specific antibody to a greater extent than the clone expressing wild-type OprP. These results suggest that either the sites at which the epitope was inserted into these mutants was not accessible to the cell surface or that the structure of the epitope was somehow distorted by the surrounding amino acid sequence into w h i c h it was inserted. In the case of the poorly expressed mutant proteins, the lack of binding of the antiepitope-specific antibody may have been due to the fact that the expression levels of these mutant proteins was below the detection sensitivity of this assay.  Figure 8. Indirect immunofluorescence of E. coli strain CE1248 containing the OprPencoding plasmid with the malarial epitope inserted at site P L 10. Cells expressing the mutant protein with the epitope inserted at site P L 11 displayed similar levels of fluorescence. The primary antibody used was the malarial epitope specific monoclonal antibody p.f. 2A.10. A l l of the other malarial epitope insertion mutant expressing cells well as cells expressing wild type OprP fluoresced at the same levels as the negative control in Figure 5B.  61  2.6 Summary A 15 amino acid peptide corresponding to an epitope of the malarial pathogen P. falciparum was inserted into the Psf I site of twelve of the thirteen linker insertion mutant forms of OprP which had previously been created (see Chapter 1). The purpose of this procedure was to both assess the permissiveness of the insertion sites for the insertion of a longer length of foreign sequence and to determine if any of these sites were located in surface-exposed regions. The presence of the epitope insertion mutants i n the outer membranes of host cells was  determined by Western immunoblotting using either anti-OprP or anti-  malarial epitope-specific antibodies. The expression of a number of these proteins was shown to be adversely affected after insertion of the epitope. This w o u l d suggest that the insertion sites of these specific mutants possess a limited degree of flexibility. The majority of the mutant proteins were expressed at levels comparable to that of w i l d type OprP, suggesting that the insertion sites of these proteins were flexible enough to accommodate the presence of the foreign peptide. The fact that all of the epitope insertion mutant proteins bound the anti-malarial epitope-specific antibody indicated that each of these constructs d i d indeed contain the epitope and that it was inserted i n both the correct orientation and i n the proper reading frame. The results of the immunofluorescence  experiments using the anti-OprP antiserum as the primary  antibody mirrored those of the Western immunoblot analysis of isolated outer membranes; those mutant proteins which were present i n the isolated outer membranes at greatly reduced levels compared to the wild-type protein were not shown to be present at detectable levels at the cell surface of whole cells. Indirect immunofluorescence  of whole cells expressing the epitope insertion  mutant proteins was used to determine if any of the insertion sites were located i n regions of the protein which are accessible to the antibodies at the cell surface. The results of these experiments indicated that two insertion sites of the seven efficiently  62  expressed mutants were surface exposed. While these results could be interpreted as meaning that only a low proportion of the sites into w h i c h the epitope was inserted were surface localized, a negative immunofluorescence result does not necessarily mean that a particular insertion site was not exposed to the cell surface. Both steric hindrance imposed by the presence of other sections of the protein as w e l l as a lack of local flexibility could have resulted i n a negative result for a particular mutant even though the epitope was indeed inserted into a site which faces the cell surface.  63  Chapter 3. Modeling of the OprP monomer  3.1 Introduction The results of both the linker insertion and epitope insertion mutagenesis of O p r P were used to create a topological model of the structure of OprP. Certain data regarding the structure of previously examined porins were used to help create a framework into which these results could be placed. The general amphipathic nature of the fJ-strand regions as well as the actual number of trans-membrane strands exhibited i n the structural models of the other porin proteins was maintained i n the proposed model of OprP. The orientation of the various loop regions of the O p r P model were partially determined by the results of the immunofluorescence experiments w i t h the anti-malarial epitope-specific antibody. After establishing an initial model for the folding pattern of the O p r P monomers, this model was tested by the site-directed insertion of the malarial epitope into the proposed site of a surface-exposed loop as well as by the deletion of sections of another proposed surface-exposed loop.  3.2 Structural implications of linker insertion mutagenesis The analysis of a number of bacterial porins by the method of linker insertion mutagenesis has revealed the fact that the regions of these proteins w h i c h are the most amenable to the insertion of foreign peptides are those w h i c h are located i n the loop regions. These same studies showed that insertions occurring i n the trans-membrane (3strand regions of these proteins served to inhibit expression and/or export to the outer membrane. W i t h these findings i n mind, the amino acid sequence of O p r P has been overlaid w i t h the results of the linker insertion mutagenesis experiments (Figure 9). The insertion sites of mutants whose expression levels were significantly reduced by the presence of the four amino acid insert were assumed to be located i n a trans-membrane  64  (ff)  10  E K D T T  8 F K L Q Q B IL Q IA D IY Q  E L Y A R 2  ®,  ~W~  K  I  I 1  G D T  A T  V T  K W  V T A L E R N  T D G D S V K R  N E S F V  Y  D  F  nsr  J_J  Y  G  G A S D  N  I  N  R T D V A S D E L D  D A G 8 N G N R G L  9® A  L Y  R  Q L G F L ® P H L Y H V N P G G  8 D  R  V 8 T N G G  Y  N  A  N Q  N N  Q M R P R  W  V 8 D  7  N  A Q  V  • (3  ®  A E K 11 (§) V D T R R E R D L L Y K E A S A G Q Y A 8 F A G L A  V V  I N D D E V K  I 8 D  N 3|  A G E L W  G  Gjl2|  D  V S  <g)  R L  W  Y  8 A T R E V G D A K G K T R A L G V N  W  V  Y  T  <D  IE  N  _fj  L E K A T 8  s  Q  IR  D G Y Y  G G T A Y R D  F D T Y F R G F K L N V P N F G T Y T V  G  w  W  Y A A G N 1 E E K N E P K I  N 8 1 K D T K A K V Y N A S V K V A  A  I G  L D G A K F D  Figure 9. Topological model of the monomeric structure of OprP based on linker and epitope insertion. Boxes enclose the proposed transmembrane p-strands. Circles indicate insertion sites PL 1 to P L 13 (number 1 to 13 respectively).  N G D D 8 G D G L IV Ml R L IQ Y IV F  65  p-strand. A l l of the other insertion sites were placed i n either periplasmically-exposed or surface-exposed loops. A topological model of OprP which was based on the p-turn prediction method of Paul and Rosenbusch has previously been proposed (Martin, N.,  unpublished  results). While the model proposed i n the current study differs considerably from this earlier version, the location of certain p-turn regions has been retained. In particular, the regions corresponding to the first surface-exposed loop, the second periplasmicallyexposed loop and the fourth periplasmically-exposed loop i n Figure 9 are also located in loop regions of the previously proposed model (Appendix-Figure 22). In addition, certain sections i n the current model which form the P-strands are also found to compose the P-strands of the previous model. The placement of these strands  was  partially based on the amphipathic nature of the amino acid sequences contained i n those sections of the protein (Appendix-Figure 23).  3.3 Structural implications of epitope insertion mutagenesis The insertion of foreign epitopes into the amino acid sequences of several outer membrane proteins has  successfully been used i n the past to construct three-  dimensional structures of the folding patterns of these proteins. Provided that the mutagenesis does not induce substantial changes i n the folding, this procedure can be used to differentiate between those loops which are exposed to the cell surface and those which may face the periplasmic space. The insertion of the malarial epitope into O p r P resulted i n the identification of two regions of the protein which are likely to be exposed to the cell surface (sites PL10 and PL11 i n Figure 9). These sections of O p r P were flexible enough to tolerate the insertion of the foreign peptide without manifesting profound reductions i n expression. The mutant proteins were also shown to have retained their native conformation  at the cell surface as determined by  indirect  66  immunofluorescence experiments  w i t h the anti-OprP  (trimer-specific) polyclonal  antiserum. Although only two of the linker insertion sites were identified as being exposed to the cell surface, there was reason to believe that epitopes inserted into certain other sites of O p r P were also contained i n surface exposed loops, despite the fact that they were unable to bind to the anti-malarial epitope antibody. The accessibility of these epitopes to the outer surface may have been limited if they were located at the interface between a trans-membrane (3-strand and a surface-exposed loop. The junction between these two structural features w o u l d likely be flexible enough to accommodate the insert without a substantial effect on expression. However, the epitope contained at this site might not be extended far enough from the cell surface to allow for the efficient binding of the anti-malarial epitope-specific antibody. Insertion sites PL2, PL5 and PL9 have all been assigned to surface-exposed loop regions. The  lack of accessibility of these  epitopes to the antibody could be due to the reason stated above. The lack of binding of the antibody to the epitope inserted into site PL5 can not be explain by the proposed location of the epitope w i t h i n the loop. This site has been postulated to occupy a region located i n the center of the third surface-exposed loop. Theoretically, an epitope located at this site should therefore be capable of binding the anti-epitope-specific antibody when expressed i n the outer membrane of whole cells. However the crystal structures of a number of bacterial porins have shown that the third surface-exposed loops of these proteins are folded such that they are at least partially buried w i t h i n the central channel of the native monomers. Epitopes inserted into these loops w o u l d therefore not necessarily be accessible to the outer surface despite the fact that the loops are actually facing the external surface of the cell.  3.4 Testing of the OprP topological model: i) Deletion mutagenesis of the proposed fifth surface-exposed loop  67  In order to test the validity of the proposed topological model of OprP, two sections of the proposed fifth surface-exposed  loop were both individually  and  concomitantly deleted and the mutant proteins were assessed for expression i n the outer membrane and surface exposure i n whole cells. This region was originally placed in a surface-exposed  loop because of the apparent surface-exposure  of a malarial  epitope w h i c h was cloned into this site. The section between insertion sites PL9  and  PL10 (17 amino acids) and the section between insertion site PL10 and the site i n the protein which corresponded to a downstream Ssf II restriction enzyme site i n the  DNA  sequence (16 amino acids) were deleted from the protein. In addition, the entire section of the protein encompassing the region between insertion site PL9 and the Ssf 71 site was also deleted. These three deletion mutants were expressed i n E. coli CE1248 and the outer membranes were purified and analyzed by Western immunoblotting w i t h antiOprP-specific antiserum. As shown i n Figure 10, the mutants which resulted from the individual deletion of the sections of protein sequence which border the PL10 insertion site were expressed at levels comparable to the wild-type protein. Both of these mutants also appeared to possess slightly increased electrophoretic mobilities compared to wild-type OprP. A faint band w h i c h bound the anti-OprP antibodies was shown to be present i n the lane containing the mutant protein which was generated from the deletion of both sections of OprP. The intensity of this band was significantly reduced i n comparison to either the wild-type protein or the other two deletion mutant proteins. The electrophoretic mobility of this protein was also considerably greater than that of wild-type OprP. The surface-exposure of these three mutant proteins was assessed by indirect immunofluorescence w i t h the anti-OprP antiserum. The two mutants (OprP APL9-PL10; O p r P APLIO-Ssf) which d i d not display appreciable decreases i n expression determined by Western immunoblotting were shown to be properly exported  as and  trimerized at the cell surface. However the clone expressing the mutant w i t h the largest  68  A  B  C  D  Figure 10. Whole cell Western immunoblot of E. coli CE1248 expressing the OprP mutants deleted for sections of the proposed fifth surface-exposed loop. Lane A corresponds to the mutant OprP APL9-PL10, lane B corresponds to the mutant OprP APLIO-Ssr and lane C corresponds to the mutant OprP HsPlB-Sstl. Lane D corresponds to wild-type OprP. The samples were heated at 100° C for 10 min prior to loading. Prestained molecular mass markers on the left were (from the top) 106, 80, 49.5, 32.5, 27.5 and 18.5 kDa.  69  deletion (OprP APL9-Ssf) d i d not bind the anti-OprP  specific antibodies to a  significantly higher degree than d i d the cells w h i c h served as the negative control.  3.5 Testing of the OprP topological model: ii) Epitope insertion mutagenesis of the proposed seventh surface-exposed loop A s a further test of the topological model, the malarial epitope was inserted into a site w h i c h has been proposed (based on the linker-insertion mutagenesis) as being located w i t h i n the seventh surface-exposed loop of OprP. The outer membranes from cells expressing this mutant protein were purified and examined b y Western immunoblot analysis w i t h both anti-OprP and anti-malarial specific antibodies. The protein was expressed at levels comparable to the wild-type and the malarial epitope inserted into this site was expressed i n the outer membranes (data not shown). Whole  cells expressing this mutant protein were  subjected to indirect  immunofluorescence w i t h the anti-malarial epitope-specific antibody i n order to determine if this site was accessible to the external surface of the cell. In comparison to cells expressing the wild-type protein, cells expressing the epitope insertion mutant fluoresced strongly when examined by immunofluorescence microscopy (Figure 11). This result indicated that the site at which this epitope had been inserted was contained i n a surface-exposed loop of the OprP monomer.  3.6 Summary The results of the linker and epitope insertion experiments performed o n O p r P were used to generate a topological model of the structure of the native monomer. This model was partially based on the findings of previous studies performed o n the structure of other bacterial porins. The validity of the proposed model was then tested by performing additional mutagenesis of the protein. The results of these experiments were found to be consistent w i t h the proposed structure of OprP (Figure 12).  Figure 11. Indirect immunofluorescence of E. coli strain CE1248 containing the OprPencoding plasmid with the malarial epitope inserted in the proposed seventh surfaceexposed loop. The primary antibody used was the anti-malarial epitope specific antibody p.f. 2A.10. The negative control fluoresced at levels similar to Figure 5B.  71  G  M F  E  S  K  F  E L  E  T  K  Y  A  F  T  R G  S s  F  K  K L  W  G  N  T  A  N N N E  *  T  T  s  D  D  : v  G D  s  S  |q  D  K  G  V  A  S  s  L  Y  T  P  L  F  V  T  Q  A  A  N  E  V  K  s  R  Q  R  Y  F  R  R  R  R  G  N  D  T  Q  s  N  E  G Y  G  Y  D V T  G  T  -  G  :» » G  M  A W  I  R  D  N  E  L  L  D  V  Y  K  D  G  S  N  E  A  E  D  G  A  S  V  A  I K  Q  G  K  K  D  D  A  Y  I S  G  T  S  K  K  G  D  T  A  D  R  K  S  V  W  D  S  N  K  R  A  S  N V  A  N V  D  R  L  I  A  E G W  N  S  A  V K  Y  E P  G  R  E E K N E P K  I  D D  A  W  W  G  N  V  E  T  S L  D  G  A  G  Y  G  K  A  V  N  A  F  A  D  V  L  F  Y  N  A  Y  A  L  K  Y  L  T  G  G  A  Q  G  L  P  Y  G  W  Y  G  E  R  A  G  T  R  A  L  N  T  L  3  G  V  V  V  G  R  F  E  Q I  A  T  S s  _¥_  V  V  A  G G  S  D  G  A  ®  ®  V  S  F  D  F  N  L  I  D  D  :  F  S  D  A  G  N  G  T Y  V  N  G  L  T  L  N  E  K s  D  Y  ^ T  R  N  V  Y  NS s N  : R  T  K  T  M  L  D  w  T  ^ ° N  D  Figure 12. Topological model of the monomeric structure of OprP. The sections of the fifth surface-exposed loop which were deleted are marked with hatched lines. Circles indicate the sites at which the inserted malarial epitopes were shown to be surfaceexposed.  72  B. Functional analysis of O p r P  Chapter 4. Site-directed mutagenesis of specific lysine residues of O p r P  4.1 Introduction It has been previously determined by chemical modification experiments that lysine residues present i n both OprP as well as the anion-selective E. coli p o r i n PhoE play a role i n determining the specific channel characteristics exhibited by these porins. Modifications w h i c h destroyed the positive charges of the lysine residues resulted i n porins w h i c h displayed significantly lower single-channel conductances i n comparison to the wild-type counterparts. In addition, the lysine-specific modification of OprP was shown to cause a loss i n the phosphate specificity of the modified proteins. Since the amino acids w h i c h have been identified i n other porins as being important to the establishment of specific channel characteristics tend to be located i n the aminoterminal half of these proteins, the lysine residues contained i n the amino-terminal half of O p r P were subjected to mutagenesis using a recombinant P C R method. The mutant proteins were purified and analyzed by the planar l i p i d bilayer method i n order to determine if there had been any alteration i n the channel  function. After the  identification of three specific lysine residues which appeared to play a role i n defining the channel characteristics of OprP, a triple mutant i n which all three of these lysine residues were substituted w i t h glutamate residues was created and the mutant protein was purified and analyzed i n the same manner as the single-substitution mutant proteins. In addition to the role that the amino-terminal lysine residues of O p r P were proposed to play i n determining the channel characteristics of this porin, it was proposed that certain lysine residues located i n the carboxy-terminal half of this protein might be important for the export of the newly synthesized protein to the outer  73  membrane of the host cell. The individual or concomitant mutagenesis of two lysine residues w h i c h are contained i n a four amino acid motif of a mitochondrial porin was shown to adversely affect the export of this protein (Smith et al., 1995). This particular motif occurs i n the carboxy-terminal half of a number of different porins. T w o such four amino acid sequences are located i n the carboxy-terminal half of O p r P and thus, it seemed possible that such residues might similarly be involved i n the export of this protein to the outer membrane of host cells. In order to test this hypothesis, the first lysine residue of each of these two sequences was substituted w i t h a glutamate residue by the same procedure used to mutate the amino-terminal  residues. The mutant  proteins were expressed i n E. coli and their presence i n the outer membrane was assessed by Western immunoblotting of outer membranes which had been subjected to a selective solubilization w i t h octyl-POE. Whole cells expressing the substitution mutants were also assessed for the presence of surface-exposed protein by indirect immunofluorescence.  4.2 Site-directed mutagenesis of lysine residues The substitution of lysine residues was accomplished  using a two-step P C R  method as described i n the Materials and Methods section. Mutagenic oligonucleotides w h i c h were homologous to the wild-type oprP sequence save for either one or two base pair substitutions were synthesized and used as primers for the primary amplification step. Each pair of primary P C R products was then purified, combined and amplified by a second round of P C R using primers homologous to sections of the gene which surround the mutation site. The mutagenized fragments were subcloned back into oprP, the religated plasmids were purified and the entire amplified segment was sequenced. A l l of the substitution mutants were found to contain the correct nucleotide substitutions and to be free of any random errors.  74  4.3 Expression of amino-terminal lysine substitution mutant proteins The plasmids encoding the lysine substitution mutant forms of O p r P were used to transform E. coli CE1248 and the presence of the mutant proteins was determined b y Western immunobloting of isolated outer membranes w i t h the anti-OprP-specific antiserum. A l l of the mutants appeared to be expressed at levels equal to that of w i l d type O p r P (Figure 13). The proteins were purified from the outer membrane fractions by selective detergent solubilization followed by elution from SDS-polyacrylamide gels.  4.4 Single-channel conductance of amino-terminal lysine substitution mutant proteins In order to determine whether the substitution of individual lysine residues w i t h glutamates had an effect on the conductance of the channels formed by these mutant forms of OprP, the single-channel conductance i n different concentrations of KC1 was determined for each of the mutant proteins using the planar lipid bilayer method (Table VII). The average conductance of six of the mutant proteins (Lys , Lys , Lys , 13  Lys , L y s 30  109  15  25  and Lys ) was similar to that of wild-type OprP at all tested salt 181  concentrations. In contrast, three of the mutant proteins displayed distinctly altered channel characteristics. In 1 M KC1, the Lys  74  and L y s  126  mutants exhibited levels of  conductance that were approximately one-half of that of the wild-type protein, while the L y s  121  mutant possessed a conductance of approximately one-third that of wild-type  O p r P (Figure 14). The conductance of the channels formed by these three mutants as well as w i l d type O p r P was plotted as a function of increasing salt concentration (Figure 15). A s shown previously, the single-channel conductance of wild-type OprP plateaued as the salt concentration approached 1 M indicating the presence of an anion-binding site. However, both the Lys and the Lys 74  121  mutants formed channels that displayed linear  concentration-conductance relationships at up to 3 M KC1, suggesting the free diffusion of the CI ions through the channel. The L y s -  126  mutant channel conductance, although  75  1  2  3  4  5  6  7  8  9  10 11 12 13 14  Figure 13. Whole cell lysate Western immunoblot of outer membranes of E. coli CE1248 expressing the OprP substitution mutant proteins. The lanes are labelled as follows: I) Lysl3->Glu, 2) Lys -»GIu, 3) Lys ->Glu, 4) Ly 30->Glu, 5) L y s ^ G l u , 15  25  S  6) Lys ->Gly, 7)Lys -»Glu, 8) L y s - K 3 1 u , 9) Lys -»Gly, 10) Lys -»Gln, 74  109  121  121  121  II) L y s - > G l u , 12) L y s ^ G l y , 13) L y s - > G l n , 14) pAS27-wild-type OprP. The samples were heated at 100° C for 10 min prior to loading. Prestained molecular mass markers on the left were (from the top) 106, 80, 49.5, 32.5, 27.5 and 18.5 kDa. 126  1 2 6  126  76  Table VII. Single-channel conductance of OprP Lys->Glu mutant proteins OprP mutation Average single-channel conductance (pS) ' 0.1MKC1 1MKC1 3MKC1 Wild type 103 ± 45 230 ± 42 261 ± 5 1 a  Lys ^Glu 1 3  78 ± 2 5  194 ± 3 3  220 ± 42  Lys -+Glu  74 ± 2 4  207 ± 46  N.D -  Lys ^Glu  88 ± 3 0  210± 51  236 ± 64  Lys ^Glu  93 ± 4 0  206 ± 60  308 ± 60  Lys ^Glu  41 ± 14  116 + 34  363 ± 8 3  Lys  1 0 9  ^Glu  85 ± 14  221 ± 3 4  N.D.  Lys  1 2 1  ^Glu  10 ± 2  74+11  248 ± 36  Lys  1 2 6  ^Glu  25 ± 6  99 ± 17  103 ± 23  79 ± 14  220 ± 22  N.D.  15  2 5  3 0  7 4  Lys ->Glu 181  a. Average of 100 single-channel events expressed in picoSiemens ± S.D. b. N.D.= not determined  b  Figure 14. Single-channel events of wild-type OprP and L y s  —»Glu mutant porin. Chart recorder tracings of the increasing single-channel steps resulting from the incorporation of A) wild-type OprP and B) the L y s -»Glu substitution mutant channels into lipid membranes. The bathing solution was 1 M KC1 (pH 6.0). 121  121  78  Figure 15. KC1 concentration-conductance relationship of selected OprP Lys-+Glu mutants. The single-channel conductance of three of the Lys—•Glu OprP mutants along with wild-type OprP was determined in a number of KC1 concentrations and the values were plotted as a function of the salt concentration. (•-OprP, 0-Lys -*Glu, • Lys ->Glu, •-Lys ->Glu) 74  121  126  79  significantly lower than wild-type OprP, tended to follow the same pattern as the wildtype protein, w i t h the conductance reaching a plateau as the salt concentration approached 1 M.  4.5 A n i o n selectivity of amino-terminal lysine substitution mutant proteins It has been shown previously that the lysine-specific chemical modification of O p r P resulted i n a substantial decrease i n the anion selectivity of the channels (Hancock and Benz, 1986). Unmodified OprP demonstrates a more than 100-fold preference for anions over cations as measured b y the zero-current membrane potential method. The chemically modified forms of OprP were shown to possess levels of anion selectivity that were far below that of the wild-type protein. It was therefore assumed that the modification of the lysine residues was responsible for the decreased anion selectivity of the chemically-treated proteins. In order to determine if any of the nine amino-terminal lysine residues of OprP play a role i n determining the ion selectivity of this protein, the lysine substitution mutants were subjected to analysis using the zero-current membrane potential method. A s shown i n Table VIII, the anion selectivity of the nine substitution mutants varied somewhat from that of the wild-type protein. However, these variations could not  be correlated w i t h  the previously observed  differences i n single-channel  conductance (Table VII). Even the three mutants w h i c h demonstrated distinctly different average single-channel conductances compared to the wild-type protein d i d not demonstrate any substantial decreases i n anion selectivity. In addition, not only were all of these mutants shown to possess a distinct preference for transporting anions over cations, but the selectivity displayed by these mutants was significantly greater than the anion selectivity of OprP which had been subjected to lysine-specific chemical modification (Hancock and Benz, 1986).  Table VIII. Anion selectivity of O p r P Lys->Glu substitution mutant proteins OprP mutation  Zero-current membrane potential (Vmaxf  Wild type  -36.8 ±0.08  Lys ^Glu  - 39.2 ± 0.03  Lys ->Glu  - 43.5 ± 0.05  Lys ^Glu  -41.0±0.17  Lys ^Glu  - 30.8 ± 0.40  Lys ^Glu  -42.0 ±0.10  Lys  ^Glu  -39.7 ± 0 . 5 8  Lys -+Glu  -37.3 ± 0 . 2 5  Lys ->Glu  -36.7 ± 0 . 0 8  1 3  15  2 5  3 0  7 4  1 0 9  121  126  Lvs ->Glu 181  N.D. ' b  a. Maximum potential difference obtained with a 10-fold ion gradient across the membrane ± S.D. (average of three experiments) b. N.D.= not determined  81  4.6 Phosphate specificity of amino-terminal lysine substitution mutant proteins To determine whether any of the Lys—>Glu substitutions had an effect o n the F  i  binding site of OprP, the ability of small concentrations of phosphate ions to inhibit the single-channel conductance of the mutant proteins was measured. The single-channel conductance of each mutant protein i n 0.1 M KC1 was determined prior to the addition of phosphate ions (Table VII). Increasing amounts of potassium phosphate were added to the bathing solutions, and the resultant channel conductances were measured. These data were then used to calculate the percent inhibition and the I concentration of the so  added phosphate ions (Table IX). The majority of the mutant proteins exhibited degrees of conductance inhibition similar to or greater than the wild-type protein, which displayed a 7 4 % decrease i n conductance after the addition of 3.3 m M  potassium phosphate. The L y s  74  mutant  demonstrated a slightly lowered affinity for phosphate ions, w i t h a m a x i m u m inhibition of 5 8 % and an I concentration of 1.95 m M so  type OprP. The Lys  121  compared to 0.96 m M  for wild-  substitution had a profound effect o n the ability of the protein to  b i n d phosphate ions. This mutant exhibited a maximum inhibition of 30%, and while the I for this mutant could not be measured under the conditions used to examine the 50  other mutant proteins, additional experiments revealed that it was above 10 mM. The Lys  126  mutant channel conductance, although greatly reduced compared to the w i l d -  type protein, appeared to be inhibited by the presence of phosphate ions to a similar degree. Figure 16 shows the channel conductances of the Lys , Lys 74  121  and L y s  126  mutant  proteins along w i t h wild-type OprP plotted as a function of increasing phosphate ion concentration and Figure 17 shows the percentage inhibition of these same mutants as a function of phosphate ion concentration.  Table IX. Phosphate inhibition of chloride conductance through the O p r P Lys—>GIu substitution mutant proteins OprP mutation  Phosphate inhibition of chloride conductance Maximum inhibition (%) I50 (mM)  Wild type  74  0.96  Lys ^Glu  89  0.62  Lys ^Glu  87  1.20  Lys ^Glu  79  1.00  Lys -»Glu  81  0.73  Lys ^Glu  58  1.95  Lys  1 0 9  ^Glu  79  0.84  Lys  1 2 1  ^Glu  30  >10  Lys  1 2 6  ^Glu  84  0.81  Lys  1 8 1  ^Glu  80  0.90  1 3  1 5  2 5  30  7 4  83  Figure 16. Phosphate-induced inhibition of chloride conductance. The ability of selected OprP Lys-*Glu mutants to bind phosphate ions was assessed by measuring the inhibition of single-channel conductance (in 0.1 M KC1) induced by the addition of increasing concentrations of potasium phosphate ions. Single-channel conductance was plotted as a function of phosphate ion concentration. (•-OprP, o- Lys —•Glu, •Lys ->Glu, •-Lys ->Glu) 74  121  126  84  100  0 0.00  0.50  1.00  1.50  2.00  2.50  3.00  3.50  Concentration phosphate (mM)  Figure 17. Phosphate-induced inhibition of chloride conductance. The ability of selected OprP Lys Glu mutants to bind phosphate ions was assessed by measuring the inhibition of single-channel conductance (in 0.1 M KC1) induced by the addition of increasing concentrations of phosphate ions.Percent inhibition was plotted as a function of phosphate ion concentration..(»-OprP, o- Lys ->Glu, •-Lys -*Glu,-n-Lys ->Glu) 74  m  1M  85  4.7 Channel characteristics of Lys->Gly, Lys->Gln and Lys To further examine the roles Lys , Lys 74  121  74/121,126  and L y s  126  ->Glu mutant proteins  play i n determining the  electrochemical nature of the channels formed by OprP, these amino acids were again substituted w i t h the neutrally-charged residues G l y or Gin, and the single-channel conductance was determined for each of these mutant proteins (Table X). In addition, a triple mutant w i t h Lys , Lys 74  121  and L y s  126  created and analyzed. Substituting L y s  74  all substituted w i t h glutamates was also w i t h G l y resulted i n a channel w i t h a  conductance comparable to that of the wild-type protein i n 1 M KC1. However, the channel conductance of this mutant i n 0.1 M KC1 was similar to that of the Lys —>Glu 74  mutant protein. The phosphate-induced conductance inhibition of the Lys —>Gly 74  mutant was comparable to that of wild-type OprP. Substituting the Lys  126  residue w i t h either G l y or G i n resulted i n channels that  displayed reduced levels of conductance i n comparison to the wild-type protein. In the case of the G l y substitution, the channel conductance at both 0.1 M and 1 M KC1 was lower than the Lys —>Glu mutant. Substituting Lys 126  121  w i t h either G l y or G i n resulted i n  channels w i t h reduced conductance at both 0.1 M and 1 M KC1. These mutant proteins also formed channels that were as severely impaired i n their ability to bind phosphate ions as the initial Lys ->Glu mutant protein. 121  The  single-channel conductance of the Lys  74,121,126  —>Glu triple mutant was  somewhat lower than any one of the single substitution mutants i n both 0.1 M and 1 M KC1; however, the phosphate-induced inhibition of conductance of this mutant channel was i n the range of the Lys  121  single mutant.  4.8 Expression and membrane localization of carboxy-terminal lysine substitution mutant proteins The expression and presence i n outer membranes of the Lys —>Glu and the 361  Lys —>Glu mutant proteins was assessed by SDS-polyacrylamide gel electrophoresis 385  86  Table X. Channel characteristics of OprP Lys->Gly and Lys->Gln mutant proteins OprP mutation Average single-channel conductance (pS) ' 0.1 M K C 1 1 M KC1 Maximum inhibition '' Wild type 74 % 103 ± 4 5 230 ± 4 2 a  1  Lys ^Glu  41 ± 14  116 ± 3 4  58 %  Lys ^Gly  39 ± 9  205 ± 3 5  72 %  Lys  1 2 1  ^Glu  10 ± 2  74 ± 11  30%  Lys  1 2 1  ^Gly  8 ±2  84 ± 2 6  16 %  Lys  1 2 1  ^Gln  10 ± 3  67 ± 1 8  24%  Lys  1 2 6  ^Glu  25 ± 6  99 ± 17  79%  Lys  1 2 6  ^Gly  13 ± 3  52 ± 8  N.D. -  Lys  1 2 6  ^Gln  13 ± 2  87 ± 2 8  N.D.  47 ± 9  20%  7 4  7 4  L y s  74,12 26^ U  4±1  C  a. Average of 100 single--channel events expressed in picoSiemens ± S.D. b. Maximum conductance inhibition after addition od 3.3 m M potassium phosphate c. N.D.= not determined  87  and Western immunoblot analysis of outer membranes purified from clones expressing these mutant proteins (Figure 18). Both of the substitution mutants were found to be expressed at levels comparable to the wild-type protein. In addition, not only were these proteins absent from the supernatant of outer membranes solubilized i n 0.5% octyl-POE  (a procedure  that should solubilize proteins which were produced as  inclusion bodies) but, based on the results of the immunofluorescence experiments, cells expressing these proteins bound anti-OprP antibodies to the same extent as cells expressing wild-type OprP. These findings indicate that neither of these two lysine residues are important for the biogenesis or export of OprP to the outer membrane of the host cell.  4.9 Summary A recombinant P C R site-directed mutagenesis method was used to individually mutate nine lysine residues contained i n the amino-terminal half of O p r P and two lysine residues located i n the carboxy-terminal half of the protein. The amino-terminal substitution mutants were purified and analyzed for alterations i n channel functions using the planar bilayer method. Three of these amino-terminal lysine residues (Lys , 74  Lys  121  and Lys ) were shown to be involved i n determining 126  certain  channel  characteristics of OprP. One particular residue (Lys ) which was postulated i n Chapter 121  3 to be located i n the third surface-exposed loop (Figure 19) was shown to be involved in maintaining the phosphate-specific nature of the channels formed by OprP. Two carboxy-terminal lysine residues i n OprP w h i c h were suspected of playing a role i n the export of the protein to the outer membrane were substituted w i t h glutamates. These mutant proteins were expressed i n an E. coli background and their association w i t h isolated outer membrane fractions was assessed. The results of these experiments  revealed that the substitution of these two lysine residues had no  appreciable effect on the export of OprP to the outer membrane of host cells.  88  Figure 18. Whole cell Western immunoblot of E. coli CE1248 expressing the OprP substitution mutant proteins Lys -»Giu (A) and L y s - > Glu (B). Lane C corresponds to wild-type OprP. The blots were reacted with the OprP-specific rabbit polyclonal serum. The samples were heated at 100° C for 10 min prior to loading. Prestained molecular mass markers on the left were (from the top) 106, 80, 49.5, 32.5, 27.5 and 18.5 kDa. 361  385  89  R O M  E K D T T  G D T T  V T  G  S F  E  K  L  L G G R L Q A D Y G R F D G Y Y  Y A R R  G G T A Y R D W  G  T  S R N V G  G  G  F L D E T 121 (g) Y A F T R S G S F 126 (K) K W L V N T V A P L N E F R G N T  M  N N  N E S F V  s G S  Y T V  L F A  s .  M  A E D F Y G A S D N  G G  _y_  W  v s s  N D N V G  A Q I T  Q  T D G D S V K R  R P R I R T D V A S D E L D R Y A Y  Y N L R G Q L V F G A L P H L V H V E N P G  V S T N G G N D A G S  A K V T R R L Y E A Q A S F A G L A W A G E  N  G N R G L F G G S  s A V E G L W K D  L  G W D  E R D R E D L K A S G Y Y A Q L A Y T I T G E P R  S L  V V N  D D E V K I S D Y R Y F L E W A G  I  A T R E V G D A K G K T R A L G V N  W Y A N  E E K N E P K I D G A K F D  Figure 19. Topological model of the monomeric structure of OprP. Circles indicate lysine residues shown to play a role in defining the channel characteristics of OprP.  N S  I K D T K A K V Y N A S V K V A  A  N G D D S G D G L V M  R L  Q Y V F  90  DISCUSSION  Previous  studies of the outer membranes of gram negative  bacteria have  revealed the existence of a class of proteins which have been termed porins. These proteins form trans-membrane channels which allow the exchange of small hydrophilic molecules between the external environment and the periplasmic space (Nikaido, 1992). Analysis of the structure and function of various porins has shown that they appear to possess a number of common features which are apparently  necessary for their  particular function. A l l of the porins examined to date traverse the outer membrane i n the form of a p-barrel. While the majority of porins whose structures have been determined are composed of 16 p-strands (Cowan et al., 1992), the monomers of the maltoporin LamB are i n the form of an 18-stranded p-barrel (Schirmer et al., 1995). The loop regions of all of these proteins were found to be the most variable with regard to both the individual residues and the lengths of the loops. However, the external loops were always found to be longer and more variable than the internally-exposed loops. Both the ion-selective and substrate-specific characteristics of several different porins have been determined to be due to the presence of a small number of amino acid residues located primarily i n the amino-terminal halves of these proteins. The  sites  w h i c h these residues occupy appear to be conserved within the proposed structures of the various porins. Combinatorial mutagenesis studies of O m p C and PhoE have shown that the residues involved i n determining the individual ion selective natures of these porins are concentrated i n the amino-terminal halves of the proteins (Benz et a l , 1989). Studies of PhoE have also shown that the substitution of one particular lysine residue located i n the amino-terminal end w i t h a negatively-charged  glutamate residue also  effected the ion selectivity of the channels formed by this protein (Bauer et al., 1989). The results of this study have been used to create a topological model of the O p r P monomer. Those sections of the protein which form loop regions have been  91  differentiated from those that compose the p-strands by means of linker insertion mutagenesis. Three surface-exposed loops have been positively identified by means of epitope insertion mutagenesis followed by indirect immunofluorescence using an antiepitope-specific antibody. In addition, the location of three lysine residues which have been shown to play a role i n maintaining the channel characteristics of O p r P have been identified. T w o of these residues appear to accelerate the rate at w h i c h the anions pass through the channel. The third lysine residue, which has been proposed as being located i n the third surface-exposed loop, appears to take part i n the formation of the anion binding site.  Topological model of O p r P  The proposed topological model of O p r P is similar to those of many other bacterial porins. The individual monomers are composed of a P-barrel w h i c h encloses a central channel. The most recent models of the E. coli general diffusion porins depict these proteins as possessing p-strands with amino acid sequences which alternate between hydrophobic and hydrophilic residues (Cowan et a l , 1992). This predicted amphipathicity is thought to be necessary for the simultaneous exposure of these strands to both the nonpolar outer membrane core and the internal lumen of the channel. The side chains of the amino acids which project into the aqueous channels w o u l d likely display a hydrophilic character. However, those residues exposed to the fatty acyl chains of the external lipid membrane w o u l d need to be hydrophobic. Interactions of the external surface of these proteins w i t h the surrounding l i p i d bilayer have been proposed to be facilitated by a band of hydrophobic residues which encircles the trimer. The boundaries of these bands are largely composed of phenylalanine and tyrosine residues (Cowan et a l , 1992)  92  The  general amphipathic nature of the P-strands displayed b y other porin  models has been maintained i n the model of OprP. The number of individual strands which form the p-barrel has been set at 16. This is i n keeping w i t h the number of pstrands found i n most porins, but it is less than the number of strands proposed to exist i n the structure of LamB (Schirmer et a l , 1995). The length of the amino acid sequences of both LamB and OprP exceed those of most other porins by at least 20%. It might therefore seem reasonable to assume that since the p-barrels of LamB contain 18 strands, so too must the p-barrels of OprP. The results of this study show that this is not likely to be the case. The insertion sites of the OprP linker mutant proteins PL3, PL8, PL12 and PL13 were all placed within p-strands. Incorporation of the four amino acid residue insert at these sites resulted i n a substantial decrease i n the amount of protein expressed i n the outer membranes and to a loss of detectable surface-exposed protein i n whole cells. The placement of the p-strands i n certain topological models of LamB and PhoE was partially based on the effect that the insertion of foreign sequences into these sites had on the expression of the mutant proteins (Charbit et a l , 1986; Agterberg et al., 1987). Those regions of these proteins where the insertion of the foreign sequence resulted i n a significant decrease i n protein expression were placed i n the p-strands. Subsequent models based on X-ray crystallography revealed that the assignment of these sites to the domain of the p-strands accurately reflected their true location i n the structure of these proteins (Schirmer et al., 1995; C o w a n et al., 1992). Although the expression levels of the mutant forms of OprP w i t h the foreign insert located i n proposed p-strand regions were reduced i n comparison to the w i l d type protein, all of these proteins were expressed to some extent. The residual levels of expression exhibited by these proteins could be due to the ability of the inserted sequences to maintain the amphipathic nature of the P-strand, allowing the displaced part of the strand to be forced into the adjacent loop. This could explain why,  although  93  all four mutant proteins exhibited reduced levels of expression, the inserts at sites PL12 and PL13, w h i c h consisted of the amphipathic sequence D L Q V , inhibited the expression of the mutant proteins to a lesser extent than the inserts at sites PL3 and PL8 which consisted of the nonpolar sequence PAGP. The  OprP  insertion sites PL1, PL4, PL6  and PL7 were  a l l placed i n  periplasmically-exposed loops. None of these mutant proteins displayed significant decreases i n protein expression after incorporation of the 12 base pair linker into the gene encoding OprP. However, the mutant forms of OprP resulting from the insertion of the malarial epitope into the linker sites PL6 and PL7 manifested profound decreases i n expression. These two insertion sites are located at the beginning of a stretch of 23 uncharged amino acid residues. N o such large tract of uncharged residues has ever been identified i n the structure of any other porin (Siehnel et al., 1990). The insertion of the 14 amino acid malarial epitope at these sites may have caused a disruption i n this area w h i c h resulted i n a destabilization of the folded structure and a reduction i n the amount of the mutant protein that was be able to be transported to and inserted into the outer membrane. The mutant proteins which contained inserts at sites PL1 and PL4 appeared to tolerate the inclusion of the malarial epitope as evidenced by their ability to be exported and expressed i n the outer membrane of whole cells. The use of the trimerspecific anti-OprP antiserum ensured that all the mutant proteins detected by this assay were indeed exposed at the cell surface i n the form of the native trimer. None of the mutant proteins w i t h the malarial epitope inserted into proposed periplasmicallyexposed loops were shown to display the epitope at surface-accessible regions. The proposed periplasmic loops of OprP tend to be longer than those of the other previously examined porins. The sixth periplasmic loop i n particular is more than twice the size of the largest such loops i n the E.coli porins (Cowan et al., 1992). Placement of this section of OprP i n the periplasmic space was dictated by two sets of results; i) sites surrounding  the proposed loop were nonpermissive for the insertion of foreign  94  sequences (indicating the presence of p-strands), ii) two loops surrounding this region were positively identified as being exposed at the cell surface. This second set of results fixes the position of this region of OprP; it w o u l d be difficult for the protein to cross the membrane two more times within such a short stretch of sequence. Since OprP is thought to interact with the periplasmic phosphate-binding  protein i n P. aeruginosa  (Poole and Hancock, 1984), these longer periplasmic loops may be necessary to facilitate that interaction. The long periplasmic loops i n the proposed topological models of the siderophore-iron receptors F h u A (Koebnik and Braun, 1993) and F o x A (Baumler and Hantke, 1992) may be similarly involved i n the proposed interactions between these proteins and cytoplasmic membrane associated proteins such as TonB i n other gramnegative bacteria. The linker insertion sites PL2, PL5 and PL9 were all placed i n surface-exposed loops despite the fact that none of the epitopes inserted into these sites were shown to be exposed at the cell surface. A l l three of these sites tolerated the insertion of the linker and the mutant proteins were able to incorporate the malarial epitope without suffering any major decreases i n expression. These mutant proteins were also shown to be properly exported to the cell surface. The lack of surface exposure of the malarial epitopes inserted at the PL2 and PL9 sites can be explained if these sites are presumed to occupy sites which are at the junction between loop and P-strand regions. Epitopes inserted into such sites might not be efficiently exposed at the cell surface and w o u l d therefore not be accessible to the epitope-specific antibody. Struyve et al. (1993) similarly speculated that the reason that an epitope inserted into the proposed second surface-exposed loop of PhoE (Agterberg et al. 1990) was not shown to bind antibodies at the cell surface was because it was located at the interface between a loop and an adjacent P-strand. Insertion site P L 5 was localized to the third surface-exposed  loop of the  proposed model of OprP. The analogous loops i n the Rhodobacter capsulatus porin  95  (Weiss et al., 1990)  and i n PhoE, O m p F and O m p C (Cowan et a l , 1992) have all been  shown to fold back into the channels and constrict their internal diameters. The surfaceexposure of an epitope inserted into the third loop of PhoE was not achieved until three copies of the epitope h a d been inserted (Struyve et al., 1993). These experiments suggested that the third loops of bacterial porins are somewhat shielded from the exterior: surf ace of the cell. The lack of surface-exposure  displayed b y the epitope  inserted into the proposed third loop of OprP can be similarly explained by the fact that this loop folds back d o w n into the central channel. A n epitope inserted into this site w o u l d be partially shielded from the external surface and w o u l d thus be unable to bind to the antibody. If the third loop of OprP is folded back into the channel, it w o u l d likely exist i n an environment which is different from that of the other surface-exposed loops. The location of this loop w o u l d allow for the presence i n its sequence of hydrophobic residues w h i c h could interact w i t h other hydrophobic residues protruding from the sides of the barrel wall. The sequence of the proposed third loop of OprP contains a higher amount of aromatic residues than any of the other predicted loops i n this model. These residues are located predominantly o n one side of the loop, w h i c h presumably w o u l d face the interior wall. The other side of the loop contains a number of residues w i t h polar or charged side chains. These residues, which w o u l d be expected to face the central opening of the pore, might play a role i n defining the channel characteristics exhibited b y this particular porin. T w o lysine residues contained i n this region of the third loop (Lys  121  and Lys ) have been shown to be involved i n the conductance of 126  anions through the channels formed by OprP. While the sequences of the well characterized E. coli porins have been shown to lack any long stretches of uncharged residues (Mizuno et al., 1983), there exists i n the O p r P sequence a stretch of 24 consecutive uncharged amino acid residues, as w e l l as three other tracts of 13 or more uncharged residues (Siehnel et al., 1990). The largest  96  region of uncharged amino acids has been proposed as lining the w a l l of one side of the channels formed by OprP. The section of the channel which this uncharged stretch of amino acids is thought to construct might interact w i t h that section of the third loop w h i c h contains a high proportion of aromatic residues. The presence of uncharged residues at this site might provide a means by which to stabilize the conformation of the loop which takes part i n forming the constriction zone. A s noted above, the insertion of the malarial epitope into two sites which were located at the beginning of this sequence of residues resulted i n a loss of detectable protein at the surface of whole cells. Three sites i n OprP have been positively identified as being located i n surfaceexposed regions. Epitopes presented at insertion sites PL10 and PL11 were shown to be capable of binding the anti-epitope specific antibody at the cell surface. These same epitope-containing mutant proteins were also shown to bind the trimer-specific antiO p r P antibodies, suggesting that the proteins d i d not suffer any gross alterations i n their conformations as a result of the insertion of the foreign epitope. The fact that these proteins were capable of binding to both the anti-OprP antibodies and the anti-epitopespecific antibodies on the surface of whole cells suggests that the insertion sites of these mutant proteins are located i n regions that are exposed to the cell surface i n the w i l d type protein. In addition to the PL10 and PL11 sites, another site i n OprP was identified as being located i n a surface-exposed region by means of site-directed epitope insertion mutagenesis. Based on the initial linker and epitope insertion mutagenesis experiments a preliminary topological model of OprP was proposed. To test the validity of this model, a copy of the malarial epitope was inserted into a site i n O p r P w h i c h was proposed to be located i n the seventh surface-exposed loop. Analysis of whole cells expressing this mutant protein by indirect immunofluorescence revealed that the epitope was indeed exposed at the cell surface.  97  The proposed topological model of OprP was further tested b y means of sitedirected deletion mutagenesis. Two sections of OprP which were proposed to be located i n the fifth surface-exposed loop were deleted and the mutant proteins were analyzed for changes i n expression. The section between linker insertion sites PL9 and PL10 as well as the section between insertion site PL10 and a site w h i c h corresponded to a downstream Sstll site i n the gene sequence were individually deleted and the deletion mutants were analyzed, by the electrophoresis of isolated outer membranes through SDS-polyacrylamide  gels. Both of the mutant proteins which contained deletions of  sections of the fifth loop were found to be expressed at levels comparable to the wildtype protein. I n addition, these mutant proteins were also shown to be properly localized to the cell surface. The results of the deletion mutation experiments not only served to determine the extent of the fifth surface-exposed loop, they also provided further evidence for the placement of the insertion site PL9 at the interface between a loop and a p-strand. The lengths of the surface-exposed loops i n the proposed model of O p r P tend to be longer than the external loops of the other well characterized porins. The proposed third surface-exposed loop is approximately the same size as those of the general diffusion porins. Deletion analysis of the fifth surface-exposed loop has revealed that this region is composed of at least 34 amino acid residues. This is as large as the third surface-exposed loop of the classical E. coli porins OmpF, O m p C and PhoE, w h i c h is the largest loop found i n the structures of these proteins. In addition to the fifth loop, the topological model of OprP also possesses w i t h i n its structure two other carboxyterminal loop regions (loop 6 and loop 7) which contain 26 and 27 amino acid residues, respectively. The abundance of such large loop regions may help to explain the small exclusion limit w h i c h is characteristic of wild-type OprP. These large loops might form a barrier over the external mouth of the channel which w o u l d restrain the passage of those substrates which were incapable of negotiating the narrow entrance of the pore.  98  The existence of such large loop regions also helps to explain w h y O p r P apparently contains only 16 p-strands despite the fact that the primary sequence of this protein is composed of approximately 2 0 % more amino acid residues than the sequences of other porins w h i c h are predicted to contain similar numbers of P-strand structures. A l t h o u g h the D N A sequences which control the phosphate-starvation inducible regulon i n both E. coli and P. aeruginosa share a certain degree of homology, the porins which these organisms express i n response to such conditions exhibit very little sequence similarity. A comparison of the nucleotide sequences of the genes encoding O p r P and PhoE yielded a total alignment of 53-60% with consistently l o w alignment scores (Siehnel et al., 1990). A similar comparison of the sequences of PhoE and LamB yielded comparable results. These findings might lead to the assumption that since the sequence of OprP does not resemble that of the classical E. coli porins, it must therefore retain a different conformation i n its native form from that of the other porins. However, the crystal structure of LamB shows that this protein assumes a similar conformation to that of the other previously crystallized porins, despite the fact that the sequence of LamB shares little homology w i t h those proteins (Schirmer et a l , 1995). Accordingly, it is possible that the structure of OprP resembles that of the other characterized porins despite the apparent lack of sequence homology. The lack of homology between OprP and PhoE is particularly surprising i n light of the fact that the trimeric forms of these two proteins cross-react w i t h an anti-OprP trimer-specific antiserum (Poole and Hancock, 1986). These findings suggest that these two proteins share one or more common conformation epitopes. Thus, despite the lack of homology between the amino acid sequences of these two porins, their common function i n the outer membranes of their respective hosts appears to dictate the assumption of similar folding patterns. In addition, a lysine residue which has been implicated as playing a role i n the maintenance of the anion-selective nature of PhoE (Bauer et al., 1989) was shown to occupy a position similar to that of a lysine residue i n  99  O p r P w h i c h has been shown to be involved i n forming the anion binding site of this porin. A gene located upstream of the oprP gene on the P. aeruginosa chromosome was recently cloned and expressed i n E. coli (Hancock et al., 1992). This gene was found to encode a polyphosphate-selective porin which was designated OprO. This porin, w h i c h cross-reacted w i t h OprP-specific antiserum, was shown to be highly homologous to OprP, w i t h 7 6 % identity and 1 6 % conserved substitutions (Siehnel et al., 1992). A comparison of the amino acid sequence of O p r O with the proposed topological model of O p r P reveals that 29/39 of the non-conservative substitutions and two of the three one amino acid gaps are located i n the loop regions. This w o u l d seem appropriate, since although the variability between closely related outer membrane proteins is usually concentrated  i n the loop regions, a certain amount of variation i n the amino acids  residues which line the interior of the channels formed w i t h i n these two proteins w o u l d be required to account for their differences i n substrate-specificity.  The role of specific lysine residues i n defining the channel characteristics of O p r P  It has previously been demonstrated that the channel characteristics of several general diffusion porins are dependent on the presence of one or more amino acids located i n their amino-terminal domains (Heine et a l , 1988; Benson et a l , 1988, Benz et al., 1989). In this study we have identified three amino-terminal lysine residues i n OprP w h i c h play a role i n defining the channel characteristics of this porin. A model of the structure of O p r P i n which the three monomers each contribute one lysine residue to form a single central binding site has previously been proposed. While the results of the current study do show that three different lysine residues of O p r P appear to be involved i n determining  the channel characteristics, it is now believed that the  monomers of this protein form three separate channels instead of a single central pore.  100 The substitution of Lys  121  w i t h a glutamate residue yielded a protein w h i c h  displayed channel conductances which were significantly smaller than those exhibited by wild-type OprP. These mutant forms of OprP were shown to conduct anions at a rate approximately 3-fold lower than that of the wild-type protein i n 1 M substitutions of L y s  74  and L y s  126  KC1. Similar  produced mutant proteins which displayed 2-fold and  2.5-fold decreases i n conductance, respectively. While the changes induced by these substitutions are profound, they do not approach the 10-fold reduction i n conductance exhibited by chemically-modified forms of OprP (Hancock and Benz, 1986). Even a triple mutant i n which all three of these lysine residues had been substituted w i t h glutamates displayed only a 5-fold decrease i n conductance. These findings suggest that there may  be certain lysine residues contained i n the carboxy-terminal end of O p r P  w h i c h also play a role i n determining the channel conductance. Alternately, the severe reduction i n conductance displayed by the chemically-modified forms of O p r P  may  have been due to factors unrelated to the actual loss of the positive charges of the modified lysine residues (eg. the presence of the modifying groups w i t h i n and/or around the channel). Lysine-specific acetylation of OprP was  shown to produce channels  with  conductances that were no longer saturated at high anion concentrations (Hancock and Benz, 1986). This result was proposed to be due to a modification of residues w h i c h are involved i n forming the anion-binding site. Of the 11 Lys—> G l u mutant forms of O p r P created during the course of this study, only the L y s  74  and L y s  121  mutants exhibited  losses i n the ability to saturate at KC1 concentrations above 1 M. The Lys —>Glu mutant 126  displayed saturation kinetics similar to that of wild-type OprP, despite the fact that the conductance of these channels was as severely affected at low salt concentrations as that of the L y s  74  and L y s  121  mutant porins. The conductance patterns of the other eight Lys—>  G l u mutants d i d not differ significantly from that of the wild-type protein. Apparently only the L y s and L y s 74  121  substitutions had a detrimental effect on the anion-binding site.  101  The  phosphate-induced inhibition of channel conductance of the Lys —>Glu 74  mutant was approximately 2-fold lower than that of the wild-type protein. Substituting this lysine residue w i t h a glycine resulted i n a protein w i t h a phosphate-induced inhibition of conductance which was similar to that of the wild-type protein. This result can be explained, if L y s  74  is assumed to occupy a space proximate to the Pi-binding site.  The positive charge of this residue w o u l d not be required for the formation of the binding site, however the placement of a negatively-charged residue at this location may have indirectly affected the interaction of phosphate (and chloride) ions w i t h the binding site. According to the topological model of OprP, L y s  74  is located at the top of  the fourth p-strand and w o u l d presumably face the interior of the channel. Substitution of Lys  121  w i t h glutamate, glycine or glutamine residues resulted i n  proteins w i t h channel conductances which were severely impaired i n their abilities to be inhibited b y the presence of phosphate ions. This particular residue is located i n the third surface-exposed loop according to the topological model. The placement of this residue i n the third loop is i n agreement w i t h this loop's role i n constricting the interior of the channels formed by several bacterial porins (Weiss et al., 1990; C o w a n et al., 1992; Schirmer et al., 1995). The equivalent lysine residue i n PhoE (Lys ) w h i c h has been 125  established as being responsible for determining the anion selectivity of this porin (Bauer et a l , 1989) was also shown to be located i n the third surface-exposed Substituting L y s  126  loop.  w i t h glutamate i n OprP had no apparent effect o n the Pi-binding site,  despite the fact that this residue is also predicted to be located i n the third surfaceexposed loop. The mutagenesis of Lys  361  and Lys  385  d i d not yield any useful information o n the  role of these residues i n OprP. The substitution of these amino acids w i t h glutamate residues d i d not appear to. affect the ability of the mutant proteins to be properly transported to and inserted into the outer membrane of the host cells. This is i n contrast w i t h the results obtained after the analogous mutagenesis of a mitchondrial porin.  102 Smith et al. (1995) showed that the independent substitution of two lysine residues located i n the carboxy-terminal half of a mitochondrial porin w i t h glutamates affected the ability of the mutant proteins to insert into membranes. These two residues were found to be present i n a sequence of the mitochondrial porin w h i c h appears to be conserved w i t h i n a small number of similar porins. The presence of two such lysine containing motifs i n the carboxy-terminal end of OprP suggested that they might play a similar role i n the biogenesis of this protein. While the results of this study suggest that these lysine residues do not serve such a purpose, it should be noted that the method applied i n the analysis of the mutant mitochondrial proteins involved a cell-free transcription/translation system. A n analysis of the effects of i n d i v i d u a l amino acid substitutions on membrane insertion using this procedure w o u l d be significantly more sensitive than one performed using the method employed i n the current study of OprP. It may therefore be possible that the mutation of OprP at these sites had an effect on the ability of the mutant proteins to insert into membranes w h i c h was not detected. Analysis of the anion selectivity of the amino-terminal Lys—>Glu substitution mutants using the zero-current membrane potential assay proved to be inconclusive. While there were slight variations i n the potential differences elicited by these mutants, these differences could not be  correlated with the observed changes i n anion  conductance. These findings conflict with the facts that; i) the destruction by chemical modification of the positive charges of accessible lysine residues of O p r P substantially decreased the anion selectivity of the channel (Hancock and  Benz, 1986), ii) the  substitution of a single lysine residue located i n the third surface-exposed loop of PhoE w i t h glutamate changed this porin from an anion-selective to a cation-selective channel (Bauer et al., 1989). It may be that the anion selectivity of OprP, which is much greater than that of PhoE, is due to the presence of several positively-charged residues, and thus w o u l d not be as adversely affected by the mutation of only one of these amino acids.  103  The phosphate-starvation inducible E. coli porin PhoE has not been shown to possess a substrate-specific binding site (Bauer et al., 1988), although it does maintain a slight preference for the transport of anions over cations. Lysine-specific chemical modifications of PhoE were shown to destroy the anion selectivity of the channels (Bauer et al., 1989). In addition, the replacement of lysine residues contained i n the amino-terminal half of this porin w i t h glutamates was also found to affect the ionselectivity of the channels formed. Four lysine residues were individually replaced and the mutant proteins were analyzed both in vivo and in vitro for changes i n channel function. Three of the substitution mutant proteins exhibited minor alterations i n anion selectivity as measured using the zero-current membrane potential assay. The fourth substitution mutant form of PhoE displayed a reversal i n ion selectivity, changing from an anion selective channel to a cation selective channel. The residue substituted i n this particular mutant protein (Lys ) is located i n the third surface-exposed loop of the 125  crystal structure of PhoE. This w o u l d be the appropriate position for a residue involved i n determining the i o n preference of a porin, owing to the fact that this loop region folds back d o w n and serves to constrict the internal diameter of the channel. A l l of the substitution mutant forms of PhoE were shown to retain single-channel conductances which were comparable to those of the wild-type protein. A recently published model of the maltose-specific porin LamB has provided an interesting rational for the substrate-specific behavior of this particular porin (Schirmer et al., 1995). In the crystal structure of this protein, a series of six aromatic residues (predominantly tyrosines and tryptophans) are arranged i n a helical pathway which extends from the external mouth of the channel through the constriction zone and out to the periplasmic side. These residues are thought to participation i n stacking interactions w i t h incoming sugar molecules. The aromatic residues at the cell surface w o u l d serve to both attract the maltose molecules to the entrance of the channel and align these molecules w i t h the aromatic pathway. The sugars w o u l d then be guided  104  through the channel by means of a series of interactions w i t h the hydrophobic faces of the aromatic side chains. The substrate specificity of this porin may therefore rely on the presence of a diffuse binding site which extends over almost the entire length of the channel. The configuration of this binding site w o u l d likely differ somewhat from that of the proposed binding site of OprP, since the structure of the phosphate ion is quite different from that of maltose. The E. coli porins O m p F and PhoE have both been determined to form nonspecific general diffusion channels (Nikaido and Rosenberg, 1983). However, a recent study of the electrostatic properties of these two porins has proposed a mechanism of transport which appears to resemble a form of facilitated diffusion (Karshikoff et al., 1994). A strong transverse electrostatic field which winds through the channels of these proteins was identified. The electrochemical nature of this field w o u l d be determined b y the nature of the individual residues involved i n creating it. Differences i n the ion selectivities displayed by these two porins are thought to be due to the differences i n the amino acid side chains of those residues w h i c h create the electrostatic fields of each of these two proteins. While a complete assessment of all 23 lysine residues contained i n O p r P has yet to be performed, the current analysis of amino-terminal residues has yielded some important information regarding the nature of the channels formed by this protein. The roles of L y s  74  and L y s  126  i n OprP appear to be to form an electrostatic funnel which  serves to focus the flow of anions toward the binding site. The Lys  121  residue seems to  render a more critical function i n this porin. Not only does the presence of this residue serve to increase the flow of anions through the channel, but it appears that this particular amino acid is required for the formation of the anion/Pi-binding site. Whether this is the only residue involved i n maintaining the P i specificity of OprP is still unknown.  105  Summary  A topological model of the P. aeruginosa porin O p r P has been proposed based on linker and epitope insertion mutagenesis (Figure 20). This model is consistent w i t h the current crystal structures of several porins isolated from the outer membranes of other gram negative bacteria. The p-barrel is proposed as being comprised of 16 strands, and the longer more variable loops of the barrel were placed at the external surface. Three of these loops were identified by means of epitope insertion. The third surface-exposed loop was shown to contain two lysine residues (Lys , Lys ) w h i c h were important for 121  126  the transport of anions through the channels formed by this protein. One of these residues (Lys ) was implicated as taking part i n the formation of the anion binding site. 121  A third lysine w h i c h also appears to affect the ability of the channels to conduct anions has been localized to the top of the fourth p-strand.  106  (g)  1D F • T F R F  K L G G  \" ^ \ R P  ©  G  F  ^ M  E © A T S  Y  s  L  N  A  D  R  V  T  A  E  I  R  T  G  0  N  E  N  D  S  L  D  V  G  N  K  0  G  N  G  N  T  E  •  A  E  0  G  L  G  V  A  S  T  P  L  F  A  A  N  E  V  R  V  F  R  R  G  0  T  G  S V K  ,  Y  A  T  T  V T  G  T  W| V  V  —ft  s  D  7 ®  D  D  G  Y  1  G  T  S  K  K  G  T  A  D  R  K  G  L H  L H  A  N  A  N  S  V  Q  V  D  0  V  G  R  Y  L A  A  L  A  E  I  E  G L  G 12  w V  P G D  G  E  L  W  1 a  T  G  A  w  A G  © R  Y  G  I  A  N  U| R  S  W  V  Y  L  IQ  K  A N  E  T~ IV  N  V  ©  D  V  L F  W V  G  D  A  •  ©  s  Y F  F G  L  P  R  a  K  K  5 5  V  A| •  3  A  K  A  Y G  M  I  •  V  G  R  V  S G  G>_  N  A  N  L 9J  N  R  R —T  N  1  D  S V  N  N  G  © D  V  Q  V  11  L  ®  1  K  N  L  G  E  A  v G G  K  R  I  s \ \ \ s  ® W  V  •  G  V  Y IV  A E  K  S  N E P K I  I G  L •  G A  K F  D  Figure 20. Topological model of the monomeric structure of OprP. Boxes enclose the proposed transmembrane P-strands. Red circles indicate the sites at which the inserted malarial epitopes were shown to be surface-exposed. Green circles indicate lysine residues shown to play a role in defining the channel characteristics of OprP. A l l other circles indicate the linker insertion sites P L 1 to PL 13 (number 1 to 13 respectively). The sections of the fifth surface-exposed loop which were deleted are marked with hatched lines.  F  107  REFERENCES  Agterberg, M . H., Adriaanse, and J. Tommassen. 1987. Use of outer membrane protein PhoE as a carrier for the transport of a foreign antigenic determinant to the cell surface of Escherichia coli K-12. Gene. 59: 145-150. Angus, B. L . and R. E. W. Hancock. 1988. Outer membrane proteins F, P and D I of Pseudomonas aeruginosa and PhoE of Escherichia coli: chemical cross-linking to reveal native oligomers. J. Bacteriol. 155: 1042-1051. Bauer, K., R. Benz, J. 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Thick arrows represent transmembrane p-strands; L, surface loops; T, periplasmic turns; N, N-terminus; C, C-terminus. Reproduced with permission from Cowan et al. 1992.  118  Figure 22. Conceptual model for the structure of OprP based upon the p-turn prediction method of Paul and Rosenbusch (1985). (Nancy L. Martin, Ph.D. thesis)  119  Figure 23. Hydropathy plots (Kyte and Doolittle, 1982) for OprP. Areas above the dotted line correspond to hydrophobic regions of the protein while those areas below the dotted line indicate hydrophilic regions (Nancy L. Martin, Ph.D thesis).  

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