A 2H-NMR STUDY OF L I P I D CHAIN DISORDER IN A MODEL MEMBRANE: EFFECT OF INTEGRAL PEPTIDE LENGTH by ROBIN MICHAEL MACQUEEN A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS.FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of P h y s i c s We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA October 15, 1986 © R o b i n M i c h a e l Macqueen, 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e - q u i r e m e n t s f o r an advanced degree a t the The U n i v e r s i t y of B r i t i s h C o lumbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head of my Depart- ment or by h i s or her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department of P h y s i c s The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date: October 15, 1986 A b s t r a c t P o l y p e p t i d e s of the form: L y s 2 - G l y - L e u ^ - L y s 2 - A l a - a m i d e w i t h «=16, 20 or 24, and of the form: A c - L e u , 0 ~ L y s 2 - A l a - a m i d e , were mixed w i t h p e r d e u t e r a t e d p o t a s s i u m p a l m i t a t e a t p e p t i d e / l i p i d r a t i o s of a p p r o x i m a t e l y 1:200 and 1:100. Po- t a s s i u m phosphate b u f f e r (50mM) was added such t h a t the w a t e r : l i p i d molar r a t i o was 7.75:1. 2H-NMR measurements were made on the samples a t 49°C and 65°C. The e f f e c t of a l l pe- p t i d e s was t o cause an i n c r e a s e i n l i p i d c h a i n o r d e r which v a r i e d l i n e a r l y w i t h c o n c e n t r a t i o n . T h i s i n c r e a s e was mani- f e s t e d by an i n c r e a s e i n average o r d e r parameter f o r the en- t i r e l i p i d c h a i n of about 20%, from about .11 t o .14. i i T a b l e of Co n t e n t s 1 . Membranes 1 1.1 A m p h i p h i l e s and s e l f - a s s e m b l y 1 1.2 Membrane p r o t e i n s 8 2. 2H-NMR Theory 12 2.1 H a m i l t o n i a n of a s t a t i c 2H n u c l e u s i n a magnet- i c f i e l d 12 2.2 The q u a d r u p o l a r echo and F o u r i e r t r a n s f o r m spe- c t r o s c o p y 16 2.3 M o t i o n a l a v e r a g i n g and the o r d e r parameter 17 2.4 The powder p a t t e r n and depaking 18 3. 2H-NMR of Membranes and Model Membranes 20 3.1 Why 2H-NMR i s u s e f u l f o r the study of membranes and model membranes 20 3.2 R e l a t i o n of b i l a y e r t h i c k n e s s t o S C D 22 3.3 L i p i d - p r o t e i n i n t e r a c t i o n s examined by 2H-NMR ...25 3.4 The m a t t r e s s model 27 3.5 P r e v i o u s work a d d r e s s i n g the m a t t r e s s model 31 4. O b j e c t i v e s of T h i s Work 38 5. E x p e r i m e n t a l P r o c e d u r e s 41 5.1 M a t e r i a l s 41 5.2 Sample p r e p a r a t i o n 42 5.3 D i f f e r e n c e s from the study of D a v i s et al ( 1 983) 46 5.4 2H-NMR s p e c t r o s c o p y 47 6. R e s u l t s 49 7. D i s c u s s i o n 69 REFERENCES 7 4 i i i L i s t of T a b l e s I . Samples p r e p a r e d 45 I I . Sample water c o n t e n t , <-J>0> and |<S C D>| 68 i v L i s t of F i g u r e s 1. Dynamic p a c k i n g p r o p e r t i e s of l i p i d s and the s t r u - c t u r e s they form. (From I s r a e l a c h v i l i et al , 1980.)..6 2. Phase diagram of the p o t a s s i u m p a l m i t a t e - w a t e r system. (From Bloom et al , 1976.) 7 3. A d e u t e r i u m powder p a t t e r n , f o r the case of a x i a l symmetry ( T?=0 ) 19 4. 2H-NMR s p e c t r a of m i x t u r e s of p e r d e u t e r a t e d p o t a s - sium p a l m i t a t e and the p e p t i d e D1 i n 30% water by we i g h t . (From D a v i s et al , 1983) 36 5. Phase diagrams of two p e p t i d e - l i p i d systems. (From H u s c h i l t et al , 1 985) 37 6. 65°C s p e c t r a : B000, D000 53 7. 65°C s p e c t r a : B000, 242, 241 54 8. 65°C s p e c t r a : B000, 202, 201 55 9. 65°C s p e c t r a : B000, 162, 161 56 10. 65°C s p e c t r a : B000, Z102, 101 57 11. 65°C s p e c t r a : (a) B000; (b) B000, depaked 58 12. Lv0 v s . s i t e number a t 65°C: B000, D000 59 13. Av0 v s . s i t e number a t 65°C: B000, 242, 241 60 14. Ai>0 v s . s i t e number a t 65°C: B000, 202, 201 61 15. Av0 v s . s i t e number a t 65°C: B000, 162, 161 62 16. Av0 v s . s i t e number a t 65°C: B000, Z102, 101 63 17. |<S C D>| f o r a l l samples a t 65°C 64 18. B000 s p l i t t i n g s : 49°C, 65°C 65 19. 242 s p l i t t i n g s : 49°C, 65°C 66 20. 241 s p l i t t i n g s : 49°C, 65°C 67 v Acknowledgements I would l i k e t o thank my s u p e r v i s o r , Myer Bloom, f o r s u g g e s t i n g the e x p e r i m e n t s , p r o v i d i n g a d v i c e a t c r i t i c a l p o i n t s i n the s t u d y , making comments on b i t s and p i e c e s of my t h e s i s as i t took shape, and p r o v i d i n g f i n a n c i a l s u p port throughout the work. A l e x MacKay p r o v i d e d much u s e f u l a d v i c e d u r i n g the work and c r i t i c a l l y r e a d the t h e s i s . E l l i o t t Bur- n e l l p r o v i d e d a d v i c e i n the p l a n n i n g s t a g e s of the study and c a r e f u l l y read the t h e s i s . Ed S t e r n i n was c r i t i c a l , i n a c o n s t r u c t i v e way, of my e x p e r i m e n t a l p r o c e d u r e s , and p r o - v i d e d h e l p w i t h the d e t a i l s of s p e c t r o s c o p y and computing. J i m D e l i k a t n y p r o v i d e d a d v i c e on p r a c t i c a l a s p e c t s of work- i n g w i t h p o t a s s i u m p a l m i t a t e , and commiserated w i t h me when t h i n g s were g o i n g b a d l y . Frank N e z i l p r e p a r e d the perdeu- t e r a t e d p o t a s s i u m p a l m i t a t e . J u l i a W a l l a c e h e l p e d me w i t h v a r i o u s a s p e c t s of s p e c t r o s c o p y , d a t a a n a l y s i s and t h e s i s p r o d u c t i o n . F i n a l l y , I had u s e f u l d i s c u s s i o n s w i t h Gina Hoa- t s o n . v i 1 . MEMBRANES 1.1 AMPHIPHILES AND SELF-ASSEMBLY Only by c o n s i d e r i n g b o t h the m o l e c u l a r c o n s t i t u e n t s of a me- mbrane and the water w i t h which i t c o e x i s t s as a thermo- dynamic system can we u n d e r s t a n d membrane s t r u c t u r e . The me- mbrane m o l e c u l e s i n which we are i n t e r e s t e d a r e of d u a l c h a r a c t e r : they c o n s i s t of both h y d r o p h o b i c and h y d r o p h i l i c r e g i o n s . What t h i s means we s h a l l examine f i r s t by b r i e f l y c o n s i d e r i n g the n a t u r e of l i q u i d water and i t s s o l v e n t a b i - l i t y f o r m o l e c u l e s which a r e e i t h e r h ydrophobic or hydro- p h i l i c . The f o l l o w i n g d i s c u s s i o n i s l a r g e l y a b s t r a c t e d from the book by T a n f o r d (1980, c h . 4,5). The i m p o r t a n t f e a t u r e of the water m o l e c u l e which we must c o n s i d e r here i s i t s p o l a r i t y . Each water m o l e c u l e has, by v i r t u e of i t s two c o v a l e n t O-H bonds, two e l e c t r o p o s i t i v e r e g i o n s c e n t r e d about i t s hydrogen atoms. I f we c o n s i d e r the oxygen atom t o l i e a t the c e n t r e of a t e t r a h e d r o n w i t h the two hydrogen atoms l o c a t e d a t two v e r t i c e s , then two e l e - c t r o n e g a t i v e r e g i o n s a r e c e n t r e d on the o t h e r two v e r t i c e s . T h i s means t h a t each water m o l e c u l e can form hydrogen bonds t o f o u r o t h e r water m o l e c u l e s . X-ray d i f f r a c t i o n s t u d i e s show t h a t i n the l i q u i d s t a t e each water m o l e c u l e has i n f a c t 4.4 n e a r e s t n e i g h b o u r s , * on a v e r a g e : l i q u i d water d i s p l a y s r o u g h l y t e t r a h e d r a l symmetry. Raman and i n f r a r e d s p e c t r o s c o p y suggest t h a t water m o l e c u l e s * In c o m p a r i s o n , c l o s e s t - p a c k e d spheres have 12. 1 2 i n the l i q u i d s t a t e a r e hydrogen bonded t o each o t h e r , but w i t h more v a r i a t i o n i n bond l e n g t h s and a n g l e s than i n the u s u a l s o l i d s t a t e . C o n s e q u e n t l y , l i q u i d w a t e r ' s o r d e r i s s h o r t - r a n g e . A c c o r d i n g t o X-ray d i f f r a c t i o n measurements i t decays w i t h i n a r a d i u s of 8A. A l l of t h i s means t h a t l i q u i d water i s r a t h e r more o r d e r e d than many o t h e r l i q u i d s , but of c o u r s e l e s s o r d e r e d than a c r y s t a l l i n e s o l i d . Water's h i g h degree of l o c a l o r d e r l e a d s t o the hydro- p h o b i c e f f e c t , or l a c k of u n i f o r m m i x i n g of n o n p o l a r mole- c u l e s w i t h water. As d i s c u s s e d by T a n f o r d , c a l o r i m e t r i c and s o l u b i l i t y measurements of n o n p o l a r hydrocarbon s o l u t e s such as benzene i n water show t h a t the l o c a l arrangement of water m o l e c u l e s about a n o n p o l a r s o l u t e m o l e c u l e must be the im- p o r t a n t c o n s i d e r a t i o n h e r e . T h i s may be e x p l a i n e d i n terms of n°, the s t a n d a r d c h e m i c a l p o t e n t i a l per s o l u t e m o l e c u l e on the u n i t a r y s c a l e . We may w r i t e : M° = H° - TS° where H° i s the p a r t i a l m o l a l e n t h a l p y , or bond energy, S° i s the p a r t i a l m o l a l e n t r o p y , and.T i s the temperature of the system. For a nonpolar hydrocarbon s o l u t e m o l e c u l e , u° i s g r e a t e r i n water than i n a hydrocarbon s o l v e n t . T h i s means t h a t the t r a n s f e r of a hydrocarbon from hydrocarbon s o l v e n t t o water i s t h e r m o d y n a m i c a l l y u n f a v o u r a b l e . The h i g h e r v a l u e f o r tt°, however, does not r e s u l t from a d i f - f e r e n c e i n e n t h a l p i e s , which would be t r u e i f a s i g n i f i c a n t 3 number of water-water hydrogen bonds had to be broken i n order to introduce a s o l u t e molecule. Rather, the d i f f e r e n c e i s due to the f a c t t h at water molecules about a s o l u t e mole- c u l e to which they cannot bond no longer see an i s o t r o p i c environment. The o r i e n t a t i o n s i n space which remain open to them are fewer, and so the entropy of the system i s de- creased. Due to the negative sign i n the e x p r e s s i o n above, t h i s i n c r e a s e s the chemical p o t e n t i a l of such a s o l u t e i n water. Thus a hydrocarbon-water system seeks to minimize i t s f r e e energy by s e p a r a t i n g i n t o two bulk phases and minimiz- ing the i n t e r f a c i a l a r e a. In c o n t r a s t to the hydrophobic e f f e c t i s the h y d r o p h i l - i c e f f e c t . T h i s r e f e r s simply to the f a c t that water i s a' good s o l v e n t f o r other p o l a r molecules or f o r i o n s . From the preceeding d i s c u s s i o n i t i s c l e a r that t h i s must be because water can form hydrogen bonds to such s o l u t e molecules. Entropy p l a y s l i t t l e r o l e here, while the enthalpy of i n t r o - ducing such a s o l u t e molecule i n t o water may be n e g l i g i b l e or f a v o u r a b l e . The molecules which w i l l concern us here c o n s i s t of both a hydrophobic region and a h y d r o p h i l i c r e g i o n . Such mo- l e c u l e s are s a i d to be " a m p h i p h i l i c . " T h i s category i n c l u d e s the soaps and d e t e r g e n t s , which have an i o n i c or p o l a r hy- d r o p h i l i c "head group" and a hydrophobic hydrocarbon c h a i n " t a i l . " Included a l s o , as we s h a l l see i n the f o l l o w i n g sec- t i o n s , are the b i o l o g i c a l l i p i d s , which g e n e r a l l y have a l a r g e head group and two t a i l s , and c e r t a i n l y some of the 4 i n t e g r a l membrane p r o t e i n s . As a consequence of t h e i r a m p h i p h i l i c c h a r a c t e r , these m o l e c u l e s aggregate when p l a c e d i n w a t e r . The i m p o r t a n t t h i n g t o note i s t h a t a g g r e g a t i o n o c c u r s because i t i s t h e r m o d y n a m i c a l l y f a v o u r a b l e . Aggregate s t r u c t u r e s s e q u e s t e r h y d r o p h o b i c r e g i o n s t o g e t h e r , w h i l e k e e p i n g h y d r o p h i l i c r e - g i o n s i n c o n t a c t w i t h w a t e r . For a m p h i p h i l e s w i t h hydrocarbon t a i l s a number of ag- g r e g a t e s t r u c t u r e s a r e known, and which s t r u c t u r e i s p r e - f e r e n t i a l l y formed i s dependent upon the molar r a t i o of a m p h i p h i l e t o water, the t e m p e r a t u r e , and upon p r o p e r t i e s of the p a r t i c u l a r a m p h i p h i l e . These i n c l u d e the number and l e n g t h of hydrocarbon t a i l s , whether or not carbon-carbon double bonds e x i s t i n the t a i l s (a s i n g l e such bond produces a bend i n the c h a i n ) and s i z e and charge of the head group. Such q u a n t i t i e s a r e i n c o r p o r a t e d by I s r e a l a c h v i l i et al (1980) i n a g e n e r a l t h e o r y which c h a r a c t e r i z e s l i p i d s by a d i m e n s i o n l e s s " p a c k i n g f a c t o r " g i v e n by v / a 0 l . Here v i s the hydrocarbon c h a i n volume of the l i p i d m o l e c u l e , / i s the c h a i n l e n g t h , which v a r i e s w i t h t e m p e r a t u r e , and a0 i s the a r e a per h y d r o p h i l i c head group, which v a r i e s w i t h head group s i z e and charge. By u s i n g the requirement of e q u i l i - b rium thermodynamics t h a t the f r e e energy of i d e n t i c a l mole- c u l e s i n a system of a g g r e g a t e d s t r u c t u r e s be the same, the problem of which a g g r e g a t e s t r u c t u r e i s p r e f e r r e d f o r a g i - ven p a c k i n g f a c t o r and l i p i d c o n c e n t r a t i o n can be a d d r e s s e d . 5 The aggregate s t r u c t u r e s of minimum s i z e i n which a l l l i p i d m o l e c u l e s have t h e i r minimum p o s s i b l e f r e e energy u° a r e summarized i n F i g . 1 . I t s h o u l d be noted t h a t t h e s e s t r u - c t u r e s a r e the ones p r e f e r r e d a t r e l a t i v e l y low l i p i d con- c e n t r a t i o n s ? an e x a m i n a t i o n of F i g . 2 w i l l show t h a t f o r po- t a s s i u m p a l m i t a t e , a soap w i t h a s i n g l e hydrocarbon c h a i n , the phase p r e f e r e n t i a l l y formed a t low c o n c e n t r a t i o n s and b i o l o g i c a l t e mperatures i s indeed composed of s p h e r i c a l mi- c e l l e s . But as the l i p i d c o n c e n t r a t i o n i s i n c r e a s e d , new l i - q u i d c r y s t a l l i n e phases a r e formed: h e x a g o n a l , c u b i c , l a m e l - l a r and f i n a l l y g e l phase. 6 Lipid Critical packing parameter v/a0lr Critical packing shape Structures formed Single-chained lipids (detergents) with large head-group areas: NaDS in low salt Some lysophospholipids < 1 Cone Spherical micelles Single-chained lipids with small head-group areas: NaDS in high salt Non ionic lipids lysolecithin Truncated cone or wedge Globular or cylindrical micelles Double-chained lipids with large head-group areas, fluid chains: Lecithin, sphingomyelin Phosphatidylserine in water Phosphatidylglycerol Phosphatidylinositol Phosphatide acid Disugardiglyceridcs Some single-chained lipids with very small (uncharged) head- groups. 7 - 1 Truncated cone B Flexible bilayers Vesicles Double-chained lipids with small head-group areas, anionic lipids in high salt, saturated frozen chains: Phosphatidylethanolamine, Phosphatidylserine + Ca 1 * - 1 Cylinder PI 1 § yers • Double-chained lipids with small head-group areas, nonionic lipids, poly(cis) unsaturated chains, high T : Unsat phosphatidylethanolamine Cardiolipin + Cz** Phosphatide acid + Ca J * Monosugardiglycerides Cholesterol > 1 inverted truncated cone m< micelles F i g . 1 . Dynamic p a c k i n g p r o p e r t i e s of l i p i d s and the s t r u - c t u r e s they form. (From I s r a e l a c h v i l i et a l , 1980.) 7 F i g . 2 . Phase diagram of tern. (From Bloom et al, the p o t a s s i u m 1976.) p a l m i t a t e -water s y s - 8 1.2 MEMBRANE PROTEINS Wh i l e the l i p i d s of a b i o l o g i c a l membrane ar e r e s p o n s i b l e f o r the g e n e r a l s t r u c t u r e of the membrane and s t r u c t u r a l changes, i t i s a membrane's p r o t e i n s which a r e r e s p o n s i b l e f o r many of i t s more s p e c i f i c b i o c h e m i c a l f u n c t i o n s . Such f u n c t i o n s i n c l u d e the t r a n s p o r t of i o n s and macromolecules a c r o s s the membrane; enzy m a t i c a c t i v i t i e s , such as l i p i d b i - o s y n t h e s i s ; energy t r a n s d u c t i o n p r o c e s s e s , such as v i s i o n and p h o t o s y n t h e s i s ; r e c e p t i o n of hormones and v i r u s e s ; and a n c h o r i n g of the c y t o s k e l e t a l p r o t e i n network. G e n e r a l l y i t has been found, f o r d i f f e r e n t t y p e s of membranes, t h a t the more a c t i v e the membrane the h i g h e r i s i t s p r o t e i n c o n t e n t . Thus the m y e l i n membrane, which s e r v e s m a i n l y t o i n s u l a t e the nerve c e l l , c o n t a i n s about 25% p r o t e i n by w e i g h t . The m i t o c h o n d r i o n , on the o t h e r hand, which i s devoted t o energy t r a n s d u c t i o n , has as a major component a l a r g e i n n e r me- mbrane which c o n t a i n s about 70% p r o t e i n . These p r o t e i n s i n - c l u d e s p e c i f i c m e t a b o l i t e t r a n s l o c a t o r s and enzymes re s p o n s - i b l e f o r o x i d a t i o n r e a c t i o n s and f o r p r o d u c t i o n of the ener- gy s t o r a g e m o l e c u l e ATP. Membrane p r o t e i n s have been c l a s s i f i e d i n t o two groups: i n t e g r a l and p e r i p h e r a l . O p e r a t i o n a l l y t h e s e two groups may be d i s t i n g u i s h e d by the s e v e r i t y of the t e c h n i q u e r e q u i r e d t o s e p a r a t e them from the membrane. P e r i p h e r a l membrane p r o - t e i n s may be r e l e a s e d by agents such as s a l t s which s i m p l y d i s r u p t i o n i c or hydrogen bonds. I n t e g r a l membrane p r o t e i n s may o n l y be r e l e a s e d from the membrane by o r g a n i c s o l v e n t s 9 or d e t e r g e n t s which d i s r u p t the b i l a y e r s t r u c t u r e (Zubay, 1983, p.587). Our c oncern i n t h i s study w i l l be w i t h the i n - t e g r a l membrane p r o t e i n s . P r o t e i n s i n g e n e r a l c o n s i s t of l i n e a r sequences of amino a c i d s j o i n e d end-to-end by p e p t i d e bonds. Twenty v a r - i e t i e s of amino a c i d s a r e found i n l i v i n g o r g a n i s m s . The twenty amino a c i d s d i f f e r o n l y i n t h e i r s i d e c h a i n s , which may, a t b i o l o g i c a l pH, be c h a r g e d , n e u t r a l and p o l a r , or n e u t r a l and n o n p o l a r . Thus the s i d e c h a i n s may be c l a s s i f i e d as h y d r o p h i l i c or h y d r o p h o b i c . W h i l e the l i n e a r sequence of amino a c i d s s p e c i f i c t o a p r o t e i n forms i t s p r i m a r y s t r u c t u r e , r e g i o n s of the p r o t e i n a r e u s u a l l y t w i s t e d or p l e a t e d t o form secondary s t r u c t u r e s known as the a - h e l i x and the 0-sheet (Unwin and Henderson, 1984). These s t r u c t u r e s a r e s t a b i l i z e d by hydrogen bonds be- tween p o l a r groups on the p e p t i d e backbone. I f the s i d e c h a i n s of a sequence of amino a c i d s i n v o l v e d i n such a s t r u - c t u r e a r e h y d r o p h o b i c , then s i n c e a l l p o l a r groups on the backbone are hydrogen bonded, the e n t i r e segment i s hydro- p h o b i c . In a w a t e r - s o l u b l e p r o t e i n , such segments a r e hidden i n s i d e the h y d r o p h i l i c f o l d s of the t o r t u o u s t e r t i a r y p ro- t e i n s t r u c t u r e , the hydrogen and i o n i c - b o n d e d t a n g l e formed of l i n e a r amino a c i d sequences and secondary s t r u c t u r e s . In b a c t e r i o r h o d o p s i n , one of the f i r s t i n t e g r a l me- mbrane p r o t e i n s f o r which the intra-membrane t e r t i a r y s t r u - c t u r e was e l u c i d a t e d (Unwin & Henderson, 1984) seven a - h e l i c e s c l u s t e r t o g e t h e r l i k e a bundle of s t i c k s . Seven 10 sequences of amino a c i d s w i t h p r i m a r i l y h y d r o p h o b i c s i d e - c h a i n s have been i d e n t i f i e d i n the p r o t e i n ' s p r i m a r y s t r u c t u r e . These sequences, about the r i g h t l e n g t h t o pass t h r o u g h the membrane i n which the p r o t e i n o c c u r s , a r e se- p a r a t e d by s h o r t , p r i m a r i l y h y d r o p h i l i c segments. T h i s s t r o n g l y s u g g e s t s t h a t the reason why i n t e g r a l membrane p r o - t e i n s i n g e n e r a l a r e so f i r m l y bound t o membranes i s t h a t they a r e a m p h i p h i l e s t h e m s e l v e s , and s e q u e s t e r t h e i r hydro- p h o b i c r e g i o n s w i t h i n the h y d r o p h o b i c b i l a y e r i n t e r i o r be- cause i t i s t h e r m o d y n a m i c a l l y f a v o u r a b l e t o do so. W h i l e ba- c t e r i o r h o d o p s i n passes t h r o u g h the b i l a y e r seven t i m e s , o t h - er p r o t e i n s such as g l y c o p h o r i n a r e thought t o pass through o n l y once (Zubay, 1983, p.593). Henderson (1981) has argued a g a i n s t the l i k e l i h o o d of o c c u r r e n c e of p r o t e i n s which p r o j e c t o n l y halfway t h r o u g h the b i l a y e r . H i s main argument a g a i n s t t h i s i s t h a t bends of a p o l y p e p t i d e are accompanied by unbonded hydrogen-bonding s i t e s on the backbone, the presence of which i n the hydro- phobic environment of the b i l a y e r i n t e r i o r i s thermo- d y n a m i c a l l y u n f a v o u r a b l e . G r a m i c i d i n A, a c h a n n e l - f o r m i n g p r o t e i n , J_s thought t o pass o n l y h a l f w a y through the b i l a y - e r , when two are hydrogen-bonded end-to-end. Henderson a r - gues t h a t i n t h i s case the unbonded s t a t e i s p r o b a b l y o n l y a t r a n s i t o r y phenomenon. Henderson's arguments are somewhat c o n t r o v e r s i a l , ho- wever. Zubay, f o r example (1983, p.595) r e p o r t s t h a t e v i - dence e x i s t s t h a t the p r o t e i n cytochrome b 5 i s anchored i n 11 the membrane by a s e c t i o n of m o l e c u l a r weight 5000 d a l t o n s which does not p e n e t r a t e the b i l a y e r c o m p l e t e l y . As a com- p a r i s o n , the m o l e c u l a r weight of the a r t i f i c i a l h a l f - p e p t i d e we s h a l l d e s c r i b e i n the p r e s e n t s t u d y , p e p t i d e 10, i s about 1500 d a l t o n s . The i n t i m a t e r e l a t i o n s h i p which e x i s t s between p r o t e i n t e r t i a r y s t r u c t u r e and f u n c t i o n i s i l l u s t r a t e d by connexons, membrane p r o t e i n s which, p a i r w i s e , form c h a n n e l s between n e i g h b o u r i n g c e l l s a t the s o - c a l l e d "gap j u n c t i o n s . " These p r o t e i n s have been found t o be composed of s i x s u b u n i t s which may be a - h e l i c e s or /3-sheets (Unwin & Henderson, 1984). These s u b u n i t s s u r r o u n d a c e n t r a l c h a n n e l through the membrane. I t i s known t h a t gap j u n c t i o n s open or c l o s e de- pending upon the c o n c e n t r a t i o n of c a l c i u m i o n s w i t h i n the c e l l . Unwin and a coworker have demonstrated t h a t the con- f o r m a t i o n of these p r o t e i n s i n the membrane i s d i f f e r e n t de- pending on whether or not c a l c i u m i o n s are p r e s e n t . C a l c i u m s t r a i g h t e n s the s u b u n i t s , as i f they were b e i n g put under t e n s i o n from o p p o s i t e s i d e s of the membrane. T h i s r e s u l t s i n the c h a n n e l b e i n g c o n s t r i c t e d . 2 . 2H-NMR THEORY 2 . 1 HAMILTONIAN OF A STATIC 2H NUCLEUS IN A MAGNETIC FIELD A s i n g l e 2H (deuterium) n u c l e u s p l a c e d i n a magnetic f i e l d has a H a m i l t o n i a n which may be w r i t t e n : where # z i s the Zeeman H a m i l t o n i a n and HQ i s the quadrupole H a m i l t o n i a n . The Zeeman H a m i l t o n i a n # z d e s c r i b e s the energy of the n u c l e a r magnetic d i p o l e moment due t o i t s o r i e n t a t i o n i n the magnetic f i e l d : HZ = -M N-H 0 = - g 0 N r . f t o where: M N = n u c l e a r magnetic moment H 0 = magnetic f i e l d s t r e n g t h I = n u c l e a r s p i n o p e r a t o r 0 N = n u c l e a r magneton g = n u c l e a r g - f a c t o r . The quadrupole H a m i l t o n i a n HQ d e s c r i b e s t he energy of i n t e r a c t i o n of the n u c l e a r e l e c t r i c q u adrupole moment w i t h an e l e c t r i c f i e l d g r a d i e n t . T h i s f i e l d g r a d i e n t may a r i s e from a s p h e r i c a l asymmetry of the e l e c t r o n i c charge 12 13 d i s t r i b u t i o n about the n u c l e u s due t o a c h e m i c a l bond i n which the 2H atom p a r t i c i p a t e s . I n the case of t h i s work, the charge d i s t r i b u t i o n t o be c o n s i d e r e d i s t h a t of a C- 2H bond. S l i c h t e r (1978, ch .9) p r o v i d e s a c l e a r d e r i v a t i o n of the q u a d r u p o l e H a m i l t o n i a n , s t a r t i n g from a c l a s s i c a l formu- l a t i o n of the e l e c t r o s t a t i c energy of a charge d i s t r i b u t i o n i n an a r b i t r a r y e l e c t r i c p o t e n t i a l . T h i s H a m i l t o n i a n i s f o r - med of the s c a l a r p r o d u c t of two second-rank t e n s o r s , the n u c l e a r q u a d r u p o l e moment t e n s o r CK ̂  and the e l e c t r i c f i e l d g r a d i e n t t e n s o r V ^ . Choosing a c o o r d i n a t e system c e n t r e d on the c e n t r e of charge of the n u c l e u s , and w i t h an o r i e n t a t i o n such t h a t V.. i s d i a g o n a l , we can w r i t e : Q = e 2 <7<2 /4 l (2 I - l ) [ (3I 2 Z-I 2) + V ( t 2 x - l 2 y ) ] where: = n u c l e a r s p i n o p e r a t o r e = elementary charge Q = ( s c a l a r ) q u a d r u p o l e moment of the n u c l e u s and: eq = V z z = the f i e l d g r a d i e n t the e l e c t r o s t a t i c f i e l d w i t h : = 9 2V( x, y, z)/bi 2 , i=x,y,z< 14 In 2H-NMR the magnetic energy of the 2H n u c l e u s i s t y p - i c a l l y s e v e r a l o r d e r s of magnitude l a r g e r than the quadrupo- l a r energy. In the case of t h i s work, the magnetic f i e l d im- posed s e t s the f r e q u e n c y s e p a r a t i o n of Zeeman l e v e l s a t about 35 MHz, w h i l e the frequ e n c y s p l i t t i n g due t o the qua- d r u p o l e H a m i l t o n i a n i s measured i n hundreds of kHz. Thus we may c o n s i d e r the q u a d r u p o l e H a m i l t o n i a n t o be a p e r t u r b a t i o n on the Zeeman energy l e v e l s , f o r which a f i r s t - o r d e r c o r r e c - t i o n i s s u f f i c i e n t . I f we c o n s i d e r the case TJ=0, approxim- a t e l y t r u e f o r the C- 2H bonds we s h a l l be c o n s i d e r i n g (See- l i g , 1977, and r e f s . t h e r e i n ) , we o b t a i n energy l e v e l s ( S l i c h t e r , 1978, c h . 9 ) : Em = ~ y n m ° m + (^ 2?2/4I (21-1 ) ) ( (3co s 2 0 - 1 )/2) [3m 2-I (1 + 1 ) ] where: 7 n = the n u c l e a r gyromagnetic r a - t i o m =-1,0,1 f o r 2H 6 = the a n g l e between the ma- g n e t i c f i e l d d i r e c t i o n and the c o o r d i n a t e a x i s chosen above. With the s e l e c t i o n r u l e Am=±1, we o b t a i n a d o u b l e t w i t h a s e p a r a t i o n Avn known as the q u a d r u p o l a r s p l i t t i n g : Avn = (3/2)(e 2qQ/h)[(3cos 2e--\)/2]. In a C- 2H bond, w i t h the q u a d r u p o l a r c o u p l i n g c o n s t a n t e2qQ/h - 167 kHz ( D a v i s & J e f f r e y , 1977) the maximum pos- s i b l e v a l u e of t h i s s p l i t t i n g i s 250 kHz. 16 2.2 THE QUADRUPOLAR ECHO AND FOURIER TRANSFORM SPECTROSCOPY S i n c e the q u a d r u p o l a r i n t e r a c t i o n can g i v e r i s e t o a maximum s p l i t t i n g of 250 kHz, the p r e c e s s i n g m a g n e t i z a t i o n i n the xy p l a n e a f t e r a 90° p u l s e decays v e r y q u i c k l y , i n a few /as. Due t o i n s t r u m e n t a l l i m i t a t i o n s , i t i s not p o s s i b l e t o de- t e c t the p r e c e s s i n g m a g n e t i z a t i o n i m m e d i a t e l y a f t e r t u r n i n g o f f the o s c i l l a t i n g f i e l d . Thus a two-pulse sequence has been d e v i s e d t o cause a " s p i n echo" f o r systems of spin-1 n u c l e i such as 2H. T h i s i s the q u a d r u p o l a r echo sequence ( D a v i s et al , 1976). I t c o n s i s t s of a 90° p u l s e , a w a i t of T , t y p i c a l l y t e n s of us, and a 90° p u l s e a p p l i e d 90° out of phase w i t h the f i r s t . An echo o c c u r s a t a time T a f t e r the second p u l s e . T h i s i s w r i t t e n : O O ' l ^ r - O O 0 ) - r - e c h o . P r o v i d i n g t h a t n e g l i g i b l e r e l a x a t i o n has taken p l a c e the peak of the echo w i l l be of the same magnitude as the decay- i n g s i g n a l a f t e r the f i r s t p u l s e . The s i g n a l may be F o u r i e r t r a n s f o r m e d from the top of the echo t o o b t a i n the spectrum of the 2H n u c l e i i n the sample. In the case of a sample i n which a l l 2H n u c l e i are o r i e n t e d w i t h t h e i r a x i s of symmetry (the C- 2H bond) a t the same a n g l e t o the s t a t i c magnetic f i e l d , the F o u r i e r t r a n s - formed spectrum w i l l be a d o u b l e t s e p a r a t e d by the quadrupo- l a r s p l i t t i n g AI>Q c o r r e s p o n d i n g t o t h a t a n g l e . In o t h e r c a s e s the spectrum w i l l be more c o m p l i c a t e d . 17 2.3 MOTIONAL AVERAGING AND THE ORDER PARAMETER An e x p r e s s i o n has been found above f o r A V Q f o r a s t a t i c 2H n u c l e u s . I f the n u c l e u s i s r e o r i e n t i n g a t a r a t e which i s f a s t compared w i t h the g u a d r u p o l a r c o u p l i n g c o n s t a n t e2qQ/h = 167 kHz, then we must r e p l a c e t h a t e x p r e s s i o n by: LvQ = ( 3 / 2 ) ( e 2 q Q / h ) S C D where: S C D = (3<cos 20> - l ) / 2 . Here <> means a time average over o r i e n t a t i o n s . For a 2H nu- c l e u s r e o r i e n t i n g such t h a t a l l d i r e c t i o n s i n space a r e equ- a l l y p r o b a b l e ("random t u m b l i n g " ) 5"CD=0 and AVQ=0; the dou- b l e t c o l l a p s e s t o a s i n g l e l i n e . T h i s i s the case f o r 2H nu- c l e i i n l i q u i d water. For a mo l e c u l e which i s r e o r i e n t i i n g about an a x i s which i s p a r a l l e l t o the f i e l d d i r e c t i o n , 6 i s the a n g l e between t h i s a x i s and the C- 2H bond. 18 2.4 THE POWDER PATTERN AND DEPAKING I f the magnetic f i e l d d i r e c t i o n i s not p a r a l l e l t o the a x i s of symmetry f o r m o l e c u l a r motions as d i s c u s s e d above, but makes some a n g l e a w i t h t h i s a x i s , then we have ( S e e l i g , 1977) : A»>Q = (3/2) (e2qQ/h)SCD(3cos2a - D / 2 . I f i n a sample a l l o r i e n t a t i o n s of the a x i s of symmetry oc- c u r w i t h e q u a l p r o b a b i l i t y , then the p r o b a b i l i t y d i s t r i b u - t i o n f o r d i r e c t i o n s v a r i e s as s i n a , and we o b t a i n a "powder p a t t e r n " ( F i g . 3 ) . T h i s i s a sum of d o u b l e t s of d i f f e r e n t A P Q , weighted by t h e i r p r o b a b i l i t y of o c c u r r e n c e w i t h i n the sample: the maximum s p l i t t i n g o c c u r s f o r 0°, and i s l e a s t p r o b a b l e ; a s p l i t t i n g of h a l f t h i s magnitude o c c u r s f o r 90°, and i s most p r o b a b l e . A n u m e r i c a l p r o c e d u r e has been developed which c a l c u - l a t e s the o r i e n t e d spectrum, f o r a=0°, from such a powder p a t t e r n (Bloom et al , 1981; S t e r n i n et a l , 1983). T h i s me- t hod may a l s o be a p p l i e d t o a s u p e r p o s i t i o n of powder p a t - t e r n s , as i s c h a r a c t e r i s t i c of l a m e l l a r phase l i p i d s i n which a l l 1H n u c l e i have been r e p l a c e d w i t h 2H n u c l e i . An example of such a s u p e r p o s i t i o n , and the same spectrum a f t e r t h i s " d e p a k i n g " p r o c e d u r e , i s shown i n F i g . 1 1 . 19 F i g . 3 . A d e u t e r i u m powder p a t t e r n , f o r the case of a x i a l symmetry ( TJ=0 ) . 3. 2H-NMR OF MEMBRANES AND MODEL MEMBRANES 3.1 WHY 2H-NMR IS USEFUL FOR THE STUDY OF MEMBRANES AND MODEL MEMBRANES In 2H-NMR of l i p i d s i n the l a m e l l a r phase, i n t e r a c t i o n s such as the d i p o l a r and c h e m i c a l s h i f t i n t e r a c t i o n s l e a d t o s p l i t t i n g s of o n l y a few kHz ( D a v i s , 1983) and so may be i g n o r e d i n comparison w i t h the q u a d r u p o l a r i n t e r a c t i o n . T h i s makes a 2H-NMR spectrum p a r t i c u l a r l y easy t o i n t e r p r e t : S C D r e f l e c t s the o r i e n t a t i o n and dynamics of a m o l e c u l e a t the 2H n u c l e u s . 2H may be s u b s t i t u t e d f o r 1H a t any or a l l p o s i - t i o n s a l o n g the l i p i d c h a i n , a l l o w i n g the d e t e r m i n a t i o n of SQQ f o r s p e c i f i c p o s i t i o n s on the c h a i n . In c o n t r a s t t o the case of e l e c t r o n s p i n resonance (ESR), which r e q u i r e s the attachment t o l i p i d m o l e c u l e s of a bu l k y s p i n - l a b e l group, the 2H n u c l e u s p r o v i d e s a non-per- t u r b a t i v e probe of dynamics. As the Van der Waals r a d i u s of 2H i s the same as t h a t of 1H, i t i s not t o be expected t h a t l i p i d - l i p i d i n t e r a c t i o n s a f f e c t i n g l i p i d dynamics are a l t e r e d by t h i s s u b s t i t u t i o n . That t h i s i s so has been shown by Tang et al (1985). They have compared the s p e c t r a of pure p e r d e u t e r a t e d ( a l l 1H's r e p l a c e d by 2H's) p o t a s s i u m p a l m i t - a t e w i t h p e r d e u t e r a t e d p o t a s s i u m p a l m i t a t e mixed w i t h p r o - t i a t e d p o t a s s i u m p a l m i t a t e a t a molar r a t i o of 1:99. I f p e r - d e u t e r a t i o n a f f e c t e d the dynamics of the soap, we would ex- pect the s p e c t r a i n the two cas e s t o be d i f f e r e n t . They a re not , w i t h i n e x p e r i m e n t a l e r r o r . Thus u s i n g 2H-NMR we can 2 0 21 observe o r i e n t a t i o n s and motions of membrane and model me- mbrane l i p i d c h a i n s w i t h o u t a f f e c t i n g t h e s e q u a n t i t i e s . 22 3.2 RELATION OF BILAYER THICKNESS TO S c p Each o r d e r parameter 5Q D measured f o r a l i p i d c h a i n u s i n g 2H-NMR r e f l e c t s an o r i e n t a t i o n f o r a p a r t i c u l a r s i t e which i s an average over many m o l e c u l e s and over the t i m e s c a l e of the q u a d r u p o l a r i n t e r a c t i o n . Thus i t seems r e a s o n a b l e t h a t the f u l l s e t of o r d e r parameters a l o n g the c h a i n can be r e - l a t e d t o b i l a y e r t h i c k n e s s , which i s s u r e l y an average quan- t i t y . S c h i n d l e r and S e e l i g (1975) have done t h i s . Assuming t e t r a h e d r a l symmetry f o r the carbon bonds, they d e f i n e a "segment o r i e n t a t i o n " f o r each carbon s i t e by the d i r e c t i o n normal t o the p l a n e i n which the two C-H bonds l i e . The o t h e r d i r e c t i o n of i n t e r e s t i s the normal t o the b i l a y e r . Now assuming t h a t the s i t e c l o s e s t t o the l i p i d - w a t e r i n t e r f a c e i s o r i e n t e d p a r a l l e l t o the b i l a y e r n ormal, the o n l y segment o r i e n t a t i o n s /3 a l l o w e d a l o n g the c h a i n are 0°, 60° and 90°. These o r i e n t a t i o n s r e q u i r e the C-H bond v e c t o r s t o be o r i e n t e d a t 90°, 35.3° and 90°, and 35.3°, r e s p e c t i v e l y . * I f : s(e) = ( 3 c o s 2 e - 1)/2 then 5 C D f o r the deuterons a t a p a r t i c u l a r carbon s i t e can be c o n s i d e r e d as a sum of S(0) v a l u e s f o r d i f f e r e n t o r i e n t a - t i o n s , w e i g h t e d by t h e i r p r o b a b i l i t i e s of o c c u r r e n c e P^. Thus f o r each carbon s i t e : *These a n g l e s may best be a s c e r t a i n e d by examining a molecu- l a r model. 23 S C D = P„S(90) + P 6 0 [ S ( 3 5 . 3 ) + S ( 9 0 ) ] / 2 + P 9 0 S ( 3 5 . 3 ) and: P 0 + P 6 0 + P 9o = 1. Now the p r o j e c t i o n of the C-C bond l e n g t h on the b i l a y e r normal may be c a l c u l a t e d f o r each s i t e o r i e n t a t i o n j3, and w i t h the a i d of the above e q u a t i o n s the average p r o j e c t i o n f o r a p a r t i c u l a r s i t e may be d e t e r m i n e d from the o r d e r p a r a - meter S Q D f o r t h a t s i t e . Summing the s e p r o j e c t i o n s over a l l s i t e s on the c h a i n , S c h i n d l e r and S e e l i g o b t a i n a r e l a t i o n between b i l a y e r t h i c k n e s s and the average a b s o l u t e o r d e r parameter |<5 C D>| f o r the e n t i r e c h a i n : d = d m [ 0 . 5 + |<S C D>|], where the maximum b i l a y e r t h i c k n e s s f o r l i p i d s w i t h n seg- ments i s d m = 2.5(n)A. The assumption t h a t the f i r s t carbon s i t e i s o r i e n t e d p a r a l l e l t o the b i l a y e r normal has been examined f o r p o t a s - sium p a l m i t a t e by A b d o l l a l et al (1977). They have e x p l a i n e d the t emperature dependence of SQ-Q f o r the s i t e s c l o s e s t t o the l i p i d - w a t e r i n t e r f a c e i n terms of a model i n which the f i r s t s i t e can adopt two o r i e n t a t i o n s , denoted A and B. In t h i s model the obser v e d o r d e r parameters are the average over r a p i d exchange between both c o n f i g u r a t i o n s , A b e i n g f a - vou r e d a t low, and B a t h i g h e r t e m p e r a t u r e s . The o r i e n t a t i o n i n which the f i r s t s i t e i s p a r a l l e l t o the b i l a y e r normal i s 24 B, the h i g h - t e m p e r a t u r e o r i e n t a t i o n . R e f e r r i n g t o t h e i r F i g . 2 b , we see t h a t t h i s o r i e n t a t i o n i s e x p e c t e d t o be p r e - dominant above about 65 - 70°C. Thus the S c h i n d l e r - S e e l i g f o r m u l a may be most a p p l i c a b l e t o p o t a s s i u m p a l m i t a t e i n t h i s t e m p e r a t u r e regime. R e s u l t s of the S c h i n d l e r - S e e l i g f o r m u l a have been p l o t t e d v e r s u s temperature f o r p e r d e u t e r a t e d p o t a s s i u m pa- l m i t a t e by D a v i s and J e f f r e y (1977, t h e i r F i g . 6 ) . They ob- t a i n good agreement w i t h X-ray d i f f r a c t i o n d a t a f o r b i l a y e r t h i c k n e s s a t 86°C. I t i s i n t e r e s t i n g t o note t h a t they f i n d t h a t below about 65°C the fo r m u l a g i v e s s t r a n g e r e s u l t s . In l i g h t of the above, perhaps t h i s s i m p l y r e f l e c t s the i n a p - p l i c a b i l i t y of the fo r m u l a o u t s i d e the temperature regime i n which the B c o n f i g u r a t i o n i s predominant. I t s h o u l d be noted t h a t S c h i n d l e r and S e e l i g c o n c e i v e d t h e i r f o r m u l a w i t h b i l a y e r s of the b i o l o g i c a l p h o s p h o l i p i d DPPC i n mind. The s t r u c t u r e of DPPC i s q u i t e d i f f e r e n t from t h a t of p o t a s s i u m p a l m i t a t e . Most i m p o r t a n t l y f o r t h i s c a l - c u l a t i o n , the head group i s e n t i r e l y d i f f e r e n t , and the mo- l e c u l e has two hydrocarbon c h a i n s . C l e a r l y t h i s i s a s e p a r - a t e case from t h a t of p o t a s s i u m p a l m i t a t e , and the v a l i d i t y of the f o r m u l a f o r DPPC must be a s c e r t a i n e d i n d e p e n d e n t l y from p o t a s s i u m p a l m i t a t e r e s u l t s . Bloom and M o u r i t s e n (1986) have noted t h a t r e s u l t s of the fo r m u l a agree w e l l w i t h X-ray d i f f r a c t i o n d a t a f o r DPPC a t 50°C. 25 3.3 LI PIP-PROTEIN INTERACTIONS EXAMINED BY 2H-NMR Here we s h a l l c o n s i d e r l i p i d - p r o t e i n i n t e r a c t i o n s p r i m a r i l y under c o n d i t i o n s i n which the l i p i d s a r e i n the l a m e l l a r phase. T h i s t o p i c has r e c e n t l y been reviewed by Bloom and Smith (1985). They have summarized the r e s u l t s of 2H-NMR ex- pe r i m e n t s d e s i g n e d t o i n v e s t i g a t e t he e f f e c t of i n t e g r a l me- mbrane p r o t e i n s on d e u t e r i u m - l a b e l l e d a c y l c h a i n s of pho- s p h o l i p i d m o l e c u l e s . They p o i n t out t h a t the r e s u l t s of e l e - c t r o n s p i n resonance (ESR) e x p e r i m e n t s have s u c c e s s f u l l y been i n t e r p r e t e d i n terms of s i g n a l s from two d i s t i n c t l i p i d p o p u l a t i o n s , one i n c l o s e p r o x i m i t y t o p r o t e i n s and one f a r from p r o t e i n s , and t h a t t h i s has m o t i v a t e d a s e a r c h f o r a s i m i l a r e f f e c t u s i n g 2H-NMR. However they c o n c l u d e t h a t de- s p i t e s t u d i e s on a l a r g e v a r i e t y of p r o t e i n - l i p i d combina- t i o n s , t h e r e i s no e v i d e n c e f o r any such d i s t i n c t i o n on the 2H-NMR t i m e s c a l e . These r e s u l t s c a n, of c o u r s e , e a s i l y be r e c o n c i l e d w i t h the ESR r e s u l t s i f we p o s t u l a t e t h a t l i p i d s exchange between the two p o p u l a t i o n s a t a r a t e which i s r a - p i d on the 2H-NMR t i m e s c a l e (~10" 5s) but slow on the ESR t i - mescale ( ~ 1 0 _ 8 s ) . Bloom and Smith a l s o p o i n t out t h a t |<5(>D>| has not been obse r v e d t o depend much upon the presence or absence of p r o t e i n s i n p h o s p h o l i p i d systems, even f o r much h i g h e r p r o - t e i n c o n c e n t r a t i o n s than found i n b i o l o g i c a l systems. T h i s i s an i n t e r e s t i n g p o i n t , because we would expect i n t e g r a l membrane p r o t e i n s and c h o l e s t e r o l b oth t o be r e l a t i v e l y r i - g i d i n comparison w i t h l i p i d c h a i n s , which can take on many 26 t w i s t e d c o n f o r m a t i o n s . But, as they n o t e , c h o l e s t e r o l does cause a l a r g e i n c r e a s e i n I ^ Q Q H f o r p h o s p h o l i p i d a c y l c h a i n s . How. can t h e s e f a c t s be r e c o n c i l e d ? A number of sugges- t i o n s have been advanced. One i s t h a t the "roughness" of the p r o t e i n s u r f a c e due t o i t s amino a c i d s i d e c h a i n s , r a t h e r than r e s t r i c t i n g the motions of n e i g h b o u r i n g l i p i d t a i l s and " s t r a i g h t e n i n g them o u t " as c h o l e s t e r o l i s thought t o do, f o r c e s n e i g h b o u r i n g l i p i d s i n t o bent c o n f o r m a t i o n s so as t o a v o i d l e a v i n g a l a r g e d e f e c t volume ( S e e l i g & S e e l i g , 1980). Thus the o r d e r parameters of l i p i d s c l o s e t o and f a r from p r o t e i n s would be s i m i l a r . Another s u g g e s t i o n i s t h a t i n t e g r a l membrane p r o t e i n s s h o u l d not be thought of as r i g i d a t a l l . Perhaps they are w e l l matched, i n a f l u i d m e c h a n i c a l sense, t o n e i g h b o u r i n g l i p i d m o l e c u l e s (Bloom, 1979). Bloom has c a l l e d t h i s the " s q u i s h y p r o t e i n s " c o n j e c t u r e . S t i l l a n o t her p r o p o s a l , the one which our study was de- s i g n e d t o examine, i s d e s c r i b e d i n the f o l l o w i n g s e c t i o n . 27 3.4 THE MATTRESS MODEL T h i s i s a phenomenological thermodynamic model which ex- p l o r e s the p o s s i b i l i t y t h a t the mismatch between the l e n g t h of h y d r o p h o b i c segments of an i n t e g r a l membrane p r o t e i n and the w i d t h of the hydrophobic r e g i o n of the l i p i d b i l a y e r w i - th o u t p r o t e i n s i s an im p o r t a n t v a r i a b l e i n l i p i d - p r o t e i n i n - t e r a c t i o n s ( M o u r i t s e n & Bloom, 1984). The system c o n s i d e r e d i s a l i p i d b i l a y e r i n which b i l a y e r - s p a n n i n g p r o t e i n s a re d i s p e r s e d . Only the l o w - c o n c e n t r a t i o n regime f o r p r o t e i n s i s c o n s i d e r e d . Thus the p r o t e i n s a r e c o n s i d e r e d t o be a "per- t u r b a t i o n " on the pure l i p i d b i l a y e r . The system i s c h a r a c t e r i z e d by an excess e n t h a l p y , t o which t h r e e terms c o n t r i b u t e . The f i r s t term q u a n t i f i e s the e l a s t i c energy s t o r e d by l i p i d s and p r o t e i n s . The model t r e a t s them as s p r i n g s , c h a r a c t e r i z i n g each type of m o l e c u l e by an e l a s t i c c o n s t a n t . The hydrophobic e f f e c t i s t a k e n i n t o account i n a se- cond term. T h i s term i s p r o p o r t i o n a l t o the hy d r o p h o b i c s u r - f a c e a r e a of l i p i d or p r o t e i n exposed. I t s h o u l d be noted t h a t i n the f i r s t s e c t i o n of t h i s c h a p t e r the thermodynamic system c o n s i d e r e d was a two-component system of a m p h i p h i l e and water. In t h a t system i t was seen t h a t the hydrophobic e f f e c t was due t o a change i n e n t r o p y , not e n t h a l p y , of wat- er m o l e c u l e s next t o s o l u t e m o l e c u l e s . The system d e f i n e d by the m a t t r e s s model, however, i n c l u d e s o n l y l i p i d s and p r o - t e i n s . Thus t h e r e i s no problem w i t h m o d e l l i n g the hydro- p h o b i c e f f e c t by a term i n the e x c e s s e n t h a l p y f o r t h i s 28 system. F i n a l l y the Van der Waals a t t r a c t i o n between hydrophob- i c p a r t s of l i p i d and p r o t e i n m o l e c u l e s i s ta k e n i n t o a c - count. T h i s c o n t r i b u t e s a t h i r d term t o the exce s s e n t h a l p y . The f r e e energy of the system i s g i v e n by the sum of the e x c e s s e n t h a l p y and the f r e e energy of an i d e a l two-component m i x t u r e . Then the s i t u a t i o n of thermodynamic e q u i l i b r i u m i s o b t a i n e d by m i n i m i z i n g the f r e e energy w i t h r e s p e c t t o the l e n g t h s of l i p i d and p r o t e i n m o l e c u l e s . T h i s model i s w e l l - s u i t e d t o examining the m o d i f i c a - t i o n , by p r o t e i n s , of the l i p i d g e l - l a m e l l a r phase t r a n s i - t i o n . M o u r i t s e n and Bloom p r e s e n t n u m e r i c a l l y - o b t a i n e d phase diagrams f o r the r e g i o n of t h i s t r a n s i t i o n , f o r the p a r t i c u - l a r case of r i g i d p r o t e i n s embedded i n an e l a s t i c l i p i d b i - l a y e r . The m a t t r e s s model c o u l d e x p l a i n why the a d d i t i o n of p r o t e i n s has not been seen t o cause much e f f e c t on l i p i d o r d e r p a r a m e t e r s , when one a d d i t i o n a l f a c t i s noted: e x p e r i - menters have chosen c o m b i n a t i o n s of l i p i d s and p r o t e i n s s i - m i l a r t o those found i n n a t u r a l membrane systems. M o u r i t s e n and Bloom contend t h a t such systems s h o u l d be r e l a t i v e l y w e l l matched h y d r o p h o b i c a l l y . I t i s a p p r o p r i a t e here t o c o n s i d e r the q u e s t i o n : i f b i - o l o g i c a l membranes c o n s i s t of h y d r o p h o b i c a l l y w e l l matched l i p i d s and p r o t e i n s , why c o n s i d e r the e f f e c t s of a mismatch? One answer i s t h a t i n r e a l b i o l o g i c a l membranes, b i l a y e r t h i c k n e s s depends on temperature and l i p i d c o m p o s i t i o n , 29 e i t h e r of which may v a r y , g e n e r a t i n g a p r o t e i n - l i p i d hydro- p h o b i c mismatch. S m a l l e f f e c t s may be i m p o r t a n t h e r e , and may bear on s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s of i n t e g r a l me- mbrane p r o t e i n s . The problem of boundary l i p i d s c o u l d a l s o be e x p l a i n e d by the m a t t r e s s model. Two p o p u l a t i o n s of l i p i d s c o u l d ex- i s t , but f o r w e l l matched systems, the 2H-NMR o r d e r paramet- e r s would be the same f o r b o t h , making them i n d i s t i n g u i s h - a b l e w i t h t h i s t e c h n i q u e . The model a l s o p r o v i d e s a n a t u r a l e x p l a n a t i o n f o r an e f f e c t observed i n the g e l phase of p h o s p h o l i p i d - p r o t e i n systems. As p r o t e i n c o n c e n t r a t i o n i s i n c r e a s e d , a t tempera- t u r e s a t which pure l i p i d i s i n the g e l phase, | <SCjy' | de- c r e a s e s u n t i l i t rea c h e s the v a l u e which c h a r a c t e r i z e s the pure l i p i d l a m e l l a r phase. S i n c e the p r o t e i n s used are w e l l matched t o the l i p i d l a m e l l a r phase hydrophobic w i d t h , the e q u i l i b r i u m w i d t h of the l i p i d h y drophobic r e g i o n s h o u l d de- c r e a s e w i t h the a d d i t i o n of p r o t e i n u n t i l i t reaches the l a - m e l l a r v a l u e . T h i s would c o r r e s p o n d w i t h an i n c r e a s e d mo- t i o n a l freedom and d e c r e a s e d l e n g t h f o r l i p i d t a i l s , and t h u s a d e c r e a s e d |<S C D>|. I t s h o u l d be noted t h a t M o u r i t s e n and Bloom c o n s i d e r the m a t t r e s s model i n c o m p l e t e . They b e l i e v e t h a t i t p r o b a b l y i g n o r e s some im p o r t a n t i n t e r a c t i o n s i n r e a l l i p i d - p r o t e i n systems. For example, charged amino a c i d s b u r i e d w i t h i n the b i l a y e r which i n t e r a c t w i t h i n a p r o t e i n t o s t a b i l i z e i t s t e r t i a r y s t r u c t u r e c o u l d a l s o g i v e r i s e t o an i n t e r - p r o t e i n 30 i n t e r a c t i o n , which might l e a d t o p r o t e i n s e g r e g a t i o n i n a b u l k phase. Such i n t e r a c t i o n s would have t o be i n c l u d e d i n a more s o p h i s t i c a t e d model. 31 3.5 PREVIOUS WORK ADDRESSING THE,MATTRESS MODEL The work of D a v i s et al (1983) i s an i n n o v a t i v e approach t o t e s t i n g t he m a t t r e s s model d i r e c t l y u s i n g two s p e c i a l l y syn- t h e s i z e d p o l y p e p t i d e s . These were of the form: L y s 2 - L e u 2 a - L y s 2 - A l a - a m i d e p e p t i d e D1 and Lys 2-Gly-Leu 2«-Lys 2-Ala-amide p e p t i d e D2. They were i n t e n d e d as model p r o t e i n s - s i m p l e "pegs" which would t r a v e r s e a l i p i d b i l a y e r . The l e u c i n e s i d e c h a i n i s hy d r o p h o b i c , and p o l y - l e u c i n e , which forms the c e n t r a l seg- ment of the p e p t i d e s , i s known t o be a s t r o n g a - h e l i x form- e r . D a v i s et al p r e s e n t s t r o n g e v i d e n c e from c i r c u l a r d i - c h r o i s m measurements t h a t i n hydrophobic s o l v e n t the p o l y - l e u c i n e segment of these p e p t i d e s does indeed form an a - h e l i x . At 1.5A per amino a c i d , the hyd r o p h o b i c p o r t i o n of the p e p t i d e formed by the a - h e l i c a l p o l y - l e u c i n e segment would be about 36A. The l y s i n e s i d e c h a i n , on the o t h e r hand, has one p o s i - t i v e charge a t pH 7. Thus l y s i n e i s h y d r o p h i l i c , and s h o u l d anchor the two ends of the p e p t i d e i n water on o p p o s i t e s i d e s of the b i l a y e r . P e p t i d e D1 was mixed w i t h the p e r d e u t e r a t e d soap po- t a s s i u m p a l m i t a t e ( CH 3(CH 2), f lCOOK ), and 2 H 2 0 was added t o a c h i e v e a (soap + p e p t i d e ) : w a t e r weight r a t i o of 70:30. At t h i s water c o n c e n t r a t i o n and over the temperature range of i n t e r e s t t o t h i s s t u d y , pure p o t a s s i u m p a l m i t a t e i s i n the 32 l a m e l l a r phase (see F i g . 2 ) . Samples were p r e p a r e d w i t h no p e p t i d e and a t p e p t i d e : l i p i d molar r a t i o s of 1:100 and 1:200. 2H-NMR s p e c t r a were taken of the samples a t 49°C and 65°C. R e s u l t s a re shown i n F i g . 4 . C l e a r l y the presence of the p e p t i d e d r a m a t i c a l l y i n - c r e a s e s c h a i n o r d e r i n the soap. The f i r s t moment M, of the h a l f - s p e c t r u m (0-»») a t 49°C i n c r e a s e s from 3.3«10*s~ 1 f o r the z e r o - p e p t i d e sample t o 4 . 0 * l O ^ " 1 f o r the p e p t i d e : l i p i d 1:100 sample. U s i n g t h e S c h i n d l e r - S e e l i g f o r m u l a , D a v i s et al i n t e r p r e t t h i s i n c r e a s e i n M, t o r e f l e c t an i n c r e a s e i n b i l a y e r t h i c k n e s s of almost 7A, from 27.3A t o 34.OA. T h i s i s a s t r i k i n g r e s u l t i n d e e d , and i t was t h i s which m o t i v a t e d the p r e s e n t s t u d y . U n f o r t u n a t e l y , as we d i s c o v e r e d d u r i n g the a n a l y s i s of our r e s u l t s , t h e r e a r e two problems w i t h t h i s . One i s t h a t , a c c o r d i n g t o the work of A b d o l l a l et al c i t e d above, the S c h i n d l e r - S e e l i g f o r m u l a i s not v a l i d f o r p o t a s s i u m p a l m i t - a t e i n t h i s t emperature regime. The second i s t h a t t h i s t h i c k n e s s e s t i m a t e i s i n e r r o r . As may be v e r i f i e d from the above-quoted moments, the S c h i n d l e r - S e e l i g f o r m u l a a c t u a l l y i n d i c a t e s an i n c r e a s e of o n l y about 1A i n t h i s c a s e . Low-angle X-ray d i f f r a c t i o n was a l s o performed on the z e r o - p e p t i d e and 1:100 samples a t 50°C. T h i s r e v e a l e d an i n - c r e a s e i n b i l a y e r r e p e a t s p a c i n g w i t h p e p t i d e of o n l y 2.5A, from 38.2A t o 40.7A, r e s p e c t i v e l y . D a v i s et al i n t e r p r e t t h e s e r e s u l t s t o mean t h a t the presence of the p e p t i d e causes not o n l y an i n c r e a s e i n t h i c k n e s s of the b i l a y e r s , 33 but a l s o a decrease i n the t h i c k n e s s of water l a y e r s between the b i l a y e r s . In l i g h t of the above o b s e r v a t i o n s , t h i s con- c l u s i o n i s unfounded. The main p a r t of the s t u d y , however, was the i n c o r p o r a - t i o n of p e p t i d e D2 i n t o b i l a y e r s of p e r d e u t e r a t e d 1 , 2 , - d i p a l m i t o y l - s n - g l y c e r o - 3 - p h o s p h o c h o l i n e (DPPC) a b i o - l o g i c a l l y i m p o r t a n t p h o s p h o l i p i d w i t h two hydrocarbon t a i l s . The p e p t i d e : l i p i d molar r a t i o p r e p a r e d was 1:43, and t h i s was mixed w i t h an e q u a l weight of water. At t h i s water con- c e n t r a t i o n , pure DPPC i s i n the g e l phase below 37.5°C and i n the l a m e l l a r phase above t h i s t e m p e r a t u r e . In c o n t r a s t t o the case of p o t a s s i u m p a l m i t a t e , 2H-NMR r e s u l t s d i d not r e f l e c t a d r a m a t i c i n c r e a s e i n DPPC c h a i n o r d e r w i t h the a d d i t i o n of p e p t i d e . Though the p e p t i d e con- c e n t r a t i o n i n DPPC was t w i c e t h a t s t u d i e d i n the soap, |<S C D>| i n DPPC i n c r e a s e d by o n l y 5%. However, a c a r e f u l study was made of the phase b e h a v i o r of t h i s sample about the g e l - l a m e l l a r phase t r a n s i t i o n t e - mperature. T h i s r e v e a l e d t h a t the s p e c t r a between 22°C and 44°C can a l l be r e p r e s e n t e d as s u p e r p o s i t i o n s of the c h a r a - c t e r i s t i c pure g e l and pure l a m e l l a r - p h a s e s p e c t r a i n v a r y - i n g p r o p o r t i o n s . Thus, the range of c o e x i s t e n c e of the two phases i s broadened from l e s s than 1°C t o about 20°C by the a d d i t i o n of the p e p t i d e . In a c o n t i n u a t i o n of t h i s work, H u s c h i l t et al (1985) have mapped out a r e g i o n of the phase diagram of perdeu- t e r a t e d DPPC w i t h i n c o r p o r a t e d p e p t i d e s of the form of 34 p e p t i d e D2. One of t h e s e i s i d e n t i c a l t o p e p t i d e D2, and one i s i d e n t i c a l e x cept t h a t the c e n t r a l h y d r o p h o b ic segment i s formed of 16 r a t h e r than 24 l e u c i n e s , making i t s hydro- ph o b i c segment o n l y 2/3 as l o n g . H u s c h i l t et al r e f e r t o t h e s e by l e u c i n e number as p e p t i d e 24 and p e p t i d e 16. F i g . 5 i s the phase diagram they o b t a i n e d , where the c o n c e n t r a t i o n of p e p t i d e i s g i v e n by: X p = (moles p e p t i d e ) / (moles p e p t i d e + moles DPPC). The lower of the t h r e e l i n e s marks the t r a n s i t i o n between the g e l phase and the two-phase r e g i o n . Below t h i s l i n e l i e s the g e l phase r e g i o n ; above t h i s l i n e the g e l and l a m e l l a r phases c o e x i s t . The two upper l i n e s mark the boundary be- tween the two-phase r e g i o n and the l a m e l l a r phase, f o r samples c o n t a i n i n g the two d i f f e r e n t t y p e s of p e p t i d e . The lower of the two l i n e s i s the boundary f o r samples c o n t a i n - i n g p e p t i d e 16; the upper f o r t h o s e c o n t a i n i n g p e p t i d e 24. In the l a m e l l a r phase they found l i t t l e i n c r e a s e i n |<5 C D>| due t o e i t h e r p e p t i d e . The g e l phase of DPPC i s q u a l i t a t i v e l y d i f f e r e n t from t h a t of p o t a s s i u m p a l m i t a t e . In the g e l phase of p o t a s s i u m p a l m i t a t e , t a i l s a r e i n t e r l e a v e d (see F i g . 2 ) making the t h i c k n e s s of the h y d r o p h o b i c r e g i o n s m a l l e r than i t i s i n the l a m e l l a r phase. T h i s i s not the case f o r DPPC: i n the l a m e l l a r phase, t a i l s a r e d i s o r d e r e d and the t h i c k n e s s of the h y d r o p h o b i c r e g i o n i s 33A; i n the g e l phase, t a i l s a r e 35 o r d e r e d , and t h i s t h i c k n e s s i n c r e a s e s t o 47A. T h i s l e a d s Hu- s c h i l t et al t o a t t r i b u t e the s l i g h t l y lower l a m e l l a r / t w o - p h a s e boundary f o r samples c o n t a i n i n g p e p t i d e 16 t o t h a t p e p t i d e ' s b e t t e r h y d r o p h o b i c matching t o the DPPC l a m e l l a r phase. A c c o r d i n g t o t h i s i n t e r p r e t a t i o n , the p r e s e n c e of p e p t i d e 16 i n the b i l a y e r s s h o u l d e x t e n d the r e g i o n i n which the l a m e l l a r phase i s t h e r m o d y n a m i c a l l y p r e f e r r e d t o lower t e m p e r a t u r e s , as r e s u l t s seem t o i n d i c a t e i t does. One reason why d i f f e r e n c e s between the two p e p t i d e s are not v e r y d r a m a t i c may be t h a t l y s i n e was an u n f o r t u n a t e c h o i c e f o r the h y d r o p h i l i c a n c h o r . As D a v i s et al (1983) n o t e , l y s i n e ' s p o s i t i v e charge i s l o c a t e d a t the end of a f i v e - c a r b o n hydrophobic a c y l c h a i n . T h i s means t h a t p e p t i d e 16 may be a b l e t o adopt a c o n f o r m a t i o n w i t h l y s i n e c h a i n s extended, i n which i t s hydrophobic segment i s c o n s i - d e r a b l y l o n g e r than 16*(1.5A). Thus i n l i g h t of the m a t t r e s s model, perhaps the two p e p t i d e s s h o u l d be e x p e c t e d t o have s i m i l a r e f f e c t s on c h a i n o r d e r and phase b e h a v i o r when i n c o - r p o r a t e d i n t o b i l a y e r s of DPPC. 36 F i g . 4 . 2H-NMR s p e c t r a of mixtures of perdeuterated potassium p a l m i t a t e and the peptide D1 (see t e x t ) i n 30% water, by weight, (a) P:L=0:1, 49°C; (b) 1:200, 49°C; (c) 1:100, 49°C; (d) 0:1, 65°C; (e) 1:200, 65°C; ( f ) 1:100, 65°C. (From Davis et a l , 1983.) 37 F i g . 5 . Phase diagrams of two p e p t i d e - l i p i d systems. T r i a n g l e s r e f e r t o p e p t i d e 24, c i r c l e s t o p e p t i d e 16. (From H u s c h i l t et a l , 1985. ) 4. OBJECTIVES OF THIS WORK The d r a m a t i c r e s u l t s of D a v i s et al (1983) w i t h p o t a s - sium p a l m i t a t e and a 2 4 - l e u c i n e p o l y p e p t i d e , d e s c r i b e d i n Chapter 3, p r o v i d e d m o t i v a t i o n f o r the p r e s e n t work. S i n c e the c o m p l e t i o n of t h a t s t u d y , s e v e r a l s i m i l a r p e p t i d e s w i t h h y d r o p h o b i c r e g i o n s of d i f f e r e n t l e n g t h s have been s y n t h e - s i z e d . We d e c i d e d t o study the e f f e c t s of these on c h a i n o r d e r i n the l a m e l l a r phase of p o t a s s i u m p a l m i t a t e i n hopes of p r o v i d i n g e v i d e n c e f o r or a g a i n s t the s i m p l e p i c t u r e of the m a t t r e s s model. P e p t i d e s used i n the p r e s e n t , study were of the form: L y s 2 - G l y - L e u ^ - L y s 2 - A l a - a m i d e w i t h /?=16, 20 or 24. A d o p t i n g the t e r m i n o l o g y of H u s c h i l t et al, we s h a l l r e f e r t o t h e s e as p e p t i d e 16, p e p t i d e 20 and p e p t i d e 24. The l e n g t h s of the hydrophobic p o l y - l e u c i n e a - h e l i c e s i n th e s e p e p t i d e s a r e 24A, 30A and 36A. I t s h o u l d be noted t h a t "amide" i n the above form u l a i n d i c a t e s t h a t NH 2 r e p l a c e s OH a t the C-terminus of the pe- p t i d e : 0 II -C-NH 2. T h i s means t h a t i n s o l u t i o n a t pH 7 the C-terminus i s un- c h a r g e d . 38 39 G a l l o t and S k o u l i o s (1966) r e p o r t X-ray d i f f r a c t i o n r e - s u l t s f o r p o t a s s i u m m y r i s t a t e i n the l a m e l l a r phase a t a 70:30 weight r a t i o w i t h water. T h i s m o l e c u l e i s i d e n t i c a l t o p o t a s s i u m p a l m i t a t e , e xcept t h a t i t has two fewer CH 2 groups. At 65°C they r e p o r t a b i l a y e r t h i c k n e s s of 23.8A. T h i s i s p r o b a b l y a good e s t i m a t e f o r the h y d r o p h o b i c t h i c k - ness of p o t a s s i u m p a l m i t a t e b i l a y e r s a t t h i s c o n c e n t r a t i o n and t e m p e r a t u r e . Thus the above p e p t i d e s s h o u l d a l l have hy- d r o p h o b i c r e g i o n s which a r e of the same t h i c k n e s s as the soap or g r e a t e r , under th e s e c o n d i t i o n s . In a d d i t i o n t o the above p e p t i d e s , which were e x p e c t e d t o span the b i l a y e r , an a m p h i p h i l i c p e p t i d e w i t h o n l y one h y d r o p h o b i c end and a s h o r t a - h e l i c a l p o l y - l e u c i n e segment t e r m i n a t e d w i t h an a c e t a t e group was a v a i l a b l e . I t was of the form: A c - L e u i 0 ~ L y s 2 - A l a - a m i d e , and w i l l be r e f e r r e d t o as p e p t i d e 10. The l e n g t h of the p o l y - l e u c i n e segment of p e p t i d e 10 was 15A, a l i t t l e more than h a l f t h e h y d r o p h o b i c t h i c k n e s s of the b i l a y e r quoted above. C l e a r l y the m a t t r e s s model i s not a p p l i c a b l e t o such a p e p t i d e , as i t would be anchored t o the h y d r o p h i l i c s u r - f a c e of the b i l a y e r by o n l y one end. For t h i s reason we f e l t t h a t e x a m i n i n g the e f f e c t s of t h i s p e p t i d e on c h a i n o r d e r i n p o t a s s i u m p a l m i t a t e would g i v e us i n c r e a s e d i n s i g h t i n t o the a p p l i c a b i l i t y of the m a t t r e s s model t o p o t a s s i u m p a l m i t a t e 40 w i t h the o t h e r p e p t i d e s used i n t h e s t u d y . 5. EXPERIMENTAL PROCEDURES 5.1 MATERIALS A l l p e p t i d e s were s y n t h e s i z e d i n t h e l a b o r a t o r y of R.S. Hodges a t the Department of B i o c h e m i s t r y of the U n i v e r s i t y of A l b e r t a . S y n t h e s i s of p e p t i d e D2 ( D a v i s et al , 1983) which was i d e n t i c a l t o our p e p t i d e 24 was d e s c r i b e d i n de- t a i l i n t h a t paper. I t s h o u l d be noted t h a t the p o s i t i v e c harges a t the end of the l y s i n e s i d e c h a i n s and a t the N-terminus of each of p e p t i d e 16, 20 and 24 were n e u t r a l i z e d by a c e t a t e c o u n t e r - i o n s . In b u f f e r a t pH 7 t h e s e c o u n t e r i o n s would be i n s o l u - t i o n . P a l m i t i c a c i d was o b t a i n e d from Calbiochem (La J o l l a , C a l i f o r n i a ) and p e r d e u t e r a t e d f o l l o w i n g the procedure of H s a i o et al (1974). T h i s was then used t o p r e p a r e perdeu- t e r a t e d p o t a s s i u m p a l m i t a t e . 41 42 5.2 S A M P L E P R E P A R A T I O N S a m p l e p r e p a r a t i o n w a s s i m i l a r t o t h a t d e s c r i b e d b y D a v i s et al ( 1 9 8 3 ) . D i f f e r e n c e s a r e n o t e d i n t h e n e x t s e c t i o n . P e r d e u t e r a t e d p o t a s s i u m p a l m i t a t e a n d t h e p e p t i d e o f i n t e r e s t w e r e d i s s o l v e d t o g e t h e r i n m e t h a n o l a t t h e d e s i r e d m o l a r r a t i o . T h e m e t h a n o l w a s t h e n r e m o v e d b y r o t a r y e v a - p o r a t i o n , l e a v i n g t h e p e p t i d e a n d p o t a s s i u m p a l m i t a t e t h o r - o u g h l y m i x e d . T o i n s u r e t h a t a l l m e t h a n o l w a s e v a p o r a t e d , t h e p o w d e r w a s p u t u n d e r v a c u u m f o r a t l e a s t 12 h o u r s . I n o r d e r t o o b t a i n a 2H-NMR s i g n a l f r o m t h e w a t e r i n t h e s a m p l e s , we d e c i d e d t o h y d r a t e t h e m w i t h b u f f e r w h i c h c o n t a i n e d some 2 H 2 0 . F o r t h i s p u r p o s e 2 H 2 0 a n d 1 H 2 0 w e r e m i x e d i n t h e m o l a r r a t i o 3 :4. T h i s m e a n t t h a t f o r t h e d e g r e e o f h y d r a t i o n we u s e d , t h e r e w e r e t w i c e a s many 2 H a t o m s i n t h e b u f f e r a s i n t h e m e t h y l g r o u p t e r m i n a t i n g t h e p o t a s s i u m p a l m i t a t e c h a i n s . T h u s t h e i n t e n s i t y o f t h e w a t e r s i g n a l w a s j u s t t w i c e t h a t d u e t o t h e m e t h y l g r o u p , a n d t h e s p l i t t i n g s d u e t o e a c h c o u l d g e n e r a l l y b e r e s o l v e d a n d i d e n t i f i e d . T o t h e 2 H 2 0 + 1 H 2 0 m i x t u r e w e r e a d d e d K H 2 P 0 4 a n d K 2 H P O „ , e a c h a t a 2 5 m i l l i m o l a r c o n c e n t r a t i o n . T h i s r e s u l t e d i n a 50mM p o - t a s s i u m p h o s p h a t e b u f f e r w h i c h m e a s u r e d pH 7.0 o n a d i g i t a l pH m e t e r . F o r a l l s a m p l e s , b u f f e r w a s a d d e d i n p r o p o r t i o n t o t h e w e i g h t o f p o t a s s i u m p a l m i t a t e o n l y , n o t t o t h e c o m b i n e d w e i g h t o f p o t a s s i u m p a l m i t a t e a n d p e p t i d e . T h e m o l a r r a t i o [ H 2 0 ( a l l i s o t o p e s ) ] : p o t a s s i u m p a l m i t a t e f o r a l l s a m p l e s w a s 7 . 7 5 : 1 . I n a s y s t e m o f o n l y s o a p + w a t e r t h i s w o u l d 43 c o r r e s p o n d t o 88.6 mole-% wate r ; i f n e i t h e r soap nor water c o n t a i n e d 2H atoms, the system would c o n t a i n 32.2% water by we i g h t . The powdered soap + p e p t i d e m i x t u r e was p l a c e d i n a g l a s s tube of i n t e r n a l d i a m e t e r 5mm. B u f f e r was added i n the c o r r e c t p r o p o r t i o n w i t h a v o l u m e t r i c s y r i n g e . As a check on the a c c u r a c y of the s y r i n g e , the weight of the sample was measured b e f o r e and a f t e r t he a d d i t i o n of b u f f e r . The tube was then s e a l e d under about h a l f an atmosphere of N 2 . At t h i s p o i n t the sample tube was a p p r o x i m a t e l y 30mm l o n g , w i t h a c o n s t r i c t i o n of about 1mm i n t e r n a l d i a m e t e r ha- l f w a y a l o n g i t s l e n g t h . In o r d e r t o mix the sample, the s e a l e d tube was p l a c e d i n an oven a t 100°C and removed p e r i o d i c a l l y t o c e n t r i f u g e soap, p e p t i d e and b u f f e r b a c k - a n d - f o r t h t h r o u g h the c o n s t r - i c t i o n i n the m i d d l e . The m i x t u r e c o u l d be f o r c e d through the c o n s t r i c t i o n about f o u r t i m e s b e f o r e i t began t o c o o l too much f o r good m i x i n g . Then the sample tube had t o be put back i n t o the oven f o r a t l e a s t 20 min u t e s . A f t e r b e i n g f o r c e d b a c k - a n d - f o r t h 100 tim e s the sample was c o n s i d e r e d w e l l mixed. Then the g l a s s sample tube was me l t e d a t the c o n s t r i c t i o n and the empty h a l f removed, l e a v - i n g t he sample s e a l e d i n the o t h e r end. D u r i n g t h i s o p e r a - t i o n t he end of the tube c o n t a i n i n g the sample was kept im- mersed i n c o l d water t o prev e n t e x c e s s i v e h e a t i n g . The weight of soap i n a t y p i c a l sample was 40mg. T h i s was an amount s u f f i c i e n t t o produce a s t r o n g 2H-NMR s i g n a l . 44 P e p t i d e : s o a p molar r a t i o s p r e p a r e d a r e c o n t a i n e d i n T a b l e I . Sample B 0 0 0 was p r e p a r e d e x a c t l y i n the manner of the o t h e r samples, except t h a t o n l y soap, and no p e p t i d e , was d i s s o l v e d i n methanol. Soap used f o r sample D 0 0 0 was not d i s s o l v e d i n methanol but put d i r e c t l y i n t o a sample tube, from which p o i n t i t s t r e a t m e n t was i d e n t i c a l t o t h a t of the o t h e r samples. 45 TABLE I. Samples prepared pe pt i de mol ar r at i o s ampl (pe pt i de: soap) p e p t i d e 24 1 :109 241 1:218 242 p e p t i d e 20 1:110 201 1:218 202 p e p t i d e 16 1:112 161 1 :224 162 p e p t i d e 10 1 : 1 08 101 1:216 Z1 02 none 0:1 B000 0:1 D000 46 5.3 DIFFERENCES FROM THE STUDY OF DAVIS et a I ( 1983) In the p r e s e n t s t u d y : B u f f e r was used r a t h e r than water. B u f f e r was added i n p r o p o r t i o n t o soap, r a t h e r than i n p r o p o r t i o n t o soap + p e p t i d e . We c o n s i d e r e d the p e p t i d e a p e r t u r b a t i o n on the soap + b u f f e r m i x t u r e . Samples c o r r e s p o n d e d t o 32.2% water by weight r a t h e r than 30%. A l l p e p t i d e c o n c e n t r a t i o n s were about 10% l a r g e r than those of Da- v i s et al . 47 5.4 2H-NMR SPECTROSCOPY Spectra were taken using a spectrometer b u i l t i n the UBC Phy s i c s Department e l e c t r o n i c s workshop. Our data a c q u i s i - t i o n and p r o c e s s i n g system has been d e s c r i b e d i n d e t a i l by S t e r n i n (1985). Spectra were taken f o r a l l samples at 65°C and 49°C, a f t e r a l l o w i n g at l e a s t one hour f o r temperature e q u i l i b r a - t i o n at each new temperature. As the e l e c t r i c s u s c e p t i b i l i t y of l i p i d / w a t e r samples v a r i e s with temperature, we were able to determine when the sample had a t t a i n e d thermal e q u i l i - brium by not i n g when the tuning of the 2H-NMR probe ceased to change a p p r e c i a b l y (Davis, 1983). A l l s p e c t r a were taken on resonance at about 35MHz us- ing the quadrupolar echo technique (Davis et al, 1976). The width of a 90° pulse ranged from 1.4MS to 1.8MS, the delay T between p u l s e s was 40MS, and the echo r e p e t i t i o n time was In order to e l i m i n a t e c o n t r i b u t i o n s to the s i g n a l a r i s - ing from p u l s e s which were not e x a c t l y 90°, a c y c l e of p a i r s of p u l s e s with phases ( 0 1 f 0 2 ) was used (Davis, 1983): 5s. (0 , i r/2) ( + ) U,*/2) ( - ) (0,-TT/2) ( + ) (n,-v/2) ( - ) . The echo a r i s i n g from each p a i r was added to or su b t r a c t e d 48 from computer memory as i n d i c a t e d . The number of echoes c o l l e c t e d was 800 f o r a l l s p e c t r a . A f i v e - p o i n t i n t e r p o l a t i o n method ( D a v i s , 1983) was used t o s h i f t the f i r s t d a t a p o i n t t o the t o p of the echo i n o r d e r t o a v o i d d i s t o r t i o n i n the F o u r i e r t r a n s f o r m . Echoes were r e c o r d e d i n q u a d r a t u r e , and both c h a n n e l s were r e t a i n e d on F o u r i e r t r a n s f o r m a t i o n , r e s u l t i n g i n s l i g h t l y a s y m m e t r i c a l s p e c t r a . A l l d a t a p r o c e s s i n g was performed on a microVAX I com- p u t e r , except f o r the de p a k i n g a l g o r i t h m which was i m p l e - mented on the UBC Amdahl mainframe computer. 6. RESULTS S p e c t r a of a l l samples t a k e n a t 65°C appear i n F i g s . 6 - 1 0 . Fig.11 shows the spectrum of sample B000 at 65°C t o g e t h e r w i t h the same spectrum a f t e r d e p a k i n g ( S t e r n i n et al, 1983). Note t h a t the depaked spectrum appears t w i c e as wide as the powder spectrum, because peaks c o r r e s p o n d t o an o r i e n t a t i o n of 0° t o the magnetic f i e l d d i r e c t i o n r a t h e r than 90°. F i g s . 1 2 - 1 6 a r e p l o t s of Av0r the d o u b l e t s p l i t t i n g measured from the depaked s p e c t r a . These a r e 0° s p l i t t i n g s . Peaks have been a s s i g n e d t o s i t e s on the hydrocarbon c h a i n , assuming t h a t b oth d e u t e r o n s i n each methlene or a l l t h r e e d e u t e r o n s i n the methyl group e x h i b i t the same s p l i t - t i n g . Carbons are numbered from 1 t o 16, from the i o n i c head of the m o l e c u l e t o the t e r m i n a l methyl group. I t has been d e t e r m i n e d p r e v i o u s l y ( S t o c k t o n et al, 1976) t h a t i n c r e a s e d m o t i o n a l freedom towards the end of the c h a i n causes Au0 t o d e c r e a s e m o n o t o n i c a l l y w i t h s i t e number. Peaks f o r some s i t e s o v e r l a p p e d and c o u l d not be r e - s o l v e d , p a r t i c u l a r l y i n the " p l a t e a u " r e g i o n . In F i g s . 1 2 and 11, t h i s r e g i o n i n c l u d e s s i t e s 3 t o 8. In the case of o v e r - l a p p i n g peaks, the i n t e n s i t y of a combined peak was used as an a i d t o e s t i m a t i n g how many s i t e s c o n t r i b u t e d t o i t . Then a l l s i t e s i n c l u d e d were a s s i g n e d an average f r e q u e n c y s p l i t - t i n g . Note t h a t the e r r o r i n d e t e r m i n i n g f r e q u e n c y s p l i t - t i n g s was much l e s s than the s i z e of the p l o t symbols i n F i g s . 1 2 - 1 6 . 49 50 T a b l e I I c o n t a i n s an average 0° s p l i t t i n g , <AP 0>, c a l - c u l a t e d u s i n g the f o r m u l a : <L\P0> = (1/15)[ Z 5 A y 0 ( i ) + 3 A y 0 ( 1 6 ) ] 2 where the index i r e f e r s t o the s i t e number on the c h a i n . The reason f o r the f a c t o r of 3 m u l t i p l y i n g the methyl group s p l i t t i n g i s t h a t c- 2H bonds i n the methyl group a r e a t a d i f f e r e n t a n g l e t o the m o l e c u l a r a x i s of symmetry f o r r e o r - i e n t a t i o n s (the l o c a l b i l a y e r normal) from t h e i r c o u n t e r - p a r t s i n the methylene groups, and a r e undergoing an a d d i - t i o n a l r o t a t i o n about the l a s t C-C bond ( S e e l i g , 1977). Because the weight of soap i n the samples was about 40mg, the volume of b u f f e r r e q u i r e d was r a t h e r s m a l l , l e s s than 20/z/ . B u f f e r was added w i t h a v o l u m e t r i c s y r i n g e a c c u r - a t e t o ±.5ul. Thus the amount of b u f f e r added c o u l d not be c o n t r o l l e d t o b e t t e r than about ±3%. However, by w e i g h i n g the sample b e f o r e and a f t e r a d d i n g b u f f e r , the a c t u a l amount added c o u l d be d e t e r m i n e d t o w i t h i n ±.5%. The amount of wat- er i n each sample, e x p r e s s e d as a p e r c e n t a g e of the amount r e q u i r e d f o r a 7.75:1 water:soap molar r a t i o , i s l i s t e d i n T a b l e I I . S i n c e i s q u i t e s e n s i t i v e t o water c o n t e n t i n t h i s regime (Kok, 1985) we d e c i d e d t o a p p l y a c o r r e c t i o n t o <Av 0>. W i t h t h i s c o r r e c t i o n , <Av0> f o r a l l samples can be compared as i f a l l had e x a c t l y the water:soap molar r a t i o g i v e n above. The c o r r e c t e d v a l u e s , a l s o l i s t e d i n T a b l e I I , 5 1 have been c a l c u l a t e d u s i n g the d a t a of Kok (1985). She s t u - d i e d p e r d e u t e r a t e d p o t a s s i u m p a l m i t a t e a t c o n c e n t r a t i o n s of 85.7 and 87.6mole-%, both a t 55°C. Our c o r r e c t i o n was a mu- l t i p l i c a t i v e f a c t o r , o b t a i n e d by c a l c u l a t i n g the f r a c t i o n a l change i n <Av 0> w i t h water c o n t e n t . We chose t o c a l c u l a t e t h e c o r r e c t i o n as a f r a c t i o n a l change r a t h e r than an abso- l u t e change because the magnitude of the s p l i t t i n g s she mea- su r e d was about 10-15% l a r g e r than the s p l i t t i n g s we mea- su r e d f o r z e r o - p e p t i d e samples B000 and D000. From the above d i s c u s s i o n , i t i s c l e a r t h a t F i g s . 1 2 - 1 6 compare samples w i t h s l i g h t l y d i f f e r e n t water c o n t e n t . By r e f e r r i n g t o T a b l e I I , one can see by how much, on average, the s p l i t t i n g s f o r each sample a r e h i g h or low. The maximum average c o r r e c t i o n i s about 1kHz, about the s i z e of the p l o t symbols i n F i g s . 1 2 - 1 6 . From the c o r r e c t e d average s p l i t t i n g <AP 0> has been c a l c u l a t e d a c o r r e c t e d average a b s o l u t e o r d e r parameter |<5 C D>|, u s i n g a v a l u e of 167kHz ( D a v i s & J e f f r e y , 1977) f o r the q u a d r u p o l a r c o u p l i n g c o n s t a n t e2qQ/h. Thus: |<5 C D>| = <_i>o>/250kHz. F i g . 17 p r e s e n t s the c o r r e c t e d v a l u e s of I <^CD > I ^ R O M T a b l e I I . U n c o r r e c t e d v a l u e s a r e a l s o p l o t t e d f o r compari- son. The c o r r e c t e d v a l u e s have been g i v e n e r r o r b a r s t o r e - f l e c t the u n c e r t a i n t y i n s p l i t t i n g d e t e r m i n a t i o n . 52 F i g s . 18-20 compare s p l i t t i n g s . f o r three of the samples temperatures 49°C and 65°C. 53 1 DOOO f^KkJ I 1 1 BOOO ^ M K , 1 I I 1 1 -30 -20 -10 0 10 20 30 frequency (kHz) F i g . 6 . 65°C s p e c t r a : BOOO, DOOO. 54 241 L J 242 BOOO ^ y ^ w J I I I I I - 3 0 - 2 0 -10 0 10 20 30 frequency (kHz) F i g . 7 . 65°C s p e c t r a : BOOO, 242, 241. 55 201 ^^^J I I 202 ^ ^ A J B000 M V U J I I I I I -30 -20 -10 0 10 20 30 frequency (kHz) F i g . 8 . 65°C s p e c t r a : BOOO, 202, 201 . 56 F i g . 9 . 65°C s p e c t r a : BOOO, 162, 161. 57 -30 -20 -10 0 10 20 30 frequency (kHz) F i g . 1 0 . 65°C s p e c t r a : BOOO, Z102, 101. 58 -30 -20 -10 0 10 20 30 frequency (kHz) Fig.11- 65°C spectra: (a) BOOO; (b) BOOO, depaked. i g . 1 2 . Av0 v s . s i t e number at 65°C: BOOO, DOOO. 60 Q_j 1 ! , 1 1 1 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 site number F i g . 13. Ai>0 v s . s i t e number a t 65°C: BOOO, 242, 241 . 61 < H — i — i — i — i — i — i — i — i — i — i — i — ~ i — i — ' 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 site number F i g . 1 4 . A f 0 v s . s i t e number a t 65°C: BOOO, 202, 201. 62 6 0 * O H — i — i — i — i — i — i — i — i — i — i — i — i — i — i 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 site number F i g . 1 5 . Av0 VS. s i t e number a t 65°C: BOOO, 162, 161. 63 1 I I 1 1 1 1 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 site number F i g . 1 6 . A f 0 v s . s i t e number a t 65°C: BOOO, Z102, 101. 64 0.144 0.140- 0.136- 0.132- 0.128-I ~ 0.124- n u v 0.120- 0.116- 0.112- 0.108 0.104-j 0.100 1 4 x ^%o°° ^ ^ ^ ^ *N ^ sample F i g . 1 7 . |<5 C D>| f o r a l l samples a t 65°C. C r o s s e s r e f e r t o u n c o r r e c t e d v a l u e s , t r i a n g l e s w i t h e r r o r b a r s t o c o r r e c t e d v a l u e s . 65 F i g . 1 8 . BOOO s p l i t t i n g s : 49°C, 65°C. 66 60-a, { Legend • A24249 N I 40 H j? 30H CL W 20 H ioH A X A24265 v * — * N x N S \ — i — i — i — i — i — i — i — i — i — i — i — i — i — i 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 site number Fig.19. 242 s p l i t t i n g s : 49°C, 65°C. 67 60- I Legend 50 A 401 TP X g> 30 a 20 H 10 H w \ * B24149_ A B24165 \ \ ~~1 I 1 1 1 1 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 site number F i g . 2 0 . 241 s p l i t t i n g s : 49°C, 65°C. 68 Sample % Desired < Av Q > < A vfl > |< S C D>| Water Content Uncorrected Corrected Corrected (kHz) (kHz) 241 102.0 33.4 34.0 .136 242 96.0 32.0 30.8 .123 201 98.7 34.4 34.0 .136 202 98.0 31.5 30.9 .124 161 103.3 34.3 35.4 .142 162 100.5 31.5 31.7 .127 101 103.7 30.8 31.9 .128 Z102 98.7 33.3 32.9 .132 BOOO 102.0 27.3 27.8 .111 DOOO 101.6 28.9 29.3 .117 T a b l e I I . Sample water c o n t e n t , <Af 0 > and |<5CD>|. 7. DISCUSSION I t i s easy t o un d e r s t a n d t h e g e n e r a l c h a r a c t e r i s t i c s d i s p l a y e d by a l l powder s p e c t r a p r e s e n t e d , F i g s . 1 0 - 1 4 . A l l c o n s i s t of 16 superposed powder p a t t e r n s of d i f f e r i n g f r e - quency s p l i t t i n g , and some a l s o d i s p l a y an i s o t r o p i c water peak a t z e r o f r e q u e n c y . Each of the 14 outermost powder p a t t e r n s a r i s e s from two 2H atoms i n a methylene group a t some p o s i t i o n on the c h a i n . S p l i t t i n g s d e c r e a s e m o n o t o n i c a l l y from s i t e 2 t o s i t e 15, as mentioned i n Chapter 6. The powder p a t t e r n se- cond from the c e n t r e i s due t o t h e t h r e e 2H atoms i n the me- t h y l group. The i n t e n s i t y of t h i s p a t t e r n i s r o u g h l y 3/2 t h a t of the methylene p a t t e r n s , as e x p e c t e d . The centremost powder p a t t e r n i s due t o 2 H 2 0 m o l e c u l e s which a r e e x e c u t i n g a n i s o t r o p i c motion. I t s c e n t r e does not c o i n c i d e e x a c t l y w i t h the c e n t r e of the soap powder p a t t e r n s due t o the d i f - f e r e n t c h e m i c a l s h i f t of 2H i n 2 H 2 0 as compared t o C- 2H. The s i n g l e c e n t r a l peak i s due t o water m o l e c u l e s ( 2H 20 or 2H 1HO) which a re e x e c u t i n g i s o t r o p i c m otion, t u m b l i n g r a n - domly a t a fr e q u e n c y much g r e a t e r than t h a t c h a r a c t e r i z i n g the i n t e r a c t i o n , the q u a d r u p o l a r c o u p l i n g c o n s t a n t . The temperature b e h a v i o r of t h e s e samples ( F i g s . 1 8 - 2 0 ) i s l i t t l e m o d i f i e d by the presence of the p e p t i d e s . As i s w e l l known i n p o t a s s i u m p a l m i t a t e ( D a v i s & J e f f r e y , 1977; see e s p e c i a l l y t h e i r F i g . 3 ) the lower temperature s p e c t r a e x h i b i t a " p l a t e a u " r e g i o n of o n l y s l i g h t l y v a r y i n g s p l i t - t i n g s below about s i t e 7. T h i s p l a t e a u i s m o d i f i e d , t o a 69 70 g r e a t e r or l e s s e r degree, by an i n c r e a s e i n Av0 below s i t e 6 and an accompanying d e c r e a s e above t h i s s i t e which occur w i t h an i n c r e a s e i n t e m p e r a t u r e . F i g u r e s 10 and 16 compare s p e c t r a from samples BOOO and D000 (no m e t h a n o l ) . There a r e s l i g h t d i f f e r e n c e s , which we surmise may be due t o i n c o m p l e t e e v a p o r a t i o n of methanol from BOOO d u r i n g p r e p a r a t i o n . As BOOO was pr e p a r e d i n e x a c t - l y t he same manner as the o t h e r samples, we have s e l e c t e d i t as the z e r o - p e p t i d e s t a n d a r d w i t h which we compare the o t h e r samples. F i g u r e s 7 and 13 show the r e s u l t s of p e p t i d e 24 a t c o n c e n t r a t i o n s of about 1:200 and 1:100. Note, from Table I I , t h a t s p l i t t i n g s from sample 242 a r e about 1kHz too h i g h , w h i l e those from 241 a r e about ,5kHz t o o low. Thus the e f f e c t of p e p t i d e c o n c e n t r a t i o n on s p l i t t i n g s i s c l o s e r t o bei n g l i n e a r than i t seems from a c a s u a l i n s p e c t i o n of F i g . 1 3 . T a b l e I I shows t h a t <&v0> f o r 241 i s about 22% g r e a t e r than <Af 0> f o r BOOO, both a t 65°C. At 49°C, though b o t h v a- l u e s of M, have i n c r e a s e d , t h i s number i s s t i l l 22%. I f we c a l c u l a t e M, f o r t h e s e s p e c t r a u s i n g ( D a v i s , 1983): M, = (2ir/3v/3)<Af 0> we o b t a i n a t 49°C an i n c r e a s e i n from 3.4-10's"' t o 4.1«10*s" 1. These v a l u e s s h o u l d be compared w i t h S.S'lO's" 1 and 4.0«lO as" 1 o b t a i n e d by D a v i s et al (1983). Our 71 measurements of the e f f e c t s of p e p t i d e 24 agree w e l l w i t h t h o s e of the e a r l i e r s t u d y . In F i g u r e s 8,9,14 and 15 we see t h a t p e p t i d e s 20 and 16 a f f e c t soap s p l i t t i n g s i n much t h e same way as p e p t i d e 24 does. T h i s i s emphasized by F i g . 2 0 , which compares |<5 C D>| f o r a l l samples. A l l v a l u e s of |<5^D>| f o r the lower c o n c e n t r a t i o n s of the " f u l l p e p t i d e s " 16,20 and 24 l i e c l o s e t o a v a l u e of .125. A l l the h i g h e r c o n c e n t r a t i o n v a l u e s l i e c l o s e t o .138. Compared t o |<5^D>|=.111 o b t a i n e d f o r sample BOOO, the i n - c r e a s e i n |<5 C D>| i s a p p r o x i m a t e l y l i n e a r w i t h p e p t i d e con- c e n t r a t i o n f o r a l l of thes e p e p t i d e s . U s i n g the S c h i n d l e r - S e e l i g f o r m u l a , the above-quoted |<5 C D>| v a l u e s of .111, .125 and .138 y i e l d b i l a y e r t h i c k - ness e s t i m a t e s of 22.9A, 23.4A and 23.9A, r e s p e c t i v e l y . These v a l u e s compare f a v o u r a b l y w i t h the r e s u l t s o b t a i n e d by G a l l o t and S k o u l i o s (1966) quoted i n Chapter 4. R e f e r r i n g t o F i g . 2 0 , we see t h a t j u d g i n g from sample 101, the e f f e c t of the " h a l f p e p t i d e " 10 i s t o i n c r e a s e | < S Q D > | by o n l y about h a l f the amount caused by the o t h e r p e p t i d e s a t the same c o n c e n t r a t i o n . R e s u l t s from sample 102 ar e not i n agreement w i t h t h i s c o n c l u s i o n , however. Tog e t h e r , t h e s e r e s u l t s l e a d us t o c o n c l u d e t h a t the e f f e c t on | < ^ r j > l °^ t h i s type of p e p t i d e i s u n c o r r e l a t e d w i t h h y d r o p h o b i c mismatch, a t l e a s t f o r p e p t i d e s l o n g e r than the pure soap b i l a y e r t h i c k n e s s . W h i l e the pure soap's hy- dr o p h o b i c t h i c k n e s s i s about 24A, we f i n d t h a t the a d d i t i o n 72 of b i l a y e r - s p a n n i n g p e p t i d e s w i t h h y d r o p h o b ic r e g i o n s of 24-36A a t 1:100 p e p t i d e : l i p i d molar r a t i o i s accompanied un- i f o r m l y by an i n c r e a s e i n |<5CD>I °f about 20%. T h i s r e - f l e c t s an i n c r e a s e i n b i l a y e r t h i c k n e s s of o n l y about IA. M o u r i t s e n and Bloom (1984) i n t h e i r m a t t r e s s model pap- er suggest t h a t the model i s most a p p l i c a b l e t o the type of p e p t i d e s we have used i n the p r e s e n t s t u d y , because they a re "smoother" than p r o t e i n s and have no s p e c i f i c l i p i d b i n d i n g s i t e s . However, the e f f e c t of the s e p e p t i d e s on DPPC, a b i o - l o g i c a l p h o s p h o l i p i d , o b s e r v e d by D a v i s et al and H u s c h i l t et al , i s s i m i l a r t o t h a t of p r o t e i n s i n o t h e r p r o t e i n - 1 i p i d systems which have been s t u d i e d : c h a i n o r d e r i s not much a f - f e c t e d . T h i s s u g g e s t s t h a t the "rough p r o t e i n s u r f a c e " con- j e c t u r e ( S e e l i g & S e e l i g , 1980) d i s c u s s e d i n Chapter 3 may not be t r u e . There a r e s e v e r a l reasons why these p a r t i c u l a r p e p t i d e s were p r o b a b l y not the be s t c h o i c e f o r a t e s t of the m a t t r e s s model. The f i r s t ( D a v i s et al, 1983) i s t h a t l y s i n e , the amino a c i d chosen as h y d r o p h i l i c "anchor," has an a m p h i p h i l - i c c h a r a c t e r of i t s own, which may a l l o w the p e p t i d e s t o ad- j u s t t h e i r l e n g t h t o some degree. T h i s means t h a t a p e p t i d e of t h i s t y p e which i s t r u l y t o o s h o r t f o r the b i l a y e r would have perhaps o n l y a few l e u c i n e s . T h e r e f o r e , c o n s i d e r i n g a l s o i t s f i v e c h arges a t n e u t r a l pH, such a p e p t i d e might not be s t r o n g l y t h e r m o d y n a m i c a l l y d r i v e n t o j o i n t he b i l a y - er . 7 3 The second reason i s t h a t a p e p t i d e of t h i s type which i s too l o n g f o r the b i l a y e r , as were t h i s s t u d y ' s p e p t i d e s 16,20 and 24, c o u l d s i m p l y l i e a t an a n g l e w i t h i n the b i l a y e r i n o r d e r t o match hydrophobic and h y d r o p h i l i c r e g i o n s . T h i s might not be the case f o r a b u l k y p r o t e i n con- s i s t i n g of a number of a - h e l i c e s and/or ^ - s h e e t s . Such a p r o t e i n might in d e e d p e r t u r b b i l a y e r t h i c k n e s s i n the manner suggested by t h e m a t t r e s s model. In c o n c l u s i o n , we have found no e v i d e n c e t o a i d us i n d i s c r i m i n a t i n g between p o s s i b l e c o n j e c t u r e s r e g a r d i n g the l a c k of p e r t u r b a t i o n on b i o l o g i c a l p h o s p h o l i p i d b i l a y e r s by p r o t e i n s . The "rough p r o t e i n s u r f a c e " c o n j e c t u r e has been thrown i n t o some q u e s t i o n by the work of D a v i s et al and Hu- s c h i l t et al w i t h p e p t i d e s of the type used i n t h i s s t u d y . The " s q u i s h y p r o t e i n s " c o n j e c t u r e i s s t i l l a b l e t o e x p l a i n a l l e x t a n t r e s u l t s . The m a t t r e s s model i s not i n c o m p a t i b l e w i t h the r e s u l t s of the p r e s e n t s t u d y , and so s t i l l s t a n d s as a p o s s i b l e e x p l a n a t i o n . REFERENCES Abdollal, K.r E.E. Burnell & M.I. V a l i c (1977). The Tempera-ture dependence of water and counter ion order in soap-water mesophases. 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