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UBC Theses and Dissertations

A deuterium NMR study of gramicidin A’ Lyons, Michael James 1985

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A DEUTERIUM NMR STUDY OF GRAMICIDIN A' by MICHAEL JAMES LYONS B . S c , M c G i l l U n i v e r s i t y , 1983 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 accept 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 1985 © M i c h a e l James Lyons, 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date /$//£>/8S DE-6(3/81) A b s t r a c t T h i s t h e s i s p r e s e n t s t h e r e s u l t s of the f i r s t a p p l i c a t i o n of a n o v e l s o l i d s t a t e n u c l e a r magnetic resonance t e c h n i q u e (K. P. P a u l s e t . a l . , E u r . B i o p h y s . J . 11:1) t o a n a t u r a l l y o c c u r i n g membrane p o l y p e p t i d e . D e u t e r i u m NMR was used t o study the s t r u c t u r e and dynamics of hydrogen-exchanged g r a m i c i d i n A*, an i o n c h a n n e l , i n model membranes. The t e c h n i q u e e x p l o i t s r e c e n t l y d e v e l o p e d p r o c e d u r e s f o r s o l v e n t - s i g n a l s u p p r e s s i o n (P. T. C a l l a g h a n e t . a l . , J . Magn. Reson. 56:101), and "depakeing" powder s p e c t r a (E. S t e r n i n , M.Sc. T h e s i s , U . B . C . ) . The s p e c t r a of g r a m i c i d i n A' i n c r y s t a l l i n e form, and i n the g e l phase of the l i p i d b i l a y e r a r e s i m i l a r and i n d i c a t e l i t t l e m o l e c u l a r m o t i o n on the NMR t i m e s c a l e . In the l i q u i d c r y s t a l l i n e phase, however, the s p e c t r a suggest r a p i d u n i a x i a l r o t a t i o n of the g r a m i c i d i n about the b i l a y e r d i r e c t o r . The f r e q u e n c i e s of the l i q u i d c r y s t a l l i n e phase s p e c t r a were found t o be independent of b i l a y e r t h i c k n e s s , t e m p e r a t u r e , and t h e presence of sodium c h l o r i d e , i n the ranges i n v e s t i g a t e d . The r e s u l t s a r e d i s c u s s e d i n the c o n t e x t of the c o n d u c t i o n p r o p e r t i e s of the g r a m i c i d i n i o n c h a n n e l , o t h e r s p e c t r o s c o p i c s t u d i e s , and t h e c r e t i c a l models of the s t r u c t u r e and a c t i o n of g r a m i c i d i n . i i T a b l e of C o n t e n t s A b s t r a c t i i L i s t of T a b l e s i v L i s t of F i g u r e s v Acknowledgements v i I . I n t r o d u c t i o n 1 11 . Gramic i d i n 4 A. B i o l o g i c a l F u n c t i o n 4 B. G r a m i c i d i n : a Model System f o r the Study of Ion T r a n s p o r t 5 C. Conductance P r o p e r t i e s of G r a m i c i d i n 7 D. S t r u c t u r a l S t u d i e s of G r a m i c i d i n 9 I I I . S o l i d S t a t e NMR as a S t r u c t u r a l T o o l 14 A. D e u t e r ium NMR S p e c t r a ( 6 , 7 , 8 , 3 7 ) 15 B. A Deuterium NMR Study of a S y n t h e t i c P o l y p e p t i d e 16 IV. M a t e r i a l s and Methods 21 A. M a t e r i a l s 21 B. NMR Sample P r e p a r a t i o n 21 C. 2H NMR E x p e r i m e n t s 22 V. R e s u l t s 24 VI . D i s c u s s i o n 34 V I I . C o n c l u d i n g Remarks 40 BIBLIOGRAPHY 41 i i i L i s t of T a b l e s F l u i d S t a t e Quadrupolar S p l i t t i n g s i v L i s t of F i g u r e s 1. E x p e r i m e n t a l and S i m u l a t e d S p e c t r a of C r y s t a l l i n e P o l y p e p t i d e 17 2. G e l and F l u i d Phase S p e c t r a of the P o l y p e p t i d e 19 3. E x p e r i m e n t a l and S i m u l a t e d S p e c t r a of C r y s t a l l i n e G r a m i c i d i n 25 4. Fr e e I n d u c t i o n Decay B e f o r e and A f t e r S o l v e n t S i g n a l S u b t r a c t i o n 26 5. G e l and F l u i d Phase S p e c t r a of G r a m i c i d i n i n DPPC 27 6. F l u i d Phase Spectrum of G r a m i c i d i n i n DMPC 29 7. F l u i d Phase Spectrum of G r a m i c i d i n i n DLPC 30 8. F l u i d Phase Spectrum of G r a m i c i d i n i n the Presence of Ions 31 9. A r r h e n i u s P l o t of T 2e vs. Temperature 33 v Acknowledgements I would l i k e t o e x p r e s s my g r a t i t u d e t o the f o l l o w i n g p e o p l e : Myer Bloom, f o r s u p e r v i s i n g t h i s t h e s i s , and f o r h i s p a t i e n c e when I f o l l o w e d my own i n t e r e s t s ; A l e x MacKay f o r h e l p and encouragement, and f o r v a l u a b l e comments r e g a r d i n g the m a n u s c r i p t ; K l a a s - P e t e r Datema and P e t e r P a u l s f o r p r o v i d i n g me w i t h t h e i r u n p u b l i s h e d f i n d i n g s ; N e a l P o u l i n f o r i n t e r e s t i n g d i s c u s s i o n s . F i n a l l y , I w i s h t o d e d i c a t e t h i s t h e s i s t o my p a r e n t s James and Margaret Lyons. v i I . INTRODUCTION The attempt t o u n d e r s t a n d the b i o l o g i c a l a c t i o n of p r o t e i n s i n terms of p h y s i c a l s c i e n c e p r e s e n t s a c h a l l e n g e t o s c i e n t i s t s of many d i s c i p l i n e s . For i n t e g r a l membrane p r o t e i n s t h i s program i s a t the stage of s t r u c t u r e d e t e r m i n a t i o n . These p r o t e i n s have, u n t i l r e c e n t l y ( 1 , 2 ) , r e s i s t e d c r y s t a l l i z a t i o n , hence the c l a s s i c a l x-ray d i f f r a c t i o n t e c h n i q u e s , so u s e f u l i n s t u d i e s of s o l u b l e m a c r o m o l e c u l e s , have been i n a p p l i c a b l e . The case of g r a m i c i d i n A' 1, which can be c r y s t a l l i z e d from o r g a n i c s o l v e n t s , i l l u s t r a t e s a f u r t h e r d i s a d v a n t a g e of the x-ray d i f f r a c t i o n method. The x - r a y a n a l y s i s (3,4) of i o n bound and i o n f r e e forms of g r a m i c i d i n i n d i c a t e a major i o n - i n d u c e d change i n secondary s t r u c t u r e * of the p o l y p e p t i d e . T h i s r e s u l t i s a t v a r i a n c e w i t h the r e s u l t s of s p e c t r o s c o p i c e x p e r i m e n t s on g r a m i c i d i n i n a r t i f i c i a l membranes ( 5 ) . I t i s t h e r e f o r e f a r from c e r t a i n t h a t a g i v e n membrane p r o t e i n ' s s t r u c t u r e w i l l not be a f f e c t e d by c r y s t a l l i z a t i o n . D euterium n u c l e a r magnetic r e s o n a n c e 2 i s an e f f e c t i v e t o o l f o r s t u d y i n g t h e s t r u c t u r e and dynamics of 2H l a b e l l e d components ( f o r r e v i e w s see 6,7). The a n i s o t r o p i c r o t a t i o n s of a m o l e c u l e i n the l i q u i d c r y s t a l l i n e phase of a membrane p a r t i a l l y a verages t h e o r i e n t a t i o n dependent e l e c t r i c q u a d r u p o l e i n t e r a c t i o n , t h e r e s i d u a l q uadrupole s p l i t t i n g c o n t a i n i n g i n f o r m a t i o n about the s t r u c t u r e and r o t a t i o n of 'H e n c e f o r t h r e f e r r e d t o as g r a m i c i d i n . A b b r e v i a t e d 2H NMR. 1 2 the molecule. Under c e r t a i n c o n d i t i o n s measurement of s p e c t r a and r e l a x a t i o n times can p rovide order parameters and c o r r e l a t i o n times f o r the motion of a l a b e l l e d s i t e . When the s i t e i s attached to a molecule behaving as a r i g i d body r o t a t i n g u n i a x i a l l y , with a c o r r e l a t i o n time shorter than the a p p r o p r i a t e NMR t i m e s c a l e , the angle between the p r i n c i p l e a x i s of the e l e c t r i c f i e l d g r a d i e n t , and the a x i s of r o t a t i o n may be c a l c u l a t e d from the measured order parameter. A s y n t h e t i c p o l y p e p t i d e , 2 H - l a b e l l e d at the amide hydrogens, was s t u d i e d u sing 2H-NMR by K.P.Pauls et . al . (8). The spectrum observed in the f l u i d s t a t e of the b i l a y e r c o n s i s t e d of a powder p a t t e r n with a s i n g l e quadrupole s p l i t t i n g i n d i c a t i n g a h e l i c a l secondary s t r u c t u r e f o r the p o l y p e p t i d e r o t a t i n g about the h e l i x a x i s , c o n s i s t e n t with the expected alpha h e l i x s t r u c t u r e . Measurement of T 2 e , the r e l a x a t i o n time f o r the decay of the quadrupole echo, allowed c a l c u l a t i o n of the r o t a t i o n a l c o r r e l a t i o n time for a simple model of the molecule's r o t a t i o n a l d i f f u s i o n . F u r t h e r , the angle between the h e l i x a x i s and the amide N-D bonds was c a l c u l a t e d from the amide 2H order parameter and found to be w i t h i n the range p r e d i c t e d f o r the a - h e l i x . The success of t h i s study prompted a p p l i c a t i o n of the technique to more complicated p o l y p e p t i d e s and p r o t e i n s . G r a m i c i d i n was chosen as an i n t e r e s t i n g candidate f o r study because of i t s small s i z e (15 amino a c i d s ) , and i t s expected h e l i c a l s t r u c t u r e i n the membrane. D e s p i t e the s i m p l i c i t y of 3 i t s s t r u c t u r e , g r a m i c i d i n e x h i b i t s some f u n c t i o n a l l y i n t e r e s t i n g p r o p e r t i e s . In i o n c o n d u c t i o n e x p e r i m e n t s g r a m i c i d i n i s found t o render l i p i d membranes s e l e c t i v e l y permeable t o s m a l l monovalent c a t i o n s . The conductance p r o p e r t i e s of g r a m i c i d i n a r e w e l l c h a r a c t e r i z e d ; because of i t s l a r g e s i n g l e c h a n n e l conductance and o t h e r f e a t u r e s i t has c o n t i n u e d t o be of i n t e r e s t as a model system f o r b i o l o g i c a l i o n t r a n s p o r t . F u r t h e r m o r e , t h e r e a r e s e v e r a l groups (9,10,11) a t t e m p t i n g m o l e c u l a r dynamics s i m u l a t i o n s of g r a m i c i d i n - m e d i a t e d t r a n s p o r t . R e l i a b l e s t r u c t u r a l i n f o r m a t i o n i s needed i n t h i s a m b i t i t i o u s u n d e r t a k i n g . The r e s u l t s of t h i s 2H-NMR study of g r a m i c i d i n w i l l be d i s c u s s e d a l o n g w i t h the p o l y p e p t i d e study (8) and the work of K.P.Datema and K.P.Pauls (12) as w e l l ' as p r e v i o u s s p e c t r o s o p i c and e l e c t r i c a l measurements. "The f o l l o w i n g major c o n c l u s i o n s a re drawn: t h a t g r a m i c i d i n adopts a f a i r l y r i g i d c o n f o r m a t i o n ( s ) which does (do) not deform t o the c o n s t r a i n t s of the b i l a y e r , and t h a t i s ( a r e ) independent, of temperature i n the range i n v e s t i g a t e d ; t h a t no l a r g e change i n secondary s t r u c t u r e o c c u r s i n the presence of sodium i o n s . I I . GRAMICIDIN A. BIOLOGICAL FUNCTION G r a m i c i d i n , a l i n e a r p o l y p e p t i d e h a v i n g f i f t e e n amino a c i d s , was o r i g i n a l l y i s o l a t e d from Bacillus brevis (13) as a m o l e c u l e w i t h broad spectrum a n t i b i o t i c a c t i o n . The p r i m a r y s t r u c t u r e of the g r a m i c i d i n s were d e t e r m i n e d t o b e 3 : Formyl-L - X X X-GLY-L-ALA - D-LEU-L-ALA - D-VAL-L-VAL - D-VAL-L - T R P - D-LEU-L-YYY - D-LEU-L - T R P - D - L E U - L - T R P - E t h a n o l a m i ne where X X X denotes v a l i n e or i s o l e u c i n e , and YYY denotes t r y p t o p h a n , p h e n y l a l a n i n e , or t y r o s i n e i n g r a m i c i d i n A,B,and C r e s p e c t i v e l y ( 1 5 , o r i g i n a l r e f e r e n c e s c i t e d t h e r e i n ) . C o m m e r c i a l l y a v a i l a b l e g r a m i c i d i n A' i s a m i x t u r e of v a l i n e g r a m i c i d i n s A,B,C i n the p r o p o r t i o n s 7:1:2 ,and a s m a l l amount of the i s o l e u c i n e g r a m i c i d i n s ( 1 6 ) . G r a m i c i d i n may p r o v i d e a s e l e c t i v e advantage f o r Bacillus brevis by making a c o m p e t i t o r ' s membranes l e a k y t o c a t i o n s . Recent work (17,18) has suggested a d i f f e r e n t r o l e f o r g r a m i c i d i n , independent of i t s i o n t r a n s p o r t i n g a c t i o n . G r a m i c i d i n - l a c k i n g mutants of Bacillus brevis were found unable t o form normal spores u n l e s s p r o v i d e d w i t h g r a m i c i d i n . T h i s e f f e c t has been c o r r e l a t e d w i t h the a b i l i t y of g r a m i c i d i n t o s p e c i f i c a l l y i n h i b i t RNA t r a n s c r i p t i o n by i n h i b i t i n g b a c t e r i a l RNA polymerase. E x p e r i m e n t s w i t h s y n t h e t i c g r a m i c i d i n analogues showed t h a t o n l y t h e 3 S t a n d a r d t h r e e - l e t t e r amino a c i d a b b r e v i a t i o n s (14) a r e used h e r e . 4 5 sequence: (D-LEU-L-TRP)-Ethanolamine i s needed t o r e s t o r e s p o r u l a t i o n i n the mutants. As d i s c u s s e d below, s e v e r a l p r o p e r t i e s of g r a m i c i d i n ' s p r i m a r y s t r u c t u r e , such as the s t r i c t l y a l t e r n a t i n g c h i r a l i t y ( except f o r g l y c i n e which i s not o p t i c a l l y a c t i v e ) of the component amino a c i d s , a r e e s s e n t i a l t o i t s c h a n n e l forming p r o p e n s i t y . The au t h o r f e e l s , t h e n , t h a t i o n t r a n s p o r t s h o u l d not be d i s c a r d e d as a p o s s i b l e b i o l o g i c a l f u n c t i o n of gramic i d i n . B. GRAMICIDIN:A MODEL SYSTEM FOR THE STUDY OF ION TRANSPORT A l t h o u g h i t s n a t u r a l f u n c t i o n may be o b s c u r e , g r a m i c i d i n has s e r v e d .as an i m p o r t a n t model system i n the i n v e s t i g a t i o n of b i o l o g i c a l i o n t r a n s p o r t . I n i t i a l l y found t o decouple o x i d a t i v e p h o s p h o r y l a t i o n i n m i t o c h o n d r i a , g r a m i c i d i n was c h a r a c t e r i z e d as an ionophore s e l e c t i v e f o r monovalent c a t i o n s u s i n g the then newly de v e l o p e d b l a c k l i p i d membrane t e c h n i q u e . In a b l a c k l i p i d f i l m experiment a s m a l l amount of l i p i d d i s s o l v e d i n an o r g a n i c s o l v e n t i s p a i n t e d a c r o s s a p i n h o l e s e p a r a t i n g two compartments c o n t a i n i n g s o l u t i o n s of e l e c t r o l y t e s . The l i p i d membrane t h i n s u n t i l a s i n g l e b i l a y e r p e r s i s t s which ( b e i n g much t h i n n e r than the wavelength of v i s i b l e l i g h t ) appears b l a c k when viewed w i t h a m i c r o s c o p e . E l e c t r i c a l p r o p e r t i e s of the membrane can then be m o n i t o r e d u s i n g s e n s i t i v e v o l t a g e and c u r r e n t meters. 6 For b i l a y e r f o r m i n g l i p i d s , b l a c k membranes a r e observ e d t o be impermeable t o i o n s . A crude u n d e r s t a n d i n g of the i n s u l a t i n g p r o p e r t i e s may be had by c o n s i d e r i n g the e l e c t r o s t a t i c s e l f - e n e r g y , U, of a sodium i o n , w i t h charge Q=4.8x10~ 1 0 esu,and r a d i u s = l A , i n an i n f i n i t e medium, g i v e n i n esu u n i t s as (19) : U= Q 2/(2eR) Us i n g a p p r o x i m a t e v a l u e s f o r the d i e l e t r i c c o n s t a n t s , e=80 f o r water ,and e=2 f o r o i l , we f i n d a d i f f e r e n c e i n s e l f - e n e r g y of 3.5 eV f o r an i o n i n o i l and water . The l i p i d b i l a y e r t h u s p r e s e n t s a l a r g e p o t e n t i a l energy b a r r i e r t o i o n s . The i m p e r m e a b l i t y of the l i p i d m a t r i x of a b i o l o g i c a l membrane t o e l e c t r o l y t e s has wide r a n g i n g consequences. I t e n a b l e s the c e l l t o m a i n t a i n an asymmetry, w i t h r e s p e c t t o i o n c o n c e n t r a t i o n f o r example, a c r o s s a plasma o r o r g a n e l l e membrane. T h i s asymmetry i s a u n i v e r s a l p r o p e r t y of b i o l o g i c a l membranes. The c e l l s of the body , f o r example, have an i n t r a c e l l u l a r c o n c e n t r a t i o n of sodium of c.5-15 mM and an e x t r a c e l l u l a r c o n c e n t r a t i o n of c.140mM. For p o t a s s i u m the e x t r a - and i n t r a c e l l u l a r c o n c e n t a t i o n s , r e s p e c t i v e l y , a r e r o u g h l y t h e same ( 2 0 ) . Ion s e l e c t i v e a c t i v e "pumps" and p a s s i v e " l e a k s " a r e n e c e s s a r y i n t h e maintenance of a s t a b l e i o n g r a d i e n t . I t i s g e n e r a l l y assumed t h a t t h e s e mechanisms i n v o l v e p r o t e i n s embedded i n the membrane, however t r a n s p o r t a c t i v i t y has proven d i f f i c u l t t o i s o l a t e ( 2 1 ) . I t i s not s u r p r i s i n g t h e n t h a t the i o n t r a n s p o r t i n g p r o p e r t i e s of 7 g r a m i c i d i n , a s i m p l e p o l y p e p t i d e , e x c i t e d c o n s i d e r a b l e i n t e r e s t . C. CONDUCTANCE PROPERTIES OF GRAMICIDIN The conductance of .ions by g r a m i c i d i n has been w e l l s t u d i e d u s i n g the b l a c k l i p i d membrane t e c h n i q u e d e s c r i b e d above. An e l e g a n t experiment (22) f o l l o w e d the time c o u r s e of c u r r e n t f l u c t u a t i o n s a t c o n s t a n t v o l t a g e a c r o s s a membrane c o n t a i n i n g a s m a l l amount of g r a m i c i d i n . The f l u c t u a t i o n s were found t o be a p p r o x i m a t e l y i n t e g r a l m u l t i p l e s of a u n i t v a l u e , which was s u b s e q u e n t l y i n t e r p r e t e d as the conductance of a s i n g l e open c h a n n e l . The a u t o c o r r e l a t i o n f u n c t i o n of the c u r r e n t n o i s e was l a t e r (23) found t o be an e x p o n e n t i a l i n d i c a t i n g the c h a n n e l c l o s u r e t o be a P o i s s o n random p r o c e s s . The conductance of a s i n g l e c h a n n e l i s independent of the t h i c k n e s s of the l i p i d b i l a y e r ( 2 4 ) , however c h a n n e l l i f e t i m e has been found t o depend on the t h i c k n e s s of the b i l a y e r , the average l i f e t i m e v a r y i n g between 60 seconds i n a 64 A b i l a y e r down t o .03 seconds i n a 26 A b i l a y e r ( 2 4 ) . Such a slow r a t e s u g g e s t s t h a t c h a n n e l c l o s u r e i n v o l v e s a major s t r u c t u r a l change. The h i g h s i n g l e c h a n n e l conductance o b s e r v e d p o i n t s t o a pore model f o r the c h a n n e l , i n which i o n s move between s t a t i o n a r y b i n d i n g s i t e s of the c h a n n e l , as opposed t o a c a r r i e r model i n which the i o n - c a r r i e r complex d i f f u s e s a c r o s s the membrane. T h i s was c o r r o b o r a t e d by the r e s u l t s of (25) i n which the i n f l u e n c e of the phase of the b i l a y e r on 8 transport was investigated. No discontinuous change in the conductance of a gramicidin doped membrane was observed, while a c a r r i e r doped membrane's conductance dropped r a d i c a l l y across the l i q u i d c r y s t a l l i n e to gel phase t r a n s i t i o n . Gramicidin's conductance of various ions has been measured under otherwise i d e n t i c a l conditions (24): conductance of divalent cations and anions is n e g l i g i b l e , and conduction of monovalent cations i s (in order of decreasing conductance): H + > NH„ + > Cs + > Rb + > K+ > Na + > L i + The position of the hydrogen ion in t h i s sequence suggests i t may be transported along a f i l e of water molecules l i n i n g the lumen of the gramicidin channel. The bulk conductance of a membrane containing low concentrations of gramicidin was found to depend quadratically on the gramicidin concentration indicating the conducting channel as a dimer of the non-conducting species. A more detailed study of t h i s phenomenon (26) confirmed t h i s r e s u l t , found the non-conducting species to be a single molecule of gramicidin, and showed that most, i f not a l l , gramicidin dimers in the bilayer are conducting channels. The equilibrium between monomer and dimer gramicidin has b^en found to be s h i f t e d by factors a f f e c t i n g the thickness of the b i l a y e r : a transmembrane potential,and l i p i d s with shorter acyl chains increasing the bulk conductance, anaesthetics decreasing i t . An early experiment 9 (26) found c h o l e s t e r o l t o d e c r e a s e the b u l k conductance. T h i s may be r e l a t e d t o c h o l e s t e r o l ' s i n f l u e n c e on b i l a y e r t h i c k n e s s . D. STRUCTURAL STUDIES OF GRAMICIDIN In view of i t s c o m p a r i t i v e l y s m a l l s i z e , t h e s t r u c t u r a l b a s i s of g r a m i c i d i n ' s b i o l o g i c a l l y i n t e r e s t i n g p r o p e r t i e s p r e s e n t s an a t t r a c t i v e r e s e a r c h problem. W h i l e the c o n f o r m a t i o n of the g r a m i c i d i n channel has not been d e t e r m i n e d , a number of e x p e r i m e n t s have r e v e a l e d some a s p e c t s of i t s s t r u c t u r e , a n d s e v e r a l models have been proposed. Of t h e s e , the 7 r 6 - 3 ( L , D ) h e l i x o r i g i n a l l y proposed by D.W.Urry (28,29)(and l a t e r , i n d e p e n d e n t l y , by Ramachandran (30)) on the b a s i s of c o n f o r m a t i o n a l energy c o n s i d e r a t i o n s and model b u i l d i n g , i s the most p l a u s i b l e , and the o n l y one c o n s i s t e n t w i t h a l l of the e x i s t i n g d a t a . The 7 r 6 ' 3 ( L , D ) h e l i c a l model of the c h a n n e l i s a dimer of two l e f t handed h e l i c e s , h a v i n g a t o t a l l e n g t h of about 26 A ( 2 8 ) . The r e p e a t i n g u n i t of the h e l i x i s the L,D d i p e p t i d e . The amide c a r b o n y l C-0 bond d i r e c t i o n s of the L r e s i d u e s a r e towards the e t h a n o l a m i n e t e r m i n u s , those of the D r e s i d u e s p o i n t i n the o p p o s i t e d i r e c t i o n . T h i s unusual a l t e r n a t i o n of hydrogen bonding d i r e c t i o n s p e r m i t s head ( f o r m y l t e r m i n u s ) t o head d i m e r i z a t i o n by c o m p l e t i o n of s i x hydrogen bonds, and w i t h the a l t e r n a t i o n of a l p h a carbon c h i r a l i t y causes the s i d e c h a i n s t o l i e o u t s i d e the h e l i x i n s t e r i c a l l y uncrowded p o s i t i o n s . W i t h 6.3 r e s i d u e s / t u r n the 10 h e l i x has a 4 A bore, and l i e s i n a l o w , f l a t minimum on the Ramachandran p l o t of c o n f o r m a t i o n a l energy v e r s u s h e l i x d i h e d r a l a n g l e s ( 2 8 ) . The g r e a t e r number of c a r b o n y l groups per t u r n of the h e l i x than found i n , s a y , the a l p h a h e l i x and t h e i r a l t e r n a t i n g o r i e n t a t i o n c o u l d promote e f f i c i e n t c o m p l e x i n g of permeant i o n s . The compact N - t e r m i n a l f o r m y l groups a l l o w n e a r l y u n i n t e r u p t e d s t r u c t u r a l c o n t i n u i t y of the h e l i x a t the j u n c t i o n of the monomers. Some of the c o n d u c t i o n p r o p e r t i e s of the g r a m i c i d i n c h a n n e l may be u n d e r s t o o d i n terms of the 7 r 6 - 3 ( L , D ) s t r u c t u r e . The s e l e c t i v i t y sequence w i t h the channel s e l e c t i n g l a r g e r i o n s , may be due t o the s m a l l e r d i s t o r t i o n s the i o n s produce i n the h e l i x . The pore s e l e c t s c a t i o n s over a n i o n s perhaps because the s e can be c o o r d i n a t e d by the c a r b o n y l d i p o l e s l i n i n g the i n s i d e of the c h a n n e l , r e d u c i n g the energy b a r r i e r t o t r a n s p o r t . The c o o r d i n a t i o n of i o n s by amide c a r b o n y l s has been demonstrated u s i n g C-13 NMR of l a b e l l e d c a r b o n y l carbons i n m i c e l l e bound g r a m i c i d i n ( 3 1 ) . T h a l l i u m i o n - i n d u c e d c h e m i c a l s h i f t s were seen o n l y a t r e s i d u e s near the mouths of the c h a n n e l , i n d i c a t i n g the p r e s e nce of i o n b i n d i n g s i t e s . T h i s study a l s o r u l e d out the r i g h t handed J T 6 ' 3 ( L , D ) h e l i x w hich was i n c o n s i s t e n t w i t h the o b s e r v e d c o n t i n u i t y of c h e m i c a l s h i f t v e r s u s a l p h a carbon p o s i t i o n i n the p r i m a r y s t r u c t u r e . The g a t i n g of the c h a n n e l may be due t o the a s s o c i a t i o n of monomers and the d i s s o c i a t i o n of d i m e r s . The monotonic i n c r e a s e of b u l k conductance w i t h d e c r e a s i n g t h i c k n e s s i s c o n s i s t e n t w i t h the 11 26 A c h a n n e l b e i n g s h o r t e r than a l l of the membranes i n v e s t i g a t e d ( 2 4 ) . The 7 r 6 ' 3 ( L , D ) h e l i x i s c o n s i s t e n t w i t h the f i n d i n g s of s e v e r a l s t r u c t u r a l s t u d i e s . X-ray a n a l y s i s (3,4) of i o n bound and i o n f r e e g r a m i c i d i n showed a c y l i n d e r 32 A l o n g and 5 A wide i n the absence of s a l t , and one s h o r t e r (26 A) and w i d e r (6-8 A) i n the presence of s a l t , w i t h 2 i o n b i n d i n g s i t e s / d i m e r a t p o s i t i o n s c o n s i s t e n t w i t h the C-13 r e s u l t s . W h i l e these r e s u l t s agree v e r y w e l l w i t h the 7 r 6 ' 3 ( L , D ) model, i t has been found t h a t the c o n f o r m a t i o n of g r a m i c i d i n i n o r g a n i c s o l v e n t s (from which the c r y s t a l s were grown) i s q u i t e d i f f e r e n t from the membrane c o n f o r m a t i o n ( 5 , 3 2 ) . As d i s c u s s e d below, the c o n f o r m a t i o n a l change on i o n b i n d i n g p r o b a b l y does not o c c u r i n membrane bound g r a m i c i d i n (5,32 and t h i s s t u d y ) . F u r t h e r , the s t u d y i s a t 5 A r e s o l u t i o n , and i t i s not known whether the o b s e r v e d c y l i n d e r s are 7 r 6 ' 3 ( L , D ) h e l i c e s . The most c o n v i n c i n g e v i d e n c e f o r the 7 r 6 ' 3 ( L , D ) h e l i x came from a C-13, and F-19 NMR study of l a b e l l e d g r a m i c i d i n i n m i c e l l e s (33,34). The s h i f t r e a g e n t s Tm, and Mn and a n i t r o x i d e s p i n l a b e l a t t a c h e d t o DSPCwere used t o probe the a c c e s s i b i l i t y of l a b e l s a t t a c h e d near the f o r m y l and e t h a n o l a m i n e t e r m i n a l ends t o the aqueous i n t e r f a c e and the c e n t e r of the b i l a y e r . Based on T, and c h e m i c a l s h i f t measurements i t was found t h a t the e t h a n o l a m i n e t e r m i n u s was a c c e s s i b l e from the aqueous i n t e r f a c e but not from i n s i d e " D i s t e a r o y l p h o s p h a t i d y l c h o l i n e . 12 t h e l i p i d b i l a y e r . The o p p o s i t e was found f o r the f o r m y l t e r m i n u s . T h i s r e s u l t e l i m i n a t e d o t h e r e x i s t i n g p r o p o s a l s f o r the membrane c o n f o r m a t i o n of g r a m i c i d i n . C i r c u l a r d i c h r o i s m s p e c t r o s c o p y , s e n s i t i v e t o the secondary s t r u c t u r e of b i o m o l e c u l e s , was used t o study g r a m i c i d i n i n s o n i c a t e d v e s i c l e s ( 5 , 3 2 ) . The c i r c u l a r d i c h r o i s m of g r a m i c i d i n i n DMPC5 was found t o be i n s e n s i t i v e t o the presence of a l a r g e amount (1 M) of a cesium s a l t , e x c e p t f o r a minor change a t l o n g w a v e l e n g t h s , a t t r i b u t e d t o a s m a l l r e - o r i e n t a t i o n of the t r y p t o p h a n r e s i d u e s . The CD spectrum i n DMPC was a l s o found t o be independent of tempe r a t u r e from 4°C t o 50°C, an i n t e r v a l encompassing the g e l t o l i q u i d c r y s t a l l i n e phase t r a n s i t i o n of the membrane. The CD spectrum i n DMPC d i d not depend on c o n c e n t r a t i o n i n the range: 1:45 t o 1:15 (molar r a t i o of g r a m i c i d i n t o p h o s p h o l i p i d ) , however a t h i g h c o n c e n t r a t i o n s of g r a m i c i d i n , v e s i c l e s d i d not form. The spectrum was found t o be n e a r l y i d e n t i c a l i n DMPC amd DLPC 6, but i n DSPC and DPPC 7 i t was markedly d i f f e r e n t . The d i f f e r e n c e i n spectrum i n the t h i c k e r b i l a y e r s may be due t o a change i n p i t c h of the h e l i x or a change i n e q u i l i b r i u m between c o n f o r m a t i o n s h a v i n g d i f f e r e n t CD s p e c t r a . W h i l e the a u t h o r s of t h i s work f a v o r e d the l a t t e r e x p l a n a t i o n , they were unable t o d i s c a r d t h e former as a p o s s i b l e r e s u l t of change i n b i l a y e r t h i c k n e s s . That the s p e c t r a i n DL- and DMPC a r e the same, 5 D i m y r i s t o y l p h o s p a t i d y l c h o l i n e . 6 D i l a u r y l p h o s p h a t i d y l c h o l i n e . 7 D i p a l m i t o y I p h o s p h a t i d y l c h o l i n e . 13 and e x h i b i t no c o n c e n t r a t i o n dependance i n DMPC, i m p l i e s t h a t one of the p o s s i b l e s t r u c t u r e s predominates i n these membranes - t h e a u t h o r s i d e n t i f y i n g t h i s w i t h the c o n d u c t i n g c h a n n e l . The s t r u c t u r e of the p h o s p h o l i p i d / g r a m i c i d i n system has been s t u d i e d u s i n g P-31 NMR (12,35). I t was found t h a t s m a l l amounts of g r a m i c i d i n induced h e x a g o n a l - I I phase i n b i l a y e r s of monounsaturated and s a t u r a t e d PC's h a v i n g a c y l c h a i n l e n g t h g r e a t e r than 16 c a r b o n s . There was no i n d i c a t i o n of thes e or o t h e r n o n - b i l a y e r phases i n the s h o r t e r c h a i n membranes . I l l . SOLID STATE NMR AS A STRUCTURAL TOOL Second rank t e n s o r i n t e r a c t i o n s , such as the d i p o l a r i n t e r a c t i o n between s p i n s , c h e m i c a l s h i f t a n i s o t r o p y , and the e l e c t r i c q u a d r u p o l e c o u p l i n g a r e a f u n c t i o n of the r e l a t i v e o r i e n t a t i o n s of l a b and m o l e c u l e - f i x e d frames of r e f e r e n c e i n an NMR e x p e r i m e n t . Measurement of the magnitude of t h e s e i n t e r a c t i o n s i n an o r i e n t e d sample a l l o w s one t o de t e r m i n e the d i r e c t i o n s of the p r i n c i p l e axes of the t e n s o r . I f t h i s i n f o r m a t i o n i s o b t a i n e d f o r s e v e r a l s i t e s i n the same system t h e i r r e l a t i v e o r i e n t a t i o n s may be c a l c u l a t e d . W i t h b i o l o g i c a l m a t e r i a l s , an o r i e n t e d sample s u i t a b l e f o r NMR work i s u s u a l l y u n a v a i l a b l e . Powder s p e c t r a , however, can y i e l d some s t r u c t u r a l i n f o r m a t i o n and may be used t o t e s t e x i s t i n g models of s t r u c t u r e . An example of the a p p l i c a t i o n of s o l i d s t a t e NMR t e c h n i q u e s t o the study of a membrane p r o t e i n , i s the 1 3C-NMR stu d y of b a c t e r i o r h o d o p s i n (BR) by B.A. Lewis et. al. (36) 1 3 C l a b e l l e d l e u c i n e was i n c o r p o r a t e d i n t o BR by ad d i n g i t t o the growth medium of H. Hal obi urn. R i g i d l a t t i c e and f l u i d phase s p e c t r a of BR r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s p e r m i t t e d c a l c u l a t i o n of the range of E u l e r a n g l e s of the l e u c i n e p e p t i d e groups w i t h r e s p e c t t o the a x i s of r o t a t i o n a l d i f f u s i o n of BR i n the membrane. 14 15 A. DEUTERIUM NMR SPECTRA(6 , 7 , 8 , 3 7 ) The s i n g l e l i n e of an i s o l a t e d d e u t e r i u m n u c l e u s ' spectrum i s s p l i t by the i n t e r a c t i o n of the e l e c t r i c q u adrupole moment of the n u c l e u s w i t h a m o l e c u l a r e l e c t r i c f i e l d g r a d i e n t i n t o a d o u b l e t symmetric about the Larmor f r e q u e n c y . The quad r u p o l e s p l i t t i n g Aa>, g i v e n by the e q u a t i o n ( 8 ) : &J(6,4>)= 2OJ [ P 2 (COS0 )+7}/2SIN 20COS20] where: to =3/4(e 2qQ/h) o i n which eQ i s the q u a d r u p o l e moment of the n u c l e u s , eq i s the p r i n c i p a l v a l u e of 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 , a n d h i s P l a n c k ' s constant;*? i s the asymmetry parameter, a measure of the d e p a r t u r e of the e l e c t r i c f i e l d g r a d i e n t from c y l i n d r i c a l symmetry; 6, and 4> are the s p h e r i c a l p o l a r a n g l e s of the magnetic f i e l d v e c t o r i n the e l e c t r i c f i e l d g r a d i e n t p r i n c i p a l a x i s system. The qu a d r u p o l e s p l i t t i n g i s u s u a l l y the predominant dominant f e a t u r e of the spectrum, as o t h e r i n t e r a c t i o n s , such as the d i p o l a r c o u p l i n g between d e u t e r i u m n u c l e i , a r e s m a l l i n comparison t O CL> . a R a p i d a x i a l r e o r i e n t a t i o n (compared t o x1'AM2~1 , t he NMR t i m e s c a l e ) of the m o l e c u l e a v e r a g e s the quadrupole i n t e r a c t i o n y i e l d i n g a narrowed s p l i t t i n g of (8) : Acj(0,a)= 2 o ? [ P 2 (COS/3)+Tj/2SIN 2/3COS2a) ] P 2 (COS©) where 6 i s the a n g l e between t h e a x i s of r e o r i e n t a t i o n of the m o l e c u l e and the e x t e r n a l magnetic f i e l d , 0 and a a r e 16 s p h e r i c a l p o l a r c o o r d i n a t e s of the a x i s of r o t a t i o n i n the p r i n c i p a l a x i s c o o r d i n a t e system of the e l e c t r i c f i e l d g r a d i e n t . To p r e d i c t the e f f e c t of slower motions on the spectrum r e q u i r e s d e t a i l e d c a l c u l a t i o n . Many b i o l o g i c a l samples s u i t a b l e f o r NMR s p e c t r o s c o p y are o n l y a v a i l a b l e as "powders", c o n s i s t i n g of a l l p o s s i b l e o r i e n t a t i o n s of the m o l e c u l e s . The spectrum may then be computed u s i n g (6) : f ± ( x ) = 2 / r 9 / S I N f 3 d r 3 v / ( x ^ P 2 ( C O S 0 ) + 7 7 / 2 S I N 2 0 ) - 1 •/(-x±P 2 ( C O S 0 ) + T ? / 2 S I N 2 0 ) - 1 where x=2u/v^r f ± ( x ) a re the two branches of the spectrum, which may be e v a l u a t e d a n a l y t i c a l l y or n u m e r i c a l l y ( c o n v o l u t e d w i t h the o r i e n t e d l i n e s h a p e ) t o p r e p a r e " s i m u l a t e d " s p e c t r a . A x i a l r o t a t i o n , i n the s h o r t - c o r r e l a t i o n time l i m i t , l e a d s t o a powder spectrum h a v i n g z e r o apparent asymmetry parameter r e g a r d l e s s of the symmetry of the e l e c t r i c f i e l d g r a d i e n t , as may be seen by comparison of the above e q u a t i o n s . B. A DEUTERIUM NMR STUDY OF A SYNTHETIC POLYPEPTIDE The s y n t h e t i c p o l y p e p t i d e , w i t h p r i m a r y s t r u c t u r e : LYS 2-GLY-LEU x-LYS 2-ALA-AMIDE (where p o l y p e p t i d e s w i t h x=16,20,24 have been s y n t h e s i z e d ) was d e s i g n e d t o s y s t e m a t i c a l l y i n v e s t i g a t e l i p i d - p r o t e i n i n t e r a c t i o n s i n membranes. 17 —I 1 1 1 1 1 t i i -400 -200 0 200 400 Frequency (kHz) F i g u r e 1 (a) 2H-NMR spectrum of the c r y s t a l l i n e l a b e l l e d p olypeptide at room temperature, (b) Simulated spectrum with parameters »»g=l50 kHz, rj=. 16, T 2 e= 160iisec. Figures from reference ( 8 ) . 18 The 24 l e u c i n e p o l y p e p t i d e was the s u b j e c t of a 2H NMR study by P a u l s et. al. (8) i n which the l a b i l e hydrogens of the p o l y p e p t i d e were r e p l a c e d by d e u t e r i u m . The spectrum of c r y s t a l l i n e p o l y p e p t i d e , s h o w n i n f i g u r e 1 ( a ) , i s w e l l s i m u l a t e d ( f i g u r e 1(b)) by a s u p e r p o s i t i o n of a broad powder p a t t e r n h a v i n g parameters p^=150kHz , rp.16 due t o the amide d e u t e r o n s , and a m o t i o n a l l y narrowed one w i t h vg=36 kHz,77=0 a t t r i b u t e d t o r o t a t i n g +NH 3 groups on the l y s i n e s i d e c h a i n s . Except f o r a d i s t o r t i o n of the narrow component, caused by a s o l v e n t s u b t r a c t i o n p r o c e d u r e , the spectrum i n the g e l s t a t e of DPPC ( f i g u r e 2 ( a ) ) i s s i m i l a r t o t h a t i n the c r y s t a l . T h i s i m p l i e s t h a t the p o l y p e p t i d e has' l i t t l e m o b i l i t y i n the g e l phase of the membrane. At 42°C , i n the f l u i d s t a t e of DPPC, the spectrum ( f i g u r e 2 ( b ) ) was found t o c o n s i s t of a s i n g l e Pake d o u b l e t w e l l s i m u l a t e d u s i n g the p a r a m e t e r s : v =127 kHz, TJ=0 and the measured T 2 e=lOOMS. S i n c e the 31 amide d e u t e r o n s of the p o l y p e p t i d e a r e e q u i v a l e n t i n the spectrum the p o l y p e p t i d e must assume a h e l i c a l c o n f o r m a t i o n w i t h a x i s of m o t i o n a l n a r r o w i n g l y i n g a l o n g the h e l i c a l a x i s . The s p l i t t i n g of the m o t i o n a l l y narrowed spectrum a l l o w s c a l c u l a t i o n of t h e a n g l e between the p r i n c i p a l a x i s of the e l e c t r i c f i e l d g r a d i e n t and the h e l i x a x i s , j3, t o be I9°:within the range of the N- 2H d i r e c t i o n s e x p e c t e d f o r the a - h e l i x s t r u c t u r e . Measurement of T 2 e v e r s u s temperature e x h i b i t e d a minimum c o r r e s p o n d i n g t o t h e onset of p e p t i d e r o t a t i o n a t the temperature of the c h a i n - m e l t i n g phase t r a n s i t i o n of the b i l a y e r , and p e r m i t t e d Figure 2. 2H-NMR spectrum of the s y n t h e t i c polypeptide i n DPPC liposomes (a) at 10°C (b) at 42°C. Figures from reference ( 8 ) . 20 c a l c u l a t i o n of the r o t a t i o n a l c o r r e l a t i o n time f o r the m o t i o n , r was found t o be 2X10~ 7 sec a t 42°C. IV. MATERIALS AND METHODS A. MATERIALS G r a m i c i d i n D ( = g r a m i c i d i n A') was purchased from B o e h r i n g e r Mannheim and used w i t h o u t f u r t h e r p u r i f i c a t i o n . D i m y r i s t o y l and D i l a u r y l L - a - p h o s p h a t i d y l c h o l i n e were pur c h a s e d from Sigma Ch e m i c a l Co., which quotes them as b e i n g >98% (by w e i g h t ) p u r e , and used w i t h o u t f u r t h e r p u r i f i c a t i o n . Deuterium o x i d e (>99.8 atom %D), d i m e t h y l - d 6 s u l f o x i d e ( >99.9 atom %D), and methanol-OD were purchased from Merck,Sharpe, and Dohme I s o t o p e s . Gaseous ND 3 was o b t a i n e d from Cambridge i s o t o p e s . B. NMR SAMPLE PREPARATION The l a b i l e hydrogens of g r a m i c i d i n were d e u t e r i u m exchanged by d i s s o l v i n g the g r a m i c i d i n i n excess methanol-OD made a l k a l i n e w i t h gaseous ND 3, h e a t i n g and s t i r r i n g the s o l u t i o n a t 50°C f o r an hour, then l e t t i n g i t s i t o v e r n i g h t a t room t e m p e r a t u r e . B u l k methanol was removed u s i n g a B uchi Rotavap e v a p o r a t o r ; t r a c e q u a n t i t i e s were removed by keeping the g r a m i c i d i n under vacuum o v e r n i g h t . The degree of exchange of the l a b i l e s i t e s was checked u s i n g the 400 MHz p r o t o n NMR f a c i l i t y i n t h e C h e m i s t r y Department. The decrease i n i n t e n s i t y of the amide r e g i o n , i n the spectrum of c h e m i c a l s h i f t s , was u n i f o r m and i n d i c a t e d the degree of d e u t e r a t i o n t o be about 70%. The t r y p t o p h a n i n d o l e s i t e s showed a d e u t e r a t i o n of about 35%. These r e s u l t s a r e i n good 21 22 agreement w i t h an e a r l i e r s tudy ( 4 8 ) . 2H-NMR samples of c r y s t a l l i n e g r a m i c i d i n were pr e p a r e d by s i m p l y p a c k i n g the d r y powder i n an NMR sample tube. G r a m i c i d i n - l i p i d d i s p e r s i o n s were p r e p a r e d as f o l l o w s : l i p i d s and g r a m i c i d i n were c o d i s s o l v e d i n methanol-OD; the methanol was removed as d e s c r i b e d above; the powdered m i x t u r e was p l a c e d i n the sample t u b e ; t h e d e s i r e d amount of d e u t e r i u m o x i d e added, and the sample s t i r r e d , u s i n g a g l a s s r o d , u n t i l homogeneous. D e u t e r a t e d s o l v e n t s were used throughout the sample p r e p a r a t i o n t o p r e v e n t back-exchange the l a b i l e s i t e s . T y p i c a l l y , samples of dry,powdered g r a m i c i d i n c o n s i s t e d of 80 mgs of the p o l y p e p t i d e . The t o t a l weight of most d i s p e r s i o n s was .5 gms w i t h : .25 gms of D 20; .212 gms of l i p i d , and .038 gms of g r a m i c i d i n . Thus the c o m p o s i t i o n of the b i l a y e r s s h o u l d be 15% g r a m i c i d i n , by w e i g h t , or 1:16 moles g r a m i c i d i n t o moles of l i p i d . C. 2H NMR EXPERIMENTS 2H NMR s p e c t r a were r e c o r d e d i n q u a d r a t u r e a t 35.5 MHz u s i n g a s p e c t r o m e t e r b u i l t i n the P h y s i c s Department e l e c t r o n i c s shop, and a N a l o r a c s u p e r c o n d u c t i n g magnet. The NMR probe was tuned u s i n g a f r e q u e n c y sweeper. Care was taken w i t h the c r y s t a l l i n e samples t h a t the c o i l Q was s u f f i c i e n t l y low t o a v o i d s p e c t r a l d i s t o r t i o n due t o n o n - l i n e a r response of the probe. Data was accumulated on an I n t e l - 2 1 0 microcomputer, and p r o c e s s e d u s i n g an I n t e l - 2 3 0 microcomputer and the 23 u n i v e r s i t y mainframe. The q u a d r u p o l a r echo p u l s e sequence (50) w i t h 3,usec 7r/2 p u l s e s was used w i t h phase c y c l i n g ( 8 ) , t o o b t a i n the f r e e i n d u c t i o n decay ( F I D ) . W i t h some samples i t was not p o s s i b l e t o use 7r/2 p u l s e s w i t h o u t probe a r c i n g , i n which case 3 i u s e c p u l s e s a t lower r . f . power were used. A f t e r s o l v e n t s i g n a l s u p p r e s s i o n ( 4 0 ) , d i g i t i z a t i o n e r r o r s a t the t o p of t h e echo were c o r r e c t e d ( 6 ) , the ou t - o f - p h a s e component of the FID z e r o e d t o improve the s i g n a l t o n o i s e r a t i o by »/2, and the in-phase component F o u r i e r t r a n s f o r m e d . V. RESULTS The 2H NMR spectrum of c r y s t a l l i n e g r a m i c i d i n i s shown i n f i g u r e 3(a) . The spectrum c o n s i s t s of a s i n g l e powder p a t t e r n w i t h quadrupole c o u p l i n g v = 148 kHz and asymmetry parameter 7?= .15. The s p i n - l a t t i c e r e l a x a t i o n t i m e ,T,, and the time c o n s t a n t f o r the decay of the q u a d r u p o l a r echo, T 2 e r were 460 msec and 170 Msec r e s p e c t i v e l y . A p a r t from some l o s s of i n t e n s i t y i n the s h o u l d e r s , p o s s i b l y due t o the e f f e c t s of the f i n i t e r . f . p u l s e l e n g t h ( 3 8 ) , t h e r e i s e x c e l l e n t agreement between the e x p e r i m e n t a l spectrum and one s i m u l a t e d u s i n g the above parameters ( f i g u r e 3 (b) ). The absence of another i n e q u i v a l e n t d e u t e r i u m s i t e s u p p o r t s the p r o t o n NMR r e s u l t (see p r e v i o u s c h a p t e r ) t h a t a p p r o x i m a t e l y 90% of the d e u t e r o n s i n t h e sample a r e a t t a c h e d t o amide groups. Tryptophan i n d o l e d e u t e r o n s a r e e x p e c t e d t o have v =223 kHz (39) and would be w e l l r e s o l v e d g from the amide s p l i t t i n g . The narrow c e n t r a l peak i n the spectrum i s p r o b a b l y due t o r e s i d u a l m e t h a n o l - 0 2 H i n the sample. F i g u r e 4 (a) shows the in-phase component of the t r a n s v e r s e m a g n e t i z a t i o n a f t e r the second p u l s e of the q u a d r u p o l e echo sequence f o r a sample c o n t a i n i n g l i p o s o m e s w i t h g r a m i c i d i n . The s t r o n g , l o n g - l i v e d s i g n a l , due t o the aqueous component of the d i s p e r s i o n , i s superimposed on the weak, s h o r t - l i v e d echo from the amide d e u t e r o n s of the p e p t i d e . The s o l v e n t s i g n a l was s u b t r a c t e d f o l l o w i n g the method of C a l l a g h a n e t . a l . ( 4 0 ) , the remainder i s shown i n 24 25 -512 -256 0 256 512 FREQUENCY(kHz) -512 256 512 FREQUENCY(kHz) Figure 3. (a) 2H-NMR spectrum of 2H-exchanged gra m i c i d i n i n c r y s t a l l i n e form at 23°C. 100,000 t r a n s i e n t s were accumulated at a rate of 2/sec. (b) Powder pa t t e r n simulated using the parameters v =148 kHz, 17= .15, and T 2 e=170 jxsec. 26 (b) 1 Figure 4 . (a) Free i n d u c t i o n decay of a sample c o n t a i n i n g DLPC, deuterated gramicidin,and D 20. D i s c o n t i n u i t i e s i n the s i g n a l are a r e s u l t of computer memory overflows, (b) Figure (a) a f t e r a f i f t h order polynomial, f i t t e d to the solvent s i g n a l was subtracted.Scale:1 cm= 49.2 usee. The corresponding spectrum i s shown i n Figure 7 ( a ) . Figure 5. 2H-NMR spectrum of deuterated g r a m i c i d i n i n DPPC (a) at 20°C and (b) at 52°C. Results from Datema et.al. (12). 28 f i g u r e 4 ( b ) . F i g u r e 5 (a) shows the spectrum of quadrupole s p l i t t i n g s of 2 H - l a b e l l e d g r a m i c i d i n i n DPPC l i p o s o m e s , r e c o r d e d by K l a a s - P e t e r Datema (12) . The b i l a y e r , at 20°C, i s i n the g e l s t a t e and the spectrum i s a s i n g l e powder p a t t e r n w i t h t»^= 144 kHz , 77= .18 . In the f l u i d s t a t e of DPPC, a t 52°C, the spectrum ( f i g u r e 5(b) ) shows a number of s p l i t t i n g s (see T a b l e 1) and the shape of the s i d e s of the spectrum i n d i c a t e s t h a t rj=0 f o r the outermost l i n e s . Both s p e c t r a show a d i s t o r t i o n due t o the s o l v e n t - s u b t r a c t i o n p r o c e d u r e , as do a l l of the s p e c t r a of d i s p e r s i o n s p r e s e n t e d i n t h i s t h e s i s , ' and the r e g i o n ±25 kHz was n e g l e c t e d i n a n a l y s i s of the s p e c t r a . The f l u i d s t a t e s p e c t r a of g r a m i c i d i n i n DMPC and DLPC are g i v e n i n f i g u r e s 6(a) and 7(a) r e s p e c t i v e l y . The s p l i t t i n g s of the s p e c t r a a r e g i v e n i n T a b l e 1. A g a i n , 77=0 f o r the outermost l i n e s of the s p e c t r a . The " o r i e n t e d " s p e c t r a of f i g u r e s 6 (a) and 7(a),shown i n 6(b) and 7 ( b ) , were o b t a i n e d u s i n g a "depakeing" a l g o r i t h m d e v e l o p e d i n t h i s l a b o r a t o r y ( 4 1 ) . T h i s f a c i l i t a t e s r e s o l u t i o n of s p e c t r a l l i n e s , and p e r m i t s measurement of l i n e w i d t h s and a r e a s . The depaked s p e c t r a were t r e a t e d t o a t h r e e p o i n t b i n o m i a l smoothing r o u t i n e . L i n e w i d t h s of the s p e c t r a were found t o be i n the range 8-16 kHz. The f l u i d s t a t e spectrum of g r a m i c i d i n i n the presence of .1M NaCl i s shown i n f i g u r e 8 (a) a l o n g w i t h the depaked spectrum. An attempt t o r e c o r d a spectrum i n the presence of 29 1 (a) 1 i 1 400 -200 0 200 400 FREQUENCY(kHz) (b) i i M I i i A 1 1 l -400 -200 0 200 400 FREQUENCY(kHz) Figure 6. (a) 2H-NMR spectrum of deuterated gramicidin i n DMPC at 38°C. 122,000 t r a n s i e n t s were accumulated. (b) Figure (a) "depaked". 30 (a) -400 -200 0 200 400 FREQUENCY(kHz) I I I I L I J I I -400 -200 0 200 400 FREQUENCY(kHz) Figure 7. (a) 'H-NMR spectrum of deuterated g r a m i c i d i n i n DLPC at T= 20°C. 167,000 t r a n s i e n t s were accumulated, (b) Figure (a) ^depaked". 31 -400 -200 0 200 FREQUENCY(kHz) 400 -400 -200 0 200 FREQUENCY(kHz) 400 Figure 8. (a) 2H-NMR spectrum of deuterated g r a m i c i d i n i n DMPC at T= 40°C. The aqueous phase of the sample c o n s i s t e d of .1M NaCl i n 2H 20. 150,000 t r a n s i e n t s were accumulated, (b) Figure (a) "depaked". 32 1M NaCl was made, but the s a l i n e s o l u t i o n was found to de c r e a s e the c o i l Q - f a c t o r so much t h a t a s i g n a l was not o b s e r v a b l e . F i g u r e 9 , the r e s u l t s of K.P. Datema ( 1 2 ) , i s an A r r h e n i u s p l o t of T 2 e v e r s u s t e m p e r a t u r e . T a b l e 1 F l u i d S t a t e Quadrupolar S p l i t t i n g s 8 9 ( k H z ) DLPC 136 (4) 104 (2) 70 (1 ) DMPC 136 (2) 128 (2) 104 (2) 67 (1) 52 DPPC 134 125 1 05 96 65 49 DMPC+NaCl 134 (2) 126 (2) 100 (2) 92 70 (1) 47 U n c e r t a i n t y i n f r e q u e n c i e s ^ I k H z . 'Numbers i n p a r e n t h e s e s a r e app r o x i m a t e r e l a t i v e i n t e n s i t i e s of the l i n e s . 800 400 200 CO 3* 1 0 0 80 60 4 A A A A A A I A 40 h 20 J L I L 3.00 3.14 3.28 3.42 356 1000/T (K*1) 3.70 F i g u r e 9 A r r h e n i u s p l o t of T 2 e vs. temperature . VI . DISCUSSION In t h i s c h a p t e r , an attempt w i l l be made t o r e l a t e the 2H NMR r e s u l t s p r e s e n t e d i n c h a p t e r f o u r t o a s p e c t s of g r a m i c i d i n ' s s t r u c t u r e and a c t i o n . The v a l u e s f o r v and TJ measured from the 2H NMR o spectrum of c r y s t a l l i n e g r a m i c i d i n a r e s i m i l a r t o those found f o r d e u t e r o n s i n -N-H»««0= hydrogen bonds i n many systems ( 3 9 ) . The v a l u e of i>^=148 kHz agrees r e a s o n a b l y w e l l w i t h t h a t c a l c u l a t e d u s i n g the e m p i r i c a l r e l a t i o n ( 3 9 ) : v = 252.-522./R( 2H-•-0) 3 W i t h R=1.8 A 1 0 , e x p e c t e d f o r a T T 6 ' 3 ( L , D ) h e l i x , i> =160 kHz. W i t h 1.73 A1.1 ,the e s t i m a t e d 2H»«-0 d i s t a n c e f o r the a - h e l i x , v^=150. kHz i n e x c e l l e n t agreement w i t h the f i n d i n g s of P a u l s et . al. ( 8 ) . I t i s i n t e r e s t i n g t o note t h a t the .07 A d i f f e r e n c e i n R can make a s i g n i f i c a n t change i n f r e q u e n c y . T h i s s u g g e s t s t h a t , a t l e a s t i n p r i n c i p l e , t he 2H-NMR spectrum i s q u i t e s e n s i t i v e t o changes i n the hydrogen bonding of a m o l e c u l e . The spectrum i n the g e l s t a t e of DPPC i s a l s o a s i n g l e powder p a t t e r n , however the parameters a r e somewhat d i f f e r e n t w i t h p^=144 kHz, 77= .18. T h i s i s not s u r p r i s i n g s i n c e , as was noted i n c h a p t e r one, t h e r e i s e v i d e n c e t h a t the c o n f o r m a t i o n s of c r y s t a l and membrane-bound g r a m i c i d i n a r e not t h e same. No such change i n parameters was found f o r 1 0 Rough f i g u r e c a l c u l a t e d assuming -N-H«««0= c o l i n e a r and u s i n g the atomic c o o r d i n a t e s g i v e n by Koeppe and Kimura ( 4 2 ) . 1 1 F r o m c o o r d i n a t e s g i v e n by P a u l i n g and C o r e y ( 4 3 ) , and u s i n g the a s s u m p t i o n mentioned i n f o o t n o t e 10. 34 35 the s y n t h e t i c p o l y p e p t i d e . The f l u i d s t a t e s p e c t r a i n l i p i d b i l a y e r s c o n s i s t i n g of DPPC, DMPC, or DLPC show s e v e r a l l i n e s . W a l l a c e el. al. (5) found t h a t t h e r e was no d i f f e r e n c e between t h e g e l and f l u i d s t a t e c i r c u l a r d i c h r o i s m s p e c t r a i n DLPC,DMPC,DPPC, and DSPC b i l a y e r s . We t h e r e f o r e a t t r i b u t e the dependence of the 2H-NMR spectrum on l i p i d phase t o the e f f e c t s of m o t i o n a l n a r r o w i n g , r a t h e r than t o a change i n the s t r u c t u r e of the p o l y p e p t i d e . I t i s not p o s s i b l e t o say, by l o o k i n g a t the s p e c t r a a l o n e , what the a x i s of r o t a t i o n i s , or whether the r a t e of motion i s i n the s h o r t c o r r e l a t i o n time regime. Based on the d i s c u s s i o n of c h a p t e r one, i t i s f e l t t h a t the predominant c o n f o r m a t i o n of the g r a m i c i d i n i s a N - t e r m i n a l t o N - t e r m i n a l dimer, which i s p r o b a b l y t h e T T 6 ' 3 ( L , D ) h e l i x . That the s i n g l e c h a n n e l conductance i s u s u a l l y s t a b l e f o r a c h a n n e l ' s l i f e t i m e (24) (a t i m e s c a l e of seconds) s u g g e s t s t h a t the r o t a t i o n of the dimer i s n e a r l y about the h e l i x a x i s . O f f a x i s motions would bury the mouths of the c h a n n e l i n the b i l a y e r and p r e v e n t i o n c o n d u c t i o n . 1 2 The r o t a t i o n a l c o r r e l a t i o n time may be e s t i m a t e d u s i n g the t h e o r y p r e s e n t e d by P a u l s et . al. (8) f o r a c y l i n d r i c a l m o l e c u l e undergoing r o t a t i o n a l d i f f u s i o n about a s i n g l e a x i s . W i t h a= 7 A 1 3 , h=30 A, T?=1.1 p o i s e (8) and T=40°C : 1 2 F o r the same r e a s o n , we e x p e c t the h e l i x a x i s not t o d e v i a t e much from the b i l a y e r d i r e c t o r . 1 3 An average r a d i u s e s t i m a t e d from a space f i l l i n g model d e p i c t e d i n ( 4 4 ) . The 7 r 6 ' 3 ( L , D ) i s somewhat b u l k i e r at the C - t e r m i n a l ends than a t t h e N - t e r m i n a l ends. 36 T c = ( 4 7 r a 2 h 7 ? ) / ( k T ) = .47 usee For monomers, w i t h h=l5 A, the p r e d i c t e d v a l u e i s r c=.23 usee. The c r i t e r i o n f o r r a p i d m o t i o n a l n a r r o w i n g i s ( 8 ) : i / ( A M 2 ) - 1 » T C where AM 2, the change i n the second moment of the m o t i o n a l l y narrowed spectrum i s : AM2= 1/5w 2 { [ 1+ T ? 2/l5]-[P 2(COS/3 ) + T j/2SIN 2j3COS2a] 2} For the s m a l l e s t s p l i t t i n g s i n T a b l e 1 v = 50 kHz : i/m2~ 1 - 2.6 Msec For the l a r g e r s p l i t t i n g s , i/AMj" 1 i s g r e a t e r . F u r t h e r e v i d e n c e t h a t the motion i s i n the s h o r t c o r r e l a t i o n time l i m i t comes from comparison of the s p e c t r a r e c o r d e d i n d i f f e r i n g b i l a y e r s and a t te m p e r a t u r e s i n the range of 40-50°C ( 1 2 ) . As the r o t a t i o n a l c o r r e l a t i o n t imes under t h e s e c o n d i t i o n s a r e s u r e l y d i f f e r e n t , the near i n v a r i a n c e of the observed f r e q u e n c i e s i m p l i e s t h a t the axes of r e o r i e n t a t i o n a r e the same and t h a t the m o t i o n a l n a r r o w i n g about t h i s a x i s i s comp l e t e . We may p r e d i c t the average v a l u e of T 2 e ~ 1 ( a t 40°C), u s i n g the v a l u e s of T 2 e " 1 f o r each l i n e of the spectrum i n DMPC c a l c u l a t e d u s i n g the e q u a t i o n ( 8 ) : T 2 E = T c - ' A M 2 - 1 The p r e d i c t e d v a l u e , 50 usee, i s , a t l e a s t , t he same o r d e r of magnitude as the v a l u e s i n f i g u r e 9. Only two s e t s of d e u t e r o n s , those of the D and L r e s i d u e s of t h e d i p e p t i d e r e p e a t , a r e i n e q u i v a l e n t w i t h r e s p e c t t o m o t i o n a l n a r r o w i n g about the h e l i c a l a x i s . The 37 a n g l e s between the d i r e c t i o n s of the N-D bonds and the h e l i x a x i s a r e a p p r o x i m a t e l y 1 ' 4.5° and 19° . Assuming t h a t the p r i n c i p a l a x i s of 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 l i e s i n the N- 2H bond d i r e c t i o n , then the s p l i t t i n g s of the m o t i o n a l l y narrowed l i n e s s h o u l d b e 1 5 : v = P2(COS|3) • 1 44kHz=1 42kHz , 1 2 1 kHz . a As t h e r e i s an e q u a l number of L and D r e s i d u e s of the p o l y p e p t i d e the i n t e n s i t i e s of t h e s e two l i n e s i n the spectrum s h o u l d be e q u a l . The s p e c t r a are c l e a r l y more complex than p r e d i c t e d above. The a d d i t i o n a l l i n e s may a r i s e because of the presence of non-channel g r a m i c i d i n i n the b i l a y e r , or one l i n e of the spectrum may c o r r e s p o n d t o the t r y p t o p h a n i n d o l e d e u t e r o n s . The two l i n e s p r e d i c t e d do not c o i n c i d e " w i t h any of the l i n e s i n the e x p e r i m e n t a l spectrum. For the t h r e e l i n e s , a t 134,128,105 kHz, which have the same i n t e n s i t y i n DMPC (and DLPC, assuming t h a t the 128 and 136 kHz l i n e s are not r e s o l v e d ) , the c a l c u l a t e d a n g l e s , 0, a r e , r e s p e c t i v e l y : 0= 12.4°, 15.7°,25.1° Two of the l i n e s a r e l i k e l y t o be those a r i s i n g from d e u t e r o n s a t t a c h e d t o L and D amide bonds. I t i s not s u r p r i s i n g t h a t the a n g l e s do not agree w e l l w i t h those p r e d i c t e d above, as the d i r e c t i o n of the e l e c t r i c f i e l d g r a d i e n t p r i n c i p a l a x i s i s not known and i s d i f f i c u l t t o e s t i m a t e a c c u r a t e l y . However, the d i s c r e p a n c y i n a n g l e s may 1 " C a l c u l a t e d u s i n g p u b l i s h e d c o o r d i n a t e s (42) and the d i m e n s i o n s of the p e p t i d e bond g i v e n i n ( 4 5 ) . 1 5Terms p r o p o r t i o n a l t o TJ a r e n e g l i g i b l e . 38 a l s o r e f l e c t an e r r o r i n the 7 r 6 " 3 ( L , D ) s t r u c t u r e . The above c a l c u l a t i o n s are somewhat s p e c u l a t i v e . More d e f i n i t i v e i f l e s s d e t a i l e d s t a t e m e n t s may ' be made c o n c e r n i n g the s t r u c t u r e of g r a m i c i d i n . The e x c e l l e n t agreement of the q u a d r u p o l a r s p l i t t i n g s i n b i l a y e r s of d i f f e r i n g t h i c k n e s s 1 6 (Table 1) and a t d i f f e r e n t t e m p e r a t u r e s i m p l i e s t h a t the s t r u c t u r e of the g r a m i c i d i n p e p t i d e backbone does not deform t o adapt t o a hydrophobic mismatch w i t h the b i l a y e r . F u r t h e r , the a x i s of r o t a t i o n , as w e l l as the m o l e c u l a r o r d e r p a r a m e t e r 1 7 , of the c o n f o r m a t i o n ( s ) are a p p r o x i m a t e l y independent of t h e s e v a r i a b l e s i n the range i n v e s t i g a t e d . I f the 7 r 6 ' 3 ( L , D ) , or a n o t h e r a p p r o x i m a t e l y c y l i n d r i c a l model, i s the s t r u c t u r e of g r a m i c i d i n , i t seems l i k e l y t h a t t h e r e i s l i t t l e t i l t of the c y l i n d e r ' s a x i s w i t h r e s p e c t t o the b i l a y e r d i r e c t o r , as t h i s would would p r o b a b l y depend on the c o n s t r a i n t s of the b i l a y e r . The i n f r a r e d d i c h r o i s m r e s u l t s of Nabedryk et. al . (47) suggest t h a t the h e l i c a l a x i s of the g r a m i c i d i n c h a n n e l i n DMPC makes an a n g l e of l e s s than 15° w i t h the b i l a y e r n o r m al, i n agreement w i t h our c o n c l u s i o n s . As d i s c u s s e d i n the f i r s t c h a p t e r of t h i s t h e s i s , the conductance of a s i n g l e c h a n n e l i s independent of the t h i c k n e s s of the b i l a y e r , as one would expect f o r a r i g i d pore w i t h i t s a x i s 1 6 d, the b i l a y e r h ydrophobic t h i c k n e s s , may be e s t i m a t e d u s i n g ( 4 6 ) : d= 1.75(n-1) (where n i s the number of carbons on t h e a c y l c h a i n ) , d i s 20,23,and 26 A f o r DL,DM,and DPPC, r e s p e c t i v e l y . 1 7 We may t a k e P 2 ( C O S 8 ) , where 6 i s the a n g l e between the h e l i x a x i s and the b i l a y e r d i r e c t o r , and the average i s over the v a l u e s of $ sampled by the m o l e c u l e , weighted by the p r o b a b i l i t y w i t h which they o c c u r . 39 of r o t a t i o n a l d i f f u s i o n , the a x i s of the c y l i n d e r , p e r p e n d i c u l a r t o the p l a n e of the b i l a y e r . Our r e s u l t s a r e i n e x c e l l e n t agreement w i t h the c i r c u l a r d i c h r o i s m s t u d i e s of W a l l a c e et . al. ( 5 ) . The DMPC and DLPC depaked s p e c t r a a r e n e a r l y i d e n t i c a l , except f o r the 128 kHz l i n e , which i s not r e s o l v e d from the 134 kHz l i n e i n the DLPC spectrum, and the c i r c u l a r d i c h r o i s m s p e c t r a were i d e n t i c a l i n t h e s e b i l a y e r s . The 2H NMR spectrum i n DPPC su g g e s t s t h e r e i s no major change i n c o n f o r m a t i o n ( s ) i n t h i s b i l a y e r . The l a r g e change i n the c i r c u l a r d i c h r o i s m spectrum i n DPPC i s thus p r o b a b l y due t o a change i n e q u i l i b r i u m between c o n f o r m a t i o n s of the g r a m i c i d i n , as the a u t h o r s of t h i s paper (5) s u s p e c t e d . The presence of .1M NaCl i n the aqueous r e g i o n of the sample was found t o have l i t t l e i n f l u e n c e on the 2H NMR spectrum i n DM and DLPC, i n agreement w i t h the CD e x p e r i m e n t s . We t h e r e f o r e f e e l t h a t the r e s u l t s of Koeppe et. a/.(3,4) a r e not a p p l i c a b l e t o membrane-bound gramic i d i n . V I I . CONCLUDING REMARKS T h i s study makes i t c l e a r t h a t the membrane s t r u c t u r e of g r a m i c i d i n i s u n s o l v e d . We f e e l t h a t the r e s u l t s p r e s e n t e d i n t h i s t h e s i s have deepened our u n d e r s t a n d i n g of the gramicidin-membrane i n t e r a c t i o n , and w i l l c o n t r i b u t e t o the e v e n t u a l s o l u t i o n of the s t r u c t u r e . T h i s p r o j e c t c o u l d be extended p r o f i t a b l y i n many d i r e c t i o n s . I t would be i n t e r e s t i n g , f o r example, t o l o o k a t the e f f e c t s o t h e r i o n s , such as C a 2 + (which b l o c k s the c h a n n e l ( 4 9 ) ) , have on the spectrum. A 2H NMR study of g r a m i c i d i n s e l e c t i v e l y l a b e l l e d on the amino a c i d s i d e c h a i n s would y i e l d f u r t h e r i n f o r m a t i o n about the c o n f o r m a t i o n . The t e c h n i q u e used i n t h i s work may be a p p l i e d t o any membrane p r o t e i n amenable t o hydrogen-exchange and r o t a t i n g r a p i d l y on the NMR t i m e s c a l e . W i t h membrane p r o t e i n s c o n s t r u c t e d of the commonest secondary s t r u c t u r a l u n i t s , the a - h e l i x and /3-sheet, the s p e c t r a may prove t o be e a s i e r t o i n t e r p r e t than t h o s e r e p o r t e d h e r e . 40 BIBLIOGRAPHY 1. M i c h e l , H., and O e s t e r h e l t , D. (1980) P r o c . N a t l . Acad. S c i . USA 77:1283-85 2. - M i c h e l , H. 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