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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.Sc,  McGill University,  A THESIS SUBMITTED  1983  IN PARTIAL FULFILMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES D e p a r t m e n t 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 conforming  to the r e q u i r e d  standard  THE UNIVERSITY OF B R I T I S H COLUMBIA October  ©  1985  M i c h a e l James L y o n s ,  1985  In  presenting  this  degree at the  thesis  in  partial  fulfilment  University of  British  Columbia, I agree  freely available for reference and study. copying  of  department  this or  thesis by  for scholarly  his  publication of this thesis  or  her  Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  the  requirements  for  may  representatives.  It  be is  /$//£>/8S  advanced  that the Library shall make it  I further agree that permission  purposes  an  granted  for extensive  by the head  understood  that  for financial gain shall not be allowed without  permission.  Date  of  of  my  copying  or  my written  Abstract This  thesis  application  of  presents  a  novel  resonance  technique  11:1)  a  to  Deuterium of  was  membranes.  occuring  gramicidin  The  a l . , J.  spectra  the  lipid  motion phase, of  Magn.  on  A'  the  timescale.  gramicidin  to  about  the  be i n d e p e n d e n t presence  investigated. the  The  of  spectroscopic  the  The  sodium  results  Callaghan  spectra  of  molecular crystalline  thickness, in  rotation  director.  phase  chloride,  spectra  The were  temperature, the  ranges  a r e d i s c u s s e d i n the c o n t e x t of the  s t u d i e s , and  s t r u c t u r e and a c t i o n o f  developed  i n t h e g e l phase of  bilayer  of b i l a y e r  in  " d e p a k e i n g " powder  the l i q u i d  crystalline  c o n d u c t i o n p r o p e r t i e s of  other  T.  indicate l i t t l e In  dynamics  channel,  recently  and  form, and  a r e s i m i l a r and NMR  ion  Thesis,U.B.C.).  f r e q u e n c i e s of t h e l i q u i d  and  an  however, the s p e c t r a suggest r a p i d u n i a x i a l  the  found  magnetic  polypeptide.  s u p p r e s s i o n (P.  in crystalline  bilayer  A*,  first  Eur. Biophys. J .  membrane  Reson. 56:101),  ( E . S t e r n i n , M.Sc.  gramicidin  the  nuclear  technique e x p l o i t s  procedures for solvent-signal et.  state  of  u s e d t o s t u d y t h e s t r u c t u r e and  hydrogen-exchanged  model  solid  results  (K. P. P a u l s e t . a l . ,  naturally  NMR  the  gramicidin.  i i  gramicidin  ion  channel,  t h e c r e t i c a l models of  the  T a b l e of C o n t e n t s Abstract  i i  List  of T a b l e s  iv  List  of F i g u r e s  v  Acknowledgements I.  vi  Introduction  1  11 . G r a m i c i d i n  4  A. B i o l o g i c a l F u n c t i o n B. G r a m i c i d i n : a M o d e l Transport C. C o n d u c t a n c e D. III.  Structural  4 System  P r o p e r t i e s of G r a m i c i d i n  S o l i d S t a t e NMR  5 7  S t u d i e s of G r a m i c i d i n  A. D e u t e r i u m NMR  9  as a S t r u c t u r a l Tool  14  Spectra(6,7,8,37)  15  B. A Deuterium Polypeptide  NMR  Study  I V . M a t e r i a l s and M e t h o d s  of  a  Synthetic  16 21  A. M a t e r i a l s  21  B. NMR  21  C.  2  H  Sample P r e p a r a t i o n NMR  Experiments  22  V. R e s u l t s  24  VI . D i s c u s s i o n VII.  f o r the Study of Ion  Concluding  34 Remarks  40  BIBLIOGRAPHY  41  i ii  List Fluid  State Quadrupolar  of T a b l e s Splittings  iv  List 1.  Experimental Crystalline  of F i g u r e s  and S i m u l a t e d  S p e c t r a of  Polypeptide  17  2.  G e l and F l u i d Phase S p e c t r a  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 Crystalline Gramicidin F r e e I n d u c t i o n Decay B e f o r e a n d A f t e r  25  Solvent  26  4.  of the Polypeptide  Signal Subtraction  5.  G e l and F l u i d Phase S p e c t r a  6.  Fluid  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 in the Presence of Ions A r r h e n i u s P l o t o f T e vs. T e m p e r a t u r e  9.  2  v  of Gramicidin  i n DPPC  19  27  31 33  Acknowledgements I would l i k e people:  t o express  my g r a t i t u d e t o  interests; Alex  MacKay  a n d e n c o u r a g e m e n t , a n d f o r v a l u a b l e comments  the m a n u s c r i p t ; providing for  following  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 help  the  me  Klaas-Peter with  their  Datema  unpublished  interesting discussions. Finally,  t h i s t h e s i s t o my p a r e n t s  and  Peter  wish  James a n d M a r g a r e t  vi  regarding Pauls  f i n d i n g s ; Neal I  for  to  Lyons.  for  Poulin  dedicate  I. The  attempt t o  proteins  in  understand  terms of p h y s i c a l  t o s c i e n t i s t s o f many proteins  this  determination. resisted  INTRODUCTION  program  i s at  These p r o t e i n s  t e c h n i q u e s , so  macromolecules,  have  gramicidin  A' ,  solvents,  illustrates  a  presents a  For the  ion  free  change  polypeptide.  This  spectroscopic  hence  the  classical  x-ray  in  studies  inapplicable. be  crystallized  of The  soluble case  from  of  in  (3,4) of  gramicidin secondary  experiments  on  indicate structure  r e s u l t i s at variance  protein's  structure (1,2),  with  gramicidin  membranes ( 5 ) . I t i s t h e r e f o r e membrane  of  of  organic  f u r t h e r disadvantage of t h e x-ray  forms  ion-induced  membrane  recently  d i f f r a c t i o n m e t h o d . The x - r a y a n a l y s i s and  challenge  integral  stage  of  until  useful  can  action  have,  been  which  1  biological  science  disciplines.  crystallization,  diffraction  the  ion a * of  will  not  in  be  major the  the r e s u l t s of artificial  f a r from c e r t a i n t h a t a  structure  bound  affected  given by  c r y s t a l l i z a t ion. Deuterium tool  nuclear  f o r studying  components  magnetic resonance  2  i s an e f f e c t i v e  t h e s t r u c t u r e and dynamics o f H 2  labelled  ( f o r r e v i e w s s e e 6 , 7 ) . The a n i s o t r o p i c  rotations  of a m o l e c u l e i n t h e l i q u i d c r y s t a l l i n e phase of a  membrane  partially  electric  averages  the  quadrupole i n t e r a c t i o n , the containing  information  orientation residual  dependent quadrupole  splitting  about t h e s t r u c t u r e and r o t a t i o n of  'Henceforth r e f e r r e d t o as A b b r e v i a t e d H NMR.  gramicidin.  2  1  2  the  molecule.  spectra and  the  site  rotating  than  the  times  for  the  i s attached uniaxially,  axis  rotation  of  the  may  conditions  t i m e s can  appropriate  principle of  certain  relaxation  correlation  When body  and  Under  a  field  calculated  the  parameters site. a  time  rigid shorter  a n g l e between  gradient,  from  of  labelled  correlation  timescale,  electric  be  of  order  a m o l e c u l e b e h a v i n g as  with a  NMR  provide  motion  to  measurement  and  the  the  the  axis  measured  order  parameter. A  synthetic  h y d r o g e n s , was ( 8 ) . The  studied  of  splitting  a  powder  rotating  expected  relaxation  calculation  simple  model  of  bonds was  calculated be  The  decay  within  s u c c e s s of to  more  Gramicidin  was  c h o s e n as  because of  i t s small  structure  of  the  i n the  the  axis  2  for  consistent  and  T ,  (15  time  the  membrane. D e s p i t e  for  amide  parameter  the  and the  a  N-D and  a-helix.  candidate  amino a c i d s ) ,  echo,  diffusion.  p o l y p e p t i d e s and  interesting  the  2 e  study prompted a p p l i c a t i o n  an  the with  quadrupole  order for  bilayer  structure  rotational  helix  al .  quadrupole  correlation  amide H  complicated  size  of  amide  et .  Measurement of  range p r e d i c t e d  this  the  single  axis,  molecule's  from the  the  a  rotational  a n g l e between t h e  state  secondary  helix  at  K.P.Pauls  fluid  structure.  the  technique  helical  the  the  the  the  found to  about  of  Further,  by  with  a helical  for  allowed  i n the  pattern  alpha h e l i x  time  H-labelled  2  indicating  polypeptide  2  u s i n g H-NMR  spectrum observed  consisted  the  polypeptide,  of  the  proteins. for  study  i t s expected simplicity  of  3  its  structure,  interesting  gramicidin  properties.  exhibits In  gramicidin  i s found t o render  permeable  to  small  ion  functionally  conduction  lipid  monovalent  some  experiments  membranes  cations.  selectively  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 its has  large  s i n g l e c h a n n e l c o n d u c t a n c e and o t h e r  continued  biological groups of  ion  of  interest  transport.  (9,10,11) a t t e m p t i n g  gramicidin-mediated  information The discussed of  t o be  as  a  molecular transport.  spectrosopic  and and  major c o n c l u s i o n s  K.P.Pauls electrical  (12)  are several simulations structural  undertaking.  study as  (8) and t h e well'  measurements.  as  "The  which does  (do)  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  not  secondary  ions.  structure  occurs  in  work  following  deform  fairly to  the  i s ( a r e ) independent, of  t e m p e r a t u r e i n t h e r a n g e i n v e s t i g a t e d ; t h a t no l a r g e in  be  previous  a r e drawn: t h a t g r a m i c i d i n a d o p t s a  r i g i d conformation(s)  for  of g r a m i c i d i n w i l l  2  K.P.Datema  there  Reliable  r e s u l t s o f t h i s H-NMR s t u d y with the polypeptide  system  dynamics  i s needed i n t h i s a m b i t i t i o u s  along  features i t  model  Furthermore,  of  change  the presence of sodium  II.  A. BIOLOGICAL  GRAMICIDIN  FUNCTION  Gramicidin, a linear polypeptide was o r i g i n a l l y molecule  isolated  from  having  fifteen  Bacillus  amino a c i d s , (13)  brevis  w i t h broad spectrum a n t i b i o t i c  a c t i o n . The  as  a  primary  s t r u c t u r e o f t h e 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-XXX-GLY-L-ALA-D-LEU-L-ALA-D-VALL-VAL-D-VAL-L-TRP-D-LEU-L-YYY-D-LEUL-TRP-D-LEU-L-TRP-Ethanolami X X X denotes  where  tryptophan, C  valine  phenylalanine,  respectively(15,  or  i s o l e u c i n e , a n d YYY  denotes  or t y r o s i n e i n g r a m i c i d i n  A,B,and  original  references  Commercially  a v a i l a b l e g r a m i c i d i n A'  gramicidins  A,B,C  amount  the  proportions  a s e l e c t i v e advantage f o r  a competitor's Recent gramicidin,  7:1:2  therein). of  valine  ,and a  small  (16). Gramicidin  Bacillus  may  by m a k i n g  brevis  membranes l e a k y t o c a t i o n s .  work  ( 1 7 , 1 8 ) has s u g g e s t e d a d i f f e r e n t  independent  Gramicidin-lacking to  cited  i s a mixture  of the i s o l e u c i n e g r a m i c i d i n s  provide  unable  in  ne  form  of  i t s ion  mutants normal  of spores  role for  transporting  Bacillus  brevis  unless  action.  were  found  provided  with  g r a m i c i d i n . T h i s e f f e c t h a s been c o r r e l a t e d w i t h t h e 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  inhibit  inhibiting  polymerase.  synthetic  bacterial gramicidin  RNA  analogues  RNA  showed  transcription Experiments that  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 used here. 3  4  only (14) a r e  by with the  5 sequence: (D-LEU-L-TRP)-Ethanolamine is  needed  discussed  to  in  such  as  the  strictly  the  mutants.  chirality  active )  of  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  p r o p e n s i t y . The  author  feels,  then,  that  ion  n o t be d i s c a r d e d a s a p o s s i b l e b i o l o g i c a l  As  primary  alternating  f o r g l y c i n e which i s not o p t i c a l l y  component  should  sporulation  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  structure, (except  restore  the  forming transport  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 Although served  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 , .as an i m p o r t a n t  biological oxidative  ion  cations  pinhole  as  an  In a black  Initially in  ionophore  lipid  found  mitochondria, selective  to  solvent  decouple  g r a m i c i d i n was for  monovalent  lipid  f i l m experiment a  d i s s o l v e d i n an o r g a n i c  membrane  small  amount  i s painted across a  s e p a r a t i n g two c o m p a r t m e n t s c o n t a i n i n g s o l u t i o n s  electrolytes. bilayer  transport.  u s i n g t h e then newly developed b l a c k  technique. of l i p i d  model system i n t h e i n v e s t i g a t i o n of  phosphorylation  characterized  g r a m i c i d i n has  The  persists  wavelength  lipid which  of v i s i b l e  (being  thins  much  until  thinner  a  single  than  l i g h t ) a p p e a r s b l a c k when v i e w e d  a microscope. E l e c t r i c a l be m o n i t o r e d u s i n g  membrane  of  the with  p r o p e r t i e s o f t h e membrane c a n t h e n  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  bilayer  forming  lipids,  black  membranes  are  o b s e r v e d t o be i m p e r m e a b l e t o i o n s . A c r u d e u n d e r s t a n d i n g the  insulating  electrostatic Q=4.8x10~  properties  may  h a d by c o n s i d e r i n g t h e  s e l f - e n e r g y , U, o f a s o d i u m i o n , esu,and radius=lA,  10  be  of  with  charge  i n an i n f i n i t e medium,  given  i n esu u n i t s as (19) : U= Q / ( 2 e R ) 2  Using for  approximate values water  ,and  self-energy bilayer  e=2  f o r the d i e l e t r i c  f o r o i l , we  constants,  find  a  difference in  o f 3.5 eV f o r an i o n i n o i l and w a t e r . The  thus  presents  e=80  lipid  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  ions. The  impermeablity  of t h e l i p i d m a t r i x  membrane t o e l e c t r o l y t e s h a s w i d e r a n g i n g enables  the  cell  ion c o n c e n t r a t i o n membrane.  This  t o maintain  is  b i o l o g i c a l membranes. The c e l l s have and the are  an  intracellular  an e x t r a c e l l u l a r extraroughly  passive  and  biological  consequences.  an a s y m m e t r y , w i t h  f o r example, a c r o s s asymmetry  of a  a  of the  concentration  concentration  intracellular  respect to  a plasma o r universal body  organelle  property  ion gradient.  o f s o d i u m o f c.5-15 mM  o f c.140mM. F o r p o t a s s i u m  concentations,  respectively,  I t i s g e n e r a l l y assumed t h a t t h e s e  has proven d i f f i c u l t  surprising  then  that  the  and  i n t h e maintenance of a s t a b l e mechanisms  i n v o l v e p r o t e i n s embedded i n t h e membrane, h o w e v e r activity  of  , f o r example,  t h e same ( 2 0 ) . I o n s e l e c t i v e a c t i v e "pumps"  "leaks" are necessary  It  to ion  isolate(21).  It  transport i s not  t r a n s p o r t i n g p r o p e r t i e s of  7  gramicidin,  a  simple  polypeptide,  excited  considerable  interest.  C. CONDUCTANCE PROPERTIES OF GRAMICIDIN The  c o n d u c t a n c e o f . i o n s by g r a m i c i d i n h a s been w e l l  using  the black  elegant  lipid  experiment  fluctuations  membrane t e c h n i q u e  constant  voltage  c o n t a i n i n g a s m a l l amount o f were  f o u n d t o be a p p r o x i m a t e l y  a  single  n o i s e was l a t e r  indicating  the  channel  closure  to  be  a  Poisson is  random  independent  thickness  channel of  the  t h e a v e r a g e l i f e t i m e v a r y i n g b e t w e e n 60 s e c o n d s i n  a 64 A b i l a y e r down t o .03 s e c o n d s i n a slow  The  high  s i n g l e channel  binding  c a r r i e r model i n  bilayer(24).  closure involves a  change. conductance observed p o i n t s t o  a p o r e model f o r t h e c h a n n e l , stationary  A  26  rate suggests that channel  major s t r u c t u r a l  (25)  exponential  b i l a y e r ( 2 4 ) , however  l i f e t i m e h a s been f o u n d t o d e p e n d on t h e  across  fluctuations  ( 2 3 ) f o u n d t o be an  t h i c k n e s s of the l i p i d  a  membrane  The a u t o c o r r e l a t i o n f u n c t i o n o f  of  Such  current  i n t e g r a l m u l t i p l e s of a u n i t  The c o n d u c t a n c e o f a s i n g l e c h a n n e l  bilayer,  a  The  process. the  of  i n t e r p r e t e d as the conductance  open c h a n n e l .  the c u r r e n t  across  gramicidin.  v a l u e , w h i c h was s u b s e q u e n t l y of  d e s c r i b e d a b o v e . An  (22) f o l l o w e d t h e time c o u r s e  at  studied  sites  which  of  the  in  which  ions  the channel, ion-carrier  t h e membrane. T h i s was c o r r o b o r a t e d  move  between  as opposed t o a  complex  diffuses  by t h e r e s u l t s o f  i n w h i c h t h e i n f l u e n c e of t h e phase of t h e  bilayer  on  8  t r a n s p o r t was conductance while  a  i n v e s t i g a t e d . No of  a  gramicidin  carrier  radically  discontinuous  doped  across  the  change  doped membrane was  membrane's liquid  in  observed,  conductance  crystalline  the  to  dropped gel  phase  transition. Gramicidin's measured  under  conductance and  otherwise  of  divalent  conduction  decreasing  +  position  i t may  of  various  identical c a t i o n s and  monovalent  > NH„  +  ions  > Cs  > Rb  +  been  conditions  (24):  anions i s n e g l i g i b l e ,  cations  bulk  > K  +  along a f i l e  the lumen of the g r a m i c i d i n conductance  concentrations  is  (in  > Na  +  > Li  +  order  of  lining  channel. of  a  gramicidin  membrane was  containing  found  to  i n d i c a t i n g the  channel as a dimer of the non-conducting  found  the  non-conducting  molecule of g r a m i c i d i n , and  species  species.  showed that most,  has  b^en  e q u i l i b r i u m between monomer found  t h i c k n e s s of lipids  with  to  the  be  shifted  bilayer:  shorter  conductance, a n a e s t h e t i c s  acyl  a  by  and  if  decreasing  not  all,  channels.  dimer  gramicidin  f a c t o r s a f f e c t i n g the  transmembrane chains  this  to be a s i n g l e  g r a m i c i d i n dimers in the b i l a y e r are conducting The  low  depend  A more d e t a i l e d study of t h i s phenomenon (26) confirmed result,  of  +  of water molecules  q u a d r a t i c a l l y on the g r a m i c i d i n c o n c e n t r a t i o n conducting  has  of the hydrogen ion in t h i s sequence suggests  be t r a n s p o r t e d  The  of  conductance): H  The  conductance  potential,and  increasing i t . An  the  bulk  e a r l y experiment  9  (26)  found c h o l e s t e r o l t o  This  may  be  decrease  the  bulk  conductance.  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  bilayer  thickness.  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 of g r a m i c i d i n ' s b i o l o g i c a l l y an a t t r a c t i v e the  research problem. While  g r a m i c i d i n channel  helix  models  independently,  the  The  26  some a s p e c t s  proposed by  of  of i t s  structure,and  by  D.W.Urry  Ramachandran  the  7r  (28,29)(and  (30))  on  the  6 - 3  (L,D)  later, basis  of  e n e r g y c o n s i d e r a t i o n s and m o d e l b u i l d i n g ,  is  most p l a u s i b l e , and t h e o n l y one c o n s i s t e n t w i t h a l l o f  the e x i s t i n g  of  conformation  have been p r o p o s e d . Of t h e s e ,  originally  conformational  the  presents  h a s n o t been d e t e r m i n e d , a number o f  e x p e r i m e n t s have r e v e a l e d several  interesting properties  basis  7r ' (L,D) 6  two l e f t A  data. helical  3  model of t h e c h a n n e l  handed h e l i c e s , h a v i n g  (28).  The  repeating  unit  d i p e p t i d e . The amide c a r b o n y l C-0  a total of  length  the h e l i x  alternation  of  opposite  hydrogen  of  direction.  bonding  directions  those This  t o h e a d d i m e r i z a t i o n by c o m p l e t i o n  hydrogen  bonds,  and  chirality  causes the s i d e chains  sterically  uncrowded  the  t h e a l t e r n a t i o n of a l p h a  L  of the unusual  permits  (formyl terminus)  with  about  i s t h e L,D  bond d i r e c t i o n s o f  r e s i d u e s a r e towards the ethanolamine terminus, D residues point i n the  i s a dimer  of  head six  carbon  to l i e outside the h e l i x i n  p o s i t i o n s . W i t h 6.3  residues/turn the  10 helix  has  a 4 A b o r e , and  Ramachandran p l o t dihedral per  of  angles  alternating  i n a l o w , f l a t minimum on  conformational  ( 2 8 ) . The  t u r n of the h e l i x  their  lies  greater  allow  the h e l i x  orientation  Some channel  of  be  selecting  the  conduction  l a r g e r i o n s , may  dipoles lining  energy b a r r i e r  amide  carbonyls  Thallium residues  due  p r e s e n c e of  ion binding  observed  be  position may  be  of  i n the primary due  dissociation  of  to  the  dimers.  the  with  7r  distortions  by  study  association The  reducing  seen  ions  by  NMR  of  only  indicating  the the  inconsistent with  the  alpha  g a t i n g of the  carbon channel  of  monomers  and  monotonic  increase  of  thickness  at  a l s o r u l e d out  s h i f t versus  s t r u c t u r e . The  the  gramicidin(31).  were  channel,  s i t e s . This  chemical  (L,D)  channel  u s i n g C-13  bound  shifts  the  6 - 3  the  coordinated  demonstrated  of  conductance with decreasing  gramicidin  c o o r d i n a t i o n of  3  continuity  formyl  pore s e l e c t s c a t i o n s over  r i g h t h a n d e d J T ' ( L , D ) h e l i x w h i c h was 6  of  the  to the s m a l l e r  can  chemical  n e a r t h e mouths  N-terminal  terms  carbons i n m i c e l l e  ion-induced  efficient  i n s i d e of the c h a n n e l ,  been  and  s t r u c t u r a l c o n t i n u i t y of  t o t r a n s p o r t . The has  l a b e l l e d carbonyl  the  promote  sequence  these  helix  monomers.  in  be  alpha  p r o p e r t i e s of  selectivity  perhaps because  carbonyl  groups  could  i o n s p r o d u c e i n t h e h e l i x . The  anions  number o f c a r b o n y l  uninterupted  understood  The  helix  compact  j u n c t i o n of the  the  may  structure.  the  nearly  at the  versus  than found i n , s a y , the  c o m p l e x i n g o f p e r m e a n t i o n s . The groups  energy  the  i s consistent with  the bulk the  11 26  A  channel  investigated The  being  shorter  7r ' (L,D) 6  and  and  helix  3  ion  wider  binding  of  i s consistent with X-ray  free gramicidin  5 A wide i n the absence of  and  a l l  the  membranes  (24).  several structural studies. bound  than  (6-8  A)  sites/dimer  in  analysis  f i n d i n g s of of  ion  showed a c y l i n d e r 32 A  long  salt,  and  (3,4)  one  presence  shorter  (26  of  salt,  with  at p o s i t i o n s consistent  with  the  m o d e l , i t has  been f o u n d t h a t t h e  in organic  (from  which the  c r y s t a l s were  the  membrane  conformation  solvents  grown) i s q u i t e d i f f e r e n t (5,32).  As  binding  probably  (5,32  discussed  and  The came  7r ' (L,D) 6  not  nitroxide  ethanolamine the  conformation  of  c h a n g e on  study  whether  ion  gramicidin is  the  at  5 A  observed  labels  the  s t u d y of  shift  7r ' (L,D) 6  labelled  r e a g e n t s Tm,  and  helix  3  gramicidin Mn  and  t o DSPCwere used to probe  attached  near  the  formyl  bilayer.  Based  on  found t h a t  the  T,  and  not  from  the  the shift  ethanolamine terminus  from the aqueous i n t e r f a c e but  "Distearoylphosphatidylcholine.  chemical  a  and  t e r m i n a l e n d s t o t h e a q u e o u s i n t e r f a c e and  m e a s u r e m e n t s i t was accessible  known  NMR  spin label attached of  the  evidence for  F-19  ( 3 3 , 3 4 ) . The  accessibility  of  and  the  helices.  3  f r o m a C-13,  with  i n membrane bound  Further,  is  most c o n v i n c i n g  in micelles  center  it  well  conformational  occur  study).  and  c y l i n d e r s are  below, the  does not  this  resolution,  from  very  C-13  7r ' (L,D) gramicidin  agree  ion  While  3  results  2  A)  results. 6  these  the  the  was  inside  12 the l i p i d  b i l a y e r . The o p p o s i t e was  terminus.  This  result  found  f o r the formyl  eliminated other e x i s t i n g proposals  f o r t h e membrane c o n f o r m a t i o n o f g r a m i c i d i n . Circular secondary  dichroism  structure  gramicidin  of  biomolecules,  i n sonicated  dichroism of gramicidin to  spectroscopy,  vesicles  i n DMPC  5  sensitive  was  used  (5,32).  t o the to  The  study  circular  was f o u n d t o be i n s e n s i t i v e  t h e p r e s e n c e o f a l a r g e amount  (1 M) o f a c e s i u m 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  small  re-orientation  spectrum  i n DMPC  temperature gel  was  from  o f t h e t r y p t o p h a n r e s i d u e s . The CD also  found  to  be  4°C t o 50°C, an i n t e r v a 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  The  CD  spectrum  the range:  1:45  independent  of  encompassing the o f t h e membrane.  i n DMPC d i d n o t d e p e n d on c o n c e n t r a t i o n i n to  1:15  (molar  ratio  of  gramicidin  to  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 o f g r a m i c i d i n , v e s i c l e s d i d n o t f o r m . The s p e c t r u m was f o u n d t o identical markedly  bilayers  or  having  6  a  The  may  change  be  6 7  due  i n spectrum  i t was  7  i n the  t o a change i n p i t c h o f t h e  i n equilibrium  between  conformations  former  as  a  e x p l a n a t i o n , t h e y were u n a b l e t o possible  t h i c k n e s s . That t h e s p e c t r a  5  difference  nearly  d i f f e r e n t CD s p e c t r a . W h i l e t h e a u t h o r s o f t h i s work  favored the l a t t e r the  amd D L P C , b u t i n DSPC a n d D P P C  different.  thicker helix  i n DMPC  be  result  of  change  i n DL- a n d DMPC  Dimyristoylphospatidylcholine. Dilaurylphosphatidylcholine. DipalmitoyIphosphatidylcholine.  discard  i n bilayer  are the  same,  13 and  exhibit  that  one  no c o n c e n t r a t i o n of  dependance  in  DMPC,  implies  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 with the  conducting  s t r u c t u r e of t h e p h o s p h o l i p i d / g r a m i c i d i n  system has  channel. The  been s t u d i e d u s i n g  P-31 NMR  amounts o f g r a m i c i d i n of  monounsaturated  length greater these  or  membranes .  than  other  (12,35).  I t was f o u n d t h a t  induced hexagonal-II and  saturated  PC's  phase i n b i l a y e r s having  16 c a r b o n s . T h e r e was no  non-bilayer  phases  in  small  acyl  chain  indication  the shorter  of  chain  Ill.  SOLID STATE NMR AS A STRUCTURAL TOOL  Second rank t e n s o r interaction  between  the  quadrupole  electric  i n t e r a c t i o n s , such  spins,  the  dipolar  chemical s h i f t anisotropy,  coupling  are  a  function  of  the  orientations  reference  i n an NMR e x p e r i m e n t . M e a s u r e m e n t o f t h e m a g n i t u d e  these  interactions  determine the tensor. the  i n an o r i e n t e d  directions  If this  same  l a b and m o l e c u l e - f i x e d  and  relative  of  of  as  of  information  system  their  the  sample a l l o w s  principle  i s obtained  relative  frames of  axes  one t o of  the  for several sites i n  orientations  may  be  calculated. 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 for  NMR  work  is  however, can y i e l d  usually  unavailable.  sample  suitable  Powder  spectra,  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  techniques 13  to  of the  the study  application of  a  C-NMR s t u d y o f b a c t e r i o r h o d o p s i n  al.  (36)  adding and  1 3  C  phospholipid  the  solid  membrane (BR) by  state  B.A.  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  angles  phase  spectra  of  vesicles permitted  BR  Lewis  et.  i n t o BR by lattice  reconstituted  into  c a l c u l a t i o n of t h e range  of the l e u c i n e peptide  groups w i t h  respect  a x i s o f r o t a t i o n a l d i f f u s i o n o f BR i n t h e membrane.  14  NMR  p r o t e i n , i s the  i t t o t h e g r o w t h medium o f H. Hal obi urn. R i g i d fluid  Euler  of  of to  15 A. DEUTERIUM NMR  SPECTRA(6,7,8,37)  The  single  l i n e o f an i s o l a t e d d e u t e r i u m  is  split  by  the  moment o f  the  gradient  into  frequency.  interaction  nucleus a  The  with  a  doublet  quadrupole  of  nucleus'  the e l e c t r i c  molecular  symmetric splitting  spectrum quadrupole  electric  about Aa>,  field  the  given  Larmor  by  the  (8):  equation  2OJ [ P ( C O S 0 ) + 7 } / 2 S I N 0 C O S 2 0 ]  &J(6,4>)=  2  2  where:  to = 3 / 4 ( e q Q / h ) 2  o  in  which  the  eQ i s t h e q u a d r u p o l e moment o f t h e n u c l e u s , eq i s  principal  tensor,and parameter, gradient  value  h  is  of  the  Planck's  from c y l i n d r i c a l  gradient  splitting spectrum,  constant;*?  s y m m e t r y ; 6, and  principal  i s usually as  field  CL>  is  gradient  the  asymmetry  other  in  system.  the predominant  the  The  dominant  interactions,  field  4> a r e t h e s p h e r i c a l  vector  axis  c o u p l i n g between d e u t e r i u m n u c l e i , tO  field  a measure of t h e d e p a r t u r e of t h e e l e c t r i c  p o l a r angles of the magnetic field  electric  such  electric  quadrupole  f e a t u r e of the  as  are small  the in  dipolar  comparison  .  a Rapid a x i a l timescale)  of  reorientation the  molecule  i n t e r a c t i o n y i e l d i n g a narrowed Acj(0,a)= 2 o [ P ?  (compared  molecule  and  1  1  2  averages  the  s p l i t t i n g o f (8)  NMR  quadrupole :  (COS/3)+Tj/2SIN /3COS2a) ] P (COS©) 2  2  where 6 i s t h e a n g l e b e t w e e n t h e a x i s the  t o x 'AM ~ , t h e  2  of  the e x t e r n a l magnetic  reorientation field,  of  0 and a a r e  16 spherical  polar  principal axis gradient.  coordinates coordinate  To  predict  of the a x i s of r o t a t i o n  system  of  the e f f e c t  the e l e c t r i c  of slower  i n the field  m o t i o n s on t h e  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  biological  s a m p l e s s u i t a b l e f o r NMR  spectroscopy  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 orientations  of  computed u s i n g  the molecules.  The  possible  s p e c t r u m may t h e n be  (6) :  f (x)=2/r /SINf3dr3 /(x^P (COS0)+77/2SIN 0) ±  2  9  v  1  2  •/(-x±P ( C O S 0 ) + T ? / 2 S I N 0 ) 2  1  2  where  x=2u/v^  which  may  f ( x ) a r e t h e two b r a n c h e s o f t h e s p e c t r u m , ±  r  be  evaluated  (convoluted  with  "simulated"  spectra.  Axial  the  rotation,  analytically oriented  regardless  gradient,  as  may  numerically  lineshape)  to  i n t h e s h o r t - c o r r e l a t i o n time  l e a d s t o a powder s p e c t r u m h a v i n g parameter  or  zero  apparent  seen  by  comparison  limit,  asymmetry  o f t h e symmetry o f t h e e l e c t r i c  be  prepare  of  the  field above  equations.  B. The  A DEUTERIUM NMR STUDY OF A SYNTHETIC POLYPEPTIDE synthetic polypeptide  ,with primary  structure:  LYS - G L Y - L E U - L Y S -ALA-AMIDE 2  (where was  polypeptides  designed  interactions  to  x  2  w i t h x = 1 6 , 2 0 , 2 4 h a v e been  systematically  i n membranes.  investigate  synthesized) lipid-protein  17  —I -400  1  1  1  -200  1  1  t  0  200  i  i 400  Frequency (kHz)  F i g u r e 1 (a)H-NMR spectrum 2  of the c r y s t a l l i n e  labelled  p o l y p e p t i d e a t room t e m p e r a t u r e , (b) S i m u l a t e d spectrum w i t h parameters »» =l50 g  reference (8).  kHz, rj=. 16,  T = 160iisec. 2 e  Figures  from  18 The study  24  by  leucine polypeptide et.  Pauls  the p o l y p e p t i d e crystalline  al.  (8)  was  the  s u b j e c t of a H  i n which the  labile  were r e p l a c e d by d e u t e r i u m .  polypeptide,shown  simulated(figure  1(b))  by  in  figure  and  a m o t i o n a l l y n a r r o w e d one  attributed  to  by  (a),  +  NH  groups  3  for a d i s t o r t i o n  of  due  on  t o the  lysine  narrow  a solvent s u b t r a c t i o n procedure,  crystal.  mobility fluid  This  s t a t e of DPPC, t h e  consist  Since  v =127  kHz,  the  31  amide  equivalent  in  the  helical along  spectrum  o f a s i n g l e Pake d o u b l e t  parameters:  helical  (figure well  and  TJ=0  deuterons spectrum  conformation the  principal  helix  axis,  directions of T  2 e  to  the  be  expected  versus  f o r the  of  chain-melting  the  little  ,in  the  found  using  to the  polypeptide  splitting of  the  of the  the  field  are  must assume a  narrowing  lying  motionally  angle  between  g r a d i e n t and  range  of  the  the N- H 2  a - h e l i x s t r u c t u r e . Measurement  t e m p e r a t u r e e x h i b i t e d a minimum  o n s e t of p e p t i d e  in  2e  of the e l e c t r i c I9°:within  has'  was  simulated  the p o l y p e p t i d e  The  that  42°C  2(b))  w i t h a x i s of m o t i o n a l axis.  axis  j3, t o  to  the measured T =lOOMS.  narrowed spectrum a l l o w s c a l c u l a t i o n the  spectrum i n  i m p l i e s t h a t the p o l y p e p t i d e  i n t h e g e l p h a s e of t h e membrane. A t  side  component,  the  t h e 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 the  amide  kHz,77=0  =  g  the  the  powder  v 36  with  of  i s well  a s u p e r p o s i t i o n of a broad  deuterons,  caused  spectrum  1  p a r a m e t e r s p^=150kHz , r p . 1 6  c h a i n s . Except  hydrogens of  The  pattern having  rotating  NMR  2  r o t a t i o n at the  corresponding  t e m p e r a t u r e of  p h a s e t r a n s i t i o n of t h e b i l a y e r ,  and  the  permitted  F i g u r e 2. H-NMR spectrum of the 2  DPPC  liposomes  reference (8).  (a)  at  10°C  synthetic (b)  at  polypeptide  42°C.  in  F i g u r e s from  20 calculation motion,  r  of was  the  rotational  f o u n d t o be  2X10~  correlation 7  s e c a t 42°C.  time  f o r the  I V . MATERIALS AND  METHODS  A. MATERIALS Gramicidin  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 u s e d 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 . and  Dilauryl  L - a - p h o s p h a t i d y l c h o l i n e were  Sigma C h e m i c a l Co., weight)  pure,  Deuterium  oxide  >99.9  atom  used  (>99.8  without  atom  and  them  purchased being  further  %D),  were  >98%  (by  purification.  dimethyl-d  methanol-OD  from  sulfoxide(  6  purchased  from  was  obtained  o f g r a m i c i d i n were d e u t e r i u m  exchanged  3  isotopes.  SAMPLE PREPARATION  l a b i l e hydrogens  by d i s s o l v i n g  the  gramicidin  a l k a l i n e w i t h gaseous  ND ,  in  temperature. Bulk methanol  was  the l a b i l e  solution  i t s i t overnight  removed  c h e c k e d u s i n g t h e 400  facility  in  intensity  of t h e amide r e g i o n , was  the  by  made  at  room  Rotavap  keeping  the  vacuum o v e r n i g h t . The d e g r e e o f e x c h a n g e  s i t e s was the  methanol-OD  removed u s i n g a B u c h i  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 g r a m i c i d i n under  excess  h e a t i n g and s t i r r i n g  3  a t 50°C f o r an h o u r , t h e n l e t t i n g  shifts,  as  and Dohme I s o t o p e s . G a s e o u s ND  from Cambridge  The  and  %D),  Merck,Sharpe,  B. NMR  which quotes  Dimyristoyl  Chemistry  u n i f o r m and  t o be a b o u t  70%.  The  deuteration  of  about  Department.  MHz The  i n the spectrum  proton  NMR  decrease  in  of  i n d i c a t e d the degree of tryptophan 35%.  These 21  indole  chemical  deuteration  sites  results  of  are  showed in  a  good  22 a g r e e m e n t w i t h an e a r l i e r 2  by  H-NMR  simply  t h e d r y powder  Gramicidin-lipid  methanol mixture  was was  removed  placed  until  throughout the  i n the  the  2  H  a  H  and  NMR  NMR  was  as  tube.  follows:  methanol-OD;  above;  the  the  powdered  using a  solvents to prevent  of  glass  were  used  back-exchange  samples of dry,powdered g r a m i c i d i n c o n s i s t e d polypeptide.  was .038  .5 gms  gms  The  with:  total  .25  gms  weight  of D 0 ; 2  o f g r a m i c i d i n . Thus t h e  should  be  15%  of  .212  most gms  composition  of of  g r a m i c i d i n , by w e i g h t , o r  1:16  lipid.  EXPERIMENTS  spectrometer  built  a Nalorac  crystalline  in quadrature  superconducting  s p e c t r a l d i s t o r t i o n due  Q was  a c c u m u l a t e d on an  and  using  Intel-210  Intel-230  The  MHz  using  electronics NMR  taken  probe with  the  low  to  r e s p o n s e of  the  sufficiently  to non-linear  p r o b e . D a t a was  an  magnet.  s w e e p e r . C a r e was  samples t h a t the c o i l  processed  a t 35.5  i n the P h y s i c s Department  tuned using a frequency  avoid  prepared  Deuterated  s p e c t r a were r e c o r d e d  s h o p , and  sample  sample s t i r r e d ,  sample p r e p a r a t i o n  of the  bilayers  2  were  NMR  s a m p l e t u b e ; t h e d e s i r e d amount  m o l e s g r a m i c i d i n t o m o l e s of  C.  an  prepared  sites.  dispersions  the  in  described  homogeneous.  Typically,  lipid,  as  a d d e d , and  the  labile  of 80 mgs  dispersions  g r a m i c i d i n were  g r a m i c i d i n were c o d i s s o l v e d i n  deuterium oxide rod,  (48).  samples of c r y s t a l l i n e  packing  l i p i d s and  study  microcomputer,  microcomputer  and  the  23  university The pulses  mainframe.  quadrupolar was  used  i n d u c t i o n decay to  echo p u l s e  w i t h phase c y c l i n g ( 8 ) ,  After  signal  solvent  Fourier  signal  of  the  component  to noise  probe a r c i n g , i n which case  r . f . power were  errors at the top out-of-phase  to obtain the free  ( F I D ) . W i t h some s a m p l e s i t was n o t p o s s i b l e  use 7r/2 p u l s e s w i t h o u t  p u l s e s a t lower  ( 5 0 ) w i t h 3,usec 7 r / 2  sequence  r a t i o by  transformed.  of  used.  suppression echo the  »/2,  3iusec  were FID  and  (40),digitization  corrected zeroed  the  ( 6 ) , the  t o improve t h e  in-phase  component  V. The in  2  figure  pattern  T  2  e  with  spectrum of c r y s t a l l i n e g r a m i c i d i n . The  spectrum c o n s i s t s of  quadrupole  7 ? = .15. The  time r  NMR  3(a)  parameter the  H  RESULTS  constant  coupling  spin-lattice for  were 460 msec and  Msec  single  v = 148 kHz  and  relaxation  the decay  170  a  respectively.  r . f . pulse  one  length  due  (38),  and  (figure  3  of a n o t h e r 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  the  proton  NMR  of  (see the  previous  deuterons  a t t a c h e d t o amide g r o u p s . T r y p t o p h a n expected  t o have v =223 kHz g  f r o m t h e amide s p l i t t i n g . spectrum  is  probably  due  (b)  chapter)  in  the  indole  narrow  ).  supports that  sample  are  deuterons  are  (39) and w o u l d be w e l l  The  t o the  the e x p e r i m e n t a l spectrum  absence  90%  from  is  The  approximately  echo,  there  s i m u l a t e d u s i n g the above p a r a m e t e r s  result  and  Apart  e f f e c t s of the f i n i t e  between  asymmetry  of t h e q u a d r u p o l a r  i n the s h o u l d e r s , p o s s i b l y  agreement  powder  time,T,,  some l o s s o f i n t e n s i t y  excellent  i s shown  central  resolved  peak  in  to r e s i d u a l methanol-0 H 2  the  i n the  sample. Figure  4  (a)  shows  transverse magnetization quadrupole  echo  sequence  w i t h g r a m i c i d i n . The aqueous  the after  in-phase the  second  f o r a sample  strong, long-lived  component o f t h e d i s p e r s i o n ,  weak, s h o r t - l i v e d e c h o  from  the  component pulse  containing signal,  of  due  deuterons  the  liposomes  i s superimposed  amide  of t h e  to  the  on t h e of  the  peptide. The solvent signal was s u b t r a c t e d f o l l o w i n g t h e method o f C a l l a g h a n e t . a l . ( 4 0 ) , t h e r e m a i n d e r i s shown i n 24  25  -512  -256  0  256  512  256  512  FREQUENCY(kHz)  -512 FREQUENCY(kHz)  Figure  3. (a)H-NMR spectrum of H-exchanged g r a m i c i d i n  crystalline  2  2  form  at  23°C.  100,000  transients  accumulated a t a r a t e o f 2/sec. (b) Powder p a t t e r n  in were  simulated  u s i n g t h e parameters v =148 kHz, 17= .15, and T = 1 7 0 jxsec. 2e  26  (b)  1  Figure 4. DLPC,  (a) Free i n d u c t i o n decay of  a  sample  containing  d e u t e r a t e d g r a m i c i d i n , a n d D 0. D i s c o n t i n u i t i e s 2  i n the  s i g n a l a r e a r e s u l t of computer memory o v e r f l o w s , (b) F i g u r e (a)  after  signal  was  a  f i f t h o r d e r p o l y n o m i a l , f i t t e d t o the s o l v e n t subtracted.Scale:1  cm=  49.2  c o r r e s p o n d i n g spectrum i s shown i n F i g u r e 7 ( a ) .  usee.  The  F i g u r e 5. H-NMR spectrum of d e u t e r a t e d 2  (a) (12).  at  20°C  and  gramicidin  i n DPPC  (b) a t 52°C. R e s u l t s from Datema  et.al.  28 figure 4 ( b ) . Figure splittings recorded is  5 of  2  (a)  shows  H-labelled  by K l a a s - P e t e r  with  t»^= 144  Datema  kHz  (see  Table  show  a  of  quadrupole  DPPC  liposomes,  ( 1 2 ) . The b i l a y e r , a t 20°C, is  a  single  77= .18 . I n t h e f l u i d  (figure  powder s t a t e of  5 ( b ) ) shows a number o f  1) a n d t h e s h a p e o f t h e s i d e s o f t h e  spectrum i n d i c a t e s that spectra  in  spectrum ,  DPPC, a t 52°C, t h e s p e c t r u m splittings  spectrum  gramicidin  i n t h e g e l s t a t e and t h e  pattern  the  rj=0  f o r the  distortion  outermost  lines.  Both  due t o t h e 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 , a s do a l l o f t h e s p e c t r a  of d i s p e r s i o n s  in  ±25 kHz was n e g l e c t e d i n  this  thesis,'  and  the  region  presented  a n a l y s i s of the s p e c t r a . The are  fluid  state spectra  of g r a m i c i d i n  given  in  figures  splittings  of  the spectra are given  for  the outermost  spectra  of  this  lines  figures  were o b t a i n e d  areas. binomial  lines, The  of  the  7(a)  respectively.  i n Table  spectra.  "depakeing"  (41). This  and p e r m i t s  depaked  and  The  1. A g a i n ,  The  77=0  "oriented"  6 (a) and 7(a),shown i n 6 ( b ) and 7 ( b ) ,  using a  laboratory  spectral  6(a)  i n DMPC a n d DLPC  algorithm  facilitates  measurement  spectra  of  developed  in  resolution  of  linewidths  were t r e a t e d t o a t h r e e  smoothing r o u t i n e . L i n e w i d t h s  of t h e  spectra  and point were  f o u n d t o be i n t h e r a n g e 8-16 k H z . The f l u i d of  .1M N a C l  s t a t e spectrum of g r a m i c i d i n  i s shown i n f i g u r e 8 ( a ) a l o n g  i n the  presence  w i t h t h e depaked  s p e c t r u m . An a t t e m p t t o r e c o r d a s p e c t r u m i n t h e p r e s e n c e o f  29  1  1  (a)  400  i  -200  0  1  200  400  FREQUENCY(kHz)  (b)  A 1  M i  i -200  -400  i  I  i 0  1  l  200  400  FREQUENCY(kHz)  Figure DMPC a t  6.  (a) H-NMR spectrum of d e u t e r a t e d  38°C.  2  122,000  F i g u r e (a) "depaked".  transients  were  gramicidin i n  accumulated.  (b)  30  (a)  -400  -200  0  200  400  FREQUENCY(kHz)  I -400  I  I  -200  I  L  I  0  J  200  I  I 400  FREQUENCY(kHz)  F i g u r e 7. (a) 'H-NMR spectrum of d e u t e r a t e d DLPC  a t T=  gramicidin i n  20°C. 167,000 t r a n s i e n t s were accumulated, (b)  F i g u r e (a) ^depaked".  31  -400  -200  0  200  400  200  400  FREQUENCY(kHz)  -400  -200  0 FREQUENCY(kHz)  F i g u r e 8. (a) H-NMR spectrum of 2  DMPC  at  gramicidin  in  T= 40°C. The aqueous phase of t h e sample c o n s i s t e d  of .1M NaCl i n H 0 . 150,000 2  2  (b) F i g u r e  deuterated  (a) "depaked".  transients  were  accumulated,  32 1M  NaCl  was  made,  decrease the c o i l observable.  but  saline  Q - f a c t o r s o much t h a t  Figure  s o l u t i o n was f o u n d t o a  signal  was  2 e  versus  1 F l u i d State Quadrupolar  Splittings  8 9  (kHz)  DMPC  DPPC  DMPC+NaCl  136 ( 4 )  136 ( 2 )  134  134 (2)  128 ( 2 )  125  126 (2)  104 ( 2 )  1 05  100 (2)  96  92  67 ( 1 )  65  70 (1)  52  49  47  70 (1 )  is  temperature.  DLPC  104 ( 2 )  not  9 , t h e r e s u l t s o f K.P. Datema ( 1 2 ) ,  an A r r h e n i u s p l o t o f T  Table  the  U n c e r t a i n t y i n frequencies ^IkHz. 'Numbers i n p a r e n t h e s e s a r e a p p r o x i m a t e of t h e l i n e s .  relative  intensities  800 400 200 A  CO  3*  100  4  A  80 60  A  A A  A I  A  40 h 20  3.00  J 3.14  L 3.28  I 3.42  L 356  3.70  1000/T (K* ) 1  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 I n t h i s c h a p t e r , an a t t e m p t 2  H  NMR  results  presented  will  in  be made t o r e l a t e  chapter  the  four t o aspects 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  values  for  v  and  TJ m e a s u r e d  from t h e H 2  NMR  o spectrum of c r y s t a l l i n e found  f o r deuterons  systems  gramicidin in  are  -N-H»««0=  similar  to  those  h y d r o g e n b o n d s i n many  ( 3 9 ) . The v a l u e o f i>^=148 kHz a g r e e s  reasonably  well  with that c a l c u l a t e d using the e m p i r i c a l r e l a t i o n (39): v = 252.-522./R( H-•-0) 2  With With  R=1.8  A ,expected 1 0  fora TT ' (L,D) helix, 6  1.73 A . , t h e e s t i m a t e d 1  v^=150. Pauls  1  kHz  in  excellent  2  3  agreement  spectrum  bonding of a  powder  that,  i s quite  The  with the f i n d i n g s of  t o note  i n R c a n make a s i g n i f i c a n t  This suggests  at  i> =160 kHz.  H»«-0 d i s t a n c e f o r t h e a - h e l i x ,  et . al. ( 8 ) . I t i s i n t e r e s t i n g  difference  3  least  sensitive  in to  t h a t t h e .07  A  change i n f r e q u e n c y .  principle, changes  the  2  H-NMR  i n t h e hydrogen  molecule.  s p e c t r u m i n t h e g e l s t a t e o f DPPC i s a l s o a s i n g l e pattern,  different  with  however p^=144  the kHz,  parameters 77= .18.  are  somewhat  This i s not s u r p r i s i n g  s i n c e , a s was n o t e d  i n chapter  the  o f c r y s t a l a n d membrane-bound g r a m i c i d i n  conformations  one, there i s evidence  that  a r e n o t t h e same. No s u c h c h a n g e i n p a r a m e t e r s was f o u n d f o r Rough f i g u r e c a l c u l a t e d a s s u m i n g -N-H«««0= c o l i n e a r a n d u s i n g t h e a t o m i c c o o r d i n a t e s g i v e n by Koeppe a n d K i m u r a (42). 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 a n d C o r e y ( 4 3 ) , a n d u s i n g t h e a s s u m p t i o n m e n t i o n e d i n f o o t n o t e 10. 1 0  1 1  34  35 the  synthetic polypeptide. The  DPPC,  fluid  state spectra  DMPC, o r DLPC show s e v e r a l  found t h a t  b i l a y e r s . We H-NMR  l i n e s . Wallace  t h e r e was no d i f f e r e n c e  state circular  2  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  dichroism spectra therefore  spectrum  on  narrowing, rather  el. al. ( 5 )  between t h e g e l a n d f l u i d  i n DLPC,DMPC,DPPC, a n d DSPC  attribute  the  dependence  of  the  l i p i d phase t o t h e e f f e c t s of m o t i o n a l  than t o a change i n t h e s t r u c t u r e  of  the  polypeptide. It  i s not p o s s i b l e  t o s a y , by l o o k i n g  at  alone,  what t h e a x i s o f r o t a t i o n  i s , or whether  motion  i s i n the short c o r r e l a t i o n  time regime.  discussion  of  chapter one, i t i s f e l t  conformation of the g r a m i c i d i n dimer,  which  i s probably  s i n g l e channel conductance lifetime rotation axis  (24)  motions and  correlation Pauls  rotational  would  the  6  Based  on t h e  t h e predominant  3  stable  of seconds)  i s n e a r l y about  prevent  the rate of  TT ' (L,D) helix  i s usually  bury  spectra  i s a N-terminal to N-terminal  timescale  of t h e dimer  bilayer  by  (a  that  the  . That t h e  f o r a channel's  suggests that the  the  helix  axis.  Off  t h e mouths o f t h e c h a n n e l i n t h e  ion conduction.  1 2  The  rotational  t i m e may be e s t i m a t e d u s i n g t h e t h e o r y p r e s e n t e d 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 diffusion  about  a  undergoing  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 ) a n d T=40°C :  F o r t h e same r e a s o n , we e x p e c t t h e h e l i x a x i s n o t t o d e v i a t e much f r o m t h e b i l a y e r d i r e c t o r . An a v e r a g e r a d i u s e s t i m a t e d f r o m a s p a c e f i l l i n g model d e p i c t e d i n ( 4 4 ) . The 7 r ' ( L , D ) i s somewhat b u l k i e r a t t h e 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. 1 2  1 3  6  3  ,  36 .47 usee  T =(47ra h7?)/(kT)= 2  c  For  monomers,  with  usee. The c r i t e r i o n  h=l5  A, t h e p r e d i c t e d v a l u e i s r = . 2 3 c  f o r rapid motional narrowing i/(AM )  - 1  2  »  T  where A M , t h e c h a n g e i n t h e s e c o n d  2  2  { [ 1+T /l5]-[P (COS/3)+Tj/2SIN j3COS2a] } 2  2  ?  i n Table  i/m ~  1  2  For the l a r g e r  splittings,  Further evidence correlation recorded range  of  under  these  1 v  = 50  i/AMj"  1  the  i sgreater. motion  is  comes f r o m c o m p a r i s o n  b i l a y e r s and a t  are  i n v a r i a n c e of the observed reorientation  n a r r o w i n g about  surely  the  same  2 e  of the s p e c t r a i n the  correlation  different,  and  short  that  times  the  near  t h e axes  the  motional  i s complete.  the average  values of T  the  frequencies implies that  this axis  We may p r e d i c t the  are  in  temperatures  40-50°C ( 1 2 ) . As t h e r o t a t i o n a l conditions  kHz :  - 2.6 Msec  that  time l i m i t  in differing  2  2  For the s m a l l e s t s p l i t t i n g s  using  C  spectrum i s :  AM = 1/5w  of  (8):  moment o f t h e m o t i o n a l l y  2  narrowed  is  "  1  value  f o r each  of  line  T  ~ (at 1  2 e  40°C),  of the spectrum i n  DMPC c a l c u l a t e d u s i n g t h e e q u a t i o n ( 8 ) : T  The  2 E  =T -'AM c  1  2  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 ,  of m a g n i t u d e a s t h e v a l u e s i n f i g u r e Only residues  two s e t s o f of  the  deuterons,  dipeptide  same  order  9.  those  repeat,  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  of  the  D  and  L  are inequivalent with  the  helical  axis.  The  37 a n g l e s b e t w e e n t h e d i r e c t i o n s of t h e N-D  b o n d s and  the  a x i s are approximately '  Assuming  that  4.5°  1  principal the  and  19°  a x i s of t h e e l e c t r i c f i e l d  N- H  bond  2  direction,  m o t i o n a l l y narrowed l i n e s  then  .  the  gradient tensor l i e s  the  should  helix  splittings  in  of  the  be : 1 5  v = P (COS|3) • 1 44kHz=1 42kHz , 1 2 1 kHz . a 2  As  there  i s an  polypeptide  e q u a l number o f  the  intensities  s p e c t r u m s h o u l d be The above. presence line  are  non-channel  The  of the l i n e s lines,  at  two  two  lines  of  surprising  may  arise  i n the  because  of  g r a m i c i d i n i n the b i l a y e r , correspond  i n the e x p e r i m e n t a l  134,128,105 kHz,  the  or  to the tryptophan  one  indole  spectrum.  w h i c h have the 128  and  For  the  three  same i n t e n s i t y 136  kHz  in  lines  are  the  lines  are  attached to that  12.4°, 15.7°,25.1°  L  the  gradient  principal  likely  to  and  amide  angles  p r e d i c t e d above, as t h e axis  D do  direction is  not  be  those a r i s i n g bonds.  It  of  the  known and  from  is  not agree w e l l w i t h  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  electric  not those field  is difficult  to  i n angles  may  " 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 d i m e n s i o n s o f t h e p e p t i d e bond g i v e n i n ( 4 5 ) . T e r m s 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 . 1 5  the  r e s o l v e d ) , t h e 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 :  deuterons  1  of  l i n e s p r e d i c t e d do n o t c o i n c i d e " w i t h any  0= Two  residues  these  DMPC (and DLPC, a s s u m i n g t h a t t h e not  D  more c o m p l e x t h a n p r e d i c t e d  lines  of t h e s p e c t r u m may  deuterons.  of  clearly  additional  of  and  equal.  spectra The  L  the  38 also  reflect The  an e r r o r  above  definitive  if  concerning  differing  less  detailed  structure  the  3  statements  of  implies  splittings  (Table  1 6  that  may '  gramicidin.  quadrupolar  thickness  temperatures  6  c a l c u l a t i o n s a r e somewhat s p e c u l a t i v e .  the  agreement of  i n the 7 r " ( L , D ) s t r u c t u r e .  1)  and  t o adapt  to  made  excellent  bilayers at  of  different  the s t r u c t u r e of t h e  p e p t i d e backbone does not deform  be  The in  More  a  gramicidin hydrophobic  mismatch w i t h t h e b i l a y e r . F u r t h e r , t h e a x i s of r o t a t i o n , well  as  the  molecular  conformation(s) variables  are  order  i n the range  investigated.  gramicidin,  i t seems l i k e l y  cylinder's  axis  t h i s would  would The  (47) s u g g e s t in  DMPC  normal, the  independent If  the  of  these  7r ' (L,D), 6  that there i s l i t t l e  respect  the  or  3  model, i s the s t r u c t u r e  t o the b i l a y e r  p r o b a b l y d e p e n d on  tilt  of  director,  the c o n s t r a i n t s  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 an  a n g l e of l e s s t h a n  15° w i t h t h e  i n a g r e e m e n t w i t h o u r c o n c l u s i o n s . As chapter  s i n g l e channel  is  a s one  of  this  thesis,  independent  would  expect  of  of  the  for a r i g i d  al .  bilayer  discussed  thickness  as  channel  the conductance  the  of 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 o f N a b e d r y k et.  makes  first  bilayer,  with  of  1 7  approximately  another approximately c y l i n d r i c a l  bilayer.  parameter ,  as  of  in  of a the  pore w i t h i t s a x i s  d, t h e b i l a y e r h y d r o p h o b i c 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 t h e number o f c a r b o n s on t h e a c y l c h a i n ) , d i s 2 0 , 2 3 , a n d 26 A f o r DL,DM,and DPPC, respectively. We may t a k e P ( C O S 8 ) , where 6 i s t h e a n g l e b e t w e e n t h e h e l i x a x i s and t h e b i l a y e r d i r e c t o r , and t h e a v e r a g e i s o v e r t h e v a l u e s o f $ s a m p l e d by t h e m o l e c u l e , w e i g h t e d by t h e p r o b a b i l i t y w i t h which they occur. 1 6  1 7  2  39 of  rotational  diffusion,  the  axis  of  the  cylinder,  p e r p e n d i c u l a r t o t h e p l a n e of t h e b i l a y e r . Our  results  DLPC  the  in  depaked  in  the  spectra  were  spectrum  in  DLPC  spectrum,  identical DPPC  in  suggests  and  circular  d i c h r o i s m spectrum  change  in  equilibrium  the  sample spectrum  was in  experiments. et.  a/.(3,4)  gramic i d i n .  to  have  DM  and  We  therefore are  DLPC,  not  from  the DMPC  except f o r  the  134  kHz  c i r c u l a r dichroism  bilayers.  there  is  no  large  The  major change  2  H  NMR  change i n in  the  i n DPPC i s t h u s p r o b a b l y due t o between  o f .1M N a C l  found  with  ( 5 ) . The  these  conformations  g r a m i c i d i n , as t h e a u t h o r s of t h i s paper The p r e s e n c e  al.  identical,  i s not r e s o l v e d  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  a  agreement  spectra are nearly  128 kHz l i n e , w h i c h  line  excellent  d i c h r o i s m s t u d i e s o f W a l l a c e et .  circular and  are  in feel  the  (5) s u s p e c t e d .  i n t h e aqueous r e g i o n little  of  of  i n f l u e n c e on t h e H 2  agreement  with  the  the NMR CD  t h a t t h e r e s u l t s o f Koeppe  applicable  to  membrane-bound  V I I . CONCLUDING REMARKS This of  study  gramicidin  presented  makes i t c l e a r t h a t t h e membrane s t r u c t u r e is  unsolved.  We  feel  that  gramicidin-membrane i n t e r a c t i o n , and w i l l  the  eventual  directions.  could  to  be  extended  profitably  in  many  I t w o u l d be i n t e r e s t i n g , f o r e x a m p l e , t o l o o k a t  e f f e c t s other  channel(49)), gramicidin  contribute  s o l u t i o n of the s t r u c t u r e .  This project  chains  results  i n t h i s t h e s i s have deepened o u r u n d e r s t a n d i n g of  the  the  the  ions,  have  on  selectively  would  yield  such the  as  Ca  2 +  spectrum.  labelled  on  further  (which A  the  2  H  blocks  NMR s t u d y o f  amino  information  the  acid  side  about  the  conformation. The  technique  u s e d i n t h i s work may be a p p l i e d t o a n y  membrane p r o t e i n amenable t o h y d r o g e n - e x c h a n g e a n d  rotating  rapidly  proteins  on  constructed a-helix  the  NMR  timescale.  With  membrane  o f t h e commonest s e c o n d a r y s t r u c t u r a l u n i t s , t h e  and  /3-sheet, t h e s p e c t r a may p r o v e t o be e a s i e r t o  i n t e r p r e t than those r e p o r t e d  here.  40  BIBLIOGRAPHY 1.  M i c h e l , H., a n d O e s t e r h e l t , D. S c i . USA 77:1283-85  (1980) P r o c . N a t l .  2.  - M i c h e l , H. (1982) J . M o l . B i o l .  Acad.  158:567-572  3.  Koeppe, R. E. I I , H o d g s o n , K. 0., a n d S t r y e r , L. ( 1 9 7 8 ) J . M o l . B i o l . 121:41-54  4.  K o e p p e , R. E. I I , B e r g , J . M., Hodgson, S t r y e r , L . (1979) N a t u r e 279:723-725  5.  Wallace, B. A., V e a t c h , W. R., a n d B l o u t , E. R. B i o c h e m . 2 0 : 5754-60  6.  D a v i s , J . H. (1983) B i o c h i m . B i o p h y s . A c t a  7.  B l o o m , M., a n d S m i t h , I . C. P. (1985) I n : P r o g r e s s i n Protein-Lipid I n t e r a c t i o n s , W a t t s , A . a n d de P o n t , J . J . H. H. M. e d s . , E l s e v i e r Scienxe P u b l i s h e r s B. V. Amsterdam  8.  P a u l s , K. P. , MacKay, A. L. , Soderman, 0., B l o o m , M., T a n j e a , A. K., a n d H o d g e s , R. S. (1985) E u r . B i o p h y s . J . 11:1  9.  B e n o i t Roux, H a r v a r d U n i v e r s i t y , P e r s o n a l C o m m u n i c a t i o n  K.  0.,and (1981)  737:117-171  10. MacKay, P. H. J . , B e r e n s , P. H . , W i l s o n , K. R., a n d H a g l e r , A. T. (1984) B i o p h y s . J . 46:229-248 11. F i s c h e r , W. , B r i c k m a n n , B i o p h y s . Chem. 13:105-16 12. Datema, K. Unpublished  P., P a u l s , results.  J. K.  ,  and  P.,  13. H o t c h k i s s , R. D., Dubos, R. J . 132:791  and  (1940)  Lauger,P. B l o o m , M. J.  Biol.  (1981) (1983) Chem.  14. L e h n i n g e r , A. L . ( 1 9 8 2 ) P r i n c i p l e s o f B i o c h e m i s t r y , W o r t h Pub. I n c . , New Y o r k , p.96 15. A n d e r s o n , 0. S . , ( 1 9 8 4 ) i n : A n n u a l Review of Physiology 46, B e r n e , R. M., a n d H o f f m a n , J . F. e d s . A n n u a l R e v i e w s Inc. P a l o A l t o 16. O v c h i n n i k o v , Y. A., I v a n o v , V. T., a n d S h k o b , A. M. ( 1 9 7 4 ) Membrane A c t i v e C o m p l e x o n e s , E l s e v i e r , Amsterdam. 17. V e a t c h , W.,Sarkar, N., M u k h e r j e e , P. K., L a n g l e y , D., P a u l u a ,H. , I v a n o v , V. T. a n d S h e p e l , E. N. (1979) P r o c . Am. P e p t i d e Symp., 6 t h , R o c k f o r d , I l . : 7 0 7 - 7 1 0 41  42 18. F i s h e r , S c i . USA  R.,and B l u m e n t h a l , 79:1045-48  T.  (1982) P r o c . N a t l .  19. P a r s e g i a n , A . ( 1 9 7 4 ) i n : B i o l o g i c a l Membranes, e d . , Simon F r a s e r U n i v e r s i t y 20. A l b e r t s , B., B r a y , D., W a t s o n , J . D. (1983) G a r l a n d Pub. I n c . , New  K.  Acad. Colbow  L e w i s , J . , R u f f , M., R o b e r t s , K., Molecular B i o l o g y of the Cell York  2 1 . H a r r i s o n , R. and L u n t , G. 2nd e d i t i o n , H a l s t e a d P r e s s  ( 1 9 8 0 ) B i o l o g i c a l Membranes  22. H l a d k y ,  A.  S.  B. and  H a y d o n , D.  (1970) N a t u r e  225:451  23. L a u g e r , P. ( 1 9 8 1 ) I n : Membranes and Intercellular Communication, B a l i a n , R . , C h a b r e , M., D e v a u x , P.F. e d s . N o r t h H o l l a n d P u b l i s h i n g Co. Amsterdam 24. H a y d o n , D. A. and Hladky, B i o p h y s . 5:187-282  S.  25. K r a s n e , 174:412  and  S.  26. G o o d a l l , M.  Eisenman,G.,  r  C.  B.  (1974)  W.  Rev.  Szabo,G. ( 1 9 7 1 ) S c i e n c e  (1970) B i o c h i m . B i o p h y s . A c t a  27. V e a t c h , W. R., M a t h i e s , R., E i s e n b e r g , ( 1 9 7 5 ) J . M o l . B i o l . 99:75-92 28. U r r y , D.  Quart.  M.,  ( 1 9 7 1 ) P r o c . N a t l . A c a d . S c i . USA  219:471-8 Stryer,  L.  68:672-6  29. U r r y , D. W., G o o d a l l , M. C , G l i c k s o n , J . D., M a y e r s , F . ( 1 9 7 1 ) P r o c . N a t l . A c a d . S c i . USA 68:1907-11  D.  30. R a m a c h a n d r a n , G. N., Chandrasekaran, R. (1972) I n : Progress i n P e p t i d e R e s e a r c h , L a n d e , S. e d . Gordon and B r e a c h , New Y o r k 2:195-215 3 1 . U r r y , D. W., Trapane, S c i e n c e 221:1064-67  T.  32. W a l l a c e , B. A., V e a t c h , W. B i o p h y s . J . 37:197-199  L., R.,  Prasad,  K.  U.  and B l o u t , E. R.  (1983) (1982)  33. W e i n s t e i n , S., W a l l a c e , B. A., B l o u t , E. R., Morrow, S. Veatch, W. (1979) P r o c . N a t l . Acad. Sci. 76:4230-34 34. W e i n s t e i n , S., W a l l a c e , B. A., M o r r o w , J . S., B. ( 1 9 8 0 ) J . M o l . B i o l . 143:1-19  Veatch,  J. USA W.  35. Van E c h t e l d , G. J . A., de K r u i j f f , B., V e r k l e i j , A. J . , L e u n i s s e n - B i j v e l t , J . , de Gier, J . Biochim. Biophys. A c t a 648:1  43 36. L e w i s , B. A., H a r b i s o n , G. S., G. B i o c h e m . 24:4671-79  Herzfeld, J . , Griffen,  37. Abragam, A., Principles of Nuclear U n i v e r s i t y P r e s s , London (1961)  Magnetism  38. B l o o m , M., D a v i s , J . H., P h y s . 58:1510-17  (1980)  39. MacKay, A. L.  V a l i c , M.  ( 1 9 7 5 ) Ph.D.  I.  Thesis, Oxford  ( 1 9 8 2 ) M.Sc.  T h e s i s , U. B.  42. 4 2 . K o e p p e , R. E. I I , a n d K i m u r a , 23:23-38 43. P a u l i n g , L. and C o r e y , R. B. S c i . USA 37: 235-240  Can.  C.  M.(1984)  T.  J.  Soderman,  Biopolymers  (1951) P r o c . N a t l .  44. U r r y , D. W., P r a s a d , K. U . , T r a p a n e , N a t l . A c a d . S c i . USA 79:390-394  Oxford  Univ.  40. C a l l a g h a n , P. T., MacKay, A. L., P a u l s , K. P., O., B l o o m , M. J . Magn. R e s o n . 56:101-109 4 1 . S t e r n i n , E.  R.  L.(1982)  Acad. Proc.  45. D i c k e r s o n , R. E. a n d G e i s , I . The S t r u c t u r e a n d A c t i o n o f P r o t e i n s ( 1 9 6 9 ) W. A. B e n j a m i n I n c . M e n l o P a r k 46. M o u r i t s e n , O.  G. a n d B l o o m , M.  47. N a b e d r y k , E., G i n g o l d , M. J . 38:243-249  P.,  48. P a u l s , K. P., D a t e m a , K. P.,  ( 1 9 8 4 ) B i o p h y s . J . 46:141 Breton, J.(1982)  a n d B l o o m , M.,  Biophys.  Unpublished  49. R o e s k e , R. W. , a n d K e n n e d y , S. J . ( 1 9 8 3 ) I n : C h e m i s t r y a n d B i o c h e m i s t r y o f Amino A c i d s , P e p t i d e s , a n d P r o t e i n s B. W e i n s t e i n e d . , M a r c e l D e k k e r I n c , New Y o r k 50. D a v i s , J . H., J e f f r e y , K. R., B l o o m , M., V a l i c , M. I . , a n d H i g g s , T. P. ( 1 9 7 6 ) Chem. P h y s . L e t t . 42:390  

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