NMR Spectroscopy - American Chemical Society

12 Solid State N M R of Linear and Cyclic Peptides

Downloaded by CORNELL UNIV on August 23, 2016 | http://pubs.acs.org Publication Date: July 9, 1982 | doi: 10.1021/bk-1982-0191.ch012

L. M. GIERASCH University of Delaware, Department of Chemistry, Newark, DE 19711 M. H. FREY, J. G. HEXEM, and S. J. OPELLA University of Pennsylvania, Department of Chemistry, Philadelphia, PA 19104

Studies of synthetic model peptides can contribute to the the understanding of principles important in determining the structures of naturally occurring peptides and proteins. These tractable model systems also facilitate the development of methods for studying molecular structure and dynamics, since the methods can be verified and improved before being applied to more complex and often precious natural compounds. High resolution NMR has proved to be a very powerful approach to the analysis of solution conformations of peptides (1). The capability of obtaining high resolution NMR spectra of solid samples extends the opportunity for analysis to crystalline peptides (2-5), therefore comparisons can be made of peptide conformations in different environments and physical states. In addition, some of the information obtained from the analysis of solid-state NMR spectra is unique and provides insight into both static and dynamic aspects of peptide conformation. The experiments on peptides utilize high resolution dilute spin double resonance solid-state NMR (6, 7). In these experiments, magic angle sample spinning averages out chemical shift anisotropy and dipolar couplings, cross polarization enhances C sensitivity, and high power proton decoupling removes H- C dipolar couplings. This combination of techniques gives spectra with sufficient resolution for analysis of chemical shifts as well as lineshapes of resonances. 13

1

13

Several o f the model c y c l i c peptides used i n these s t u d i e s were designed to mimic regions o f polypeptide chains where hydrogen bonded reverse turns a r e likely to occur (£, .2, These peptides c o n t a i n p r o l i n e , an imino a c i d which has a high frequency o f occurrence i n turns ( 1 1 ) , and which i s i n t r i n s i c a l l y i n t e r e s t i n g

0097-6156/82/0191 -0233$06.00/0 © 1982 American Chemical Society

Levy; NMR Spectroscopy: New Methods and Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

N M R SPECTROSCOPY


234

because o f the impact o f i t s c y c l i c side chain on the l o c a l conformation of polypeptides* For example, various p r o l i n e r i n g geometries are p o s s i b l e Q 2 ) , and side chains o f r e s i d u e s preceding p r o l i n e s are allowed l e s s conformational freedom than i f they precede one of the amino a c i d s ( H ) * In a d d i t i o n , c i s X-Pro bonds are e n e r g e t i c a l l y competitive with the t r a n s c o n f i g u r a t i o n , i n c o n t r a s t to peptide bonds i n v o l v i n g amino a c i d s where the trans form i s s t r o n g l y favored (14)* Both c y c l i c and l i n e a r peptides have been studied by s o l i d state NMR* In some cases, comparisons can be made d i r e c t l y between s o l u t i o n and s o l i d - s t a t e conformations and i n o t h e r s , d i r e c t comparisons between X-ray d i f f r a c t i o n determined s t r u c t u r e s and NMR determined s t r u c t u r e s can be made* NMR data have also been used to describe the conformation of peptides i n the s o l i d - s t a t e i n the absence o f a p r i o r d i f f r a c t i o n a n a l y s i s * Three aspects o f s o l i d - s t a t e NMR have proved to be f r u i t f u l i n our i n i t i a l peptide s t u d i e s * F i r s t , the i s o t r o p i c chemical s h i f t s i n the s o l i d state have led to determination o f conformat i o n s taken up i n the s o l i d (when the s t r u c t u r e i s not known), and to the c o r r e l a t i o n o f expected chemical s h i f t s with p a r t i c u l a r conformational f e a t u r e s (when the s t r u c t u r e i s known from d i f f r a c t i o n data)* Second, the p o s i t i o n s and lineshapes o f resonances a r i s i n g from p o t e n t i a l l y mobile parts o f the peptide (e*g* side chains) have revealed dynamical aspects o f the s o l i d - s t a t e s t r u c t u r e s o f peptides* The a n a l y s i s o f molecular motions i s s i m p l i f i e d i n the s o l i d state by the absence o f o v e r a l l molecular tumbling, which modulates spin i n t e r a c t i o n s and leads to complex frequency -dependent s p e c t r a l responses* In p a r t i c u l a r , s i g n a l s a r i s i n g from aromatic r i n g s i d e chains are well separated from other resonances, and may be i n t e r p r e t e d i n terms o f r e o r i e n t a t i o n models o f these side chains* Such r i n g dynamics are o f great importance i n p r o t e i n s t r u c t u r e s , and s t u d i e s with model pept i d e s can help e l u c i d a t e fundamental aspects o f these processes* The combined use o f ^C, ^N, and H s p e c t r a l a n a l y s i s enhances the u t i l i t y o f s o l i d - s t a t e NMR a n a l y s i s o f r i n g dynamics* T h i r d , s i n c e magic angle sample spinning does not completely remove ^N- ^C d i p o l a r c o u p l i n g s (la), resonances from c a r bons d i r e c t l y bonded to n i t r o g e n s have an asymmetric doublet lineshape* Geometric information i s a v a i l a b l e from the a n a l y s i s o f the '^N induced s p l i t t i n g s o f resonances* 1

1

1

2

1

Conformational A n a l y s i s o f I s o t r o p i c Chemical

Shifts

Three c y c l i c peptides designed to exemplify d i f f e r e n t f e a t u r e s of peptide conformation have been studied by s o l i d - s t a t e NMR ( 3 ) , s o l u t i o n NMR (!£, 18) and X-ray d i f f r a c t i o n (!£>)* The NMR r e s u l t s described here i n b r i e f form show a v a r i e t y o f i n t e r e s t i n g phenomena f o r these peptides* Cvclo(D-Phe-Pro-Gly-DAla-Pro) i s a r i g i d c y c l i c pentapeptide which contains i n s o l u t i o n



12.

GIERASCH E T A L .

Solid State NMR

of

Peptides

235

and i n the s o l i d (from X-ray d i f f r a c t i o n a n a l y s i s ) both a 3 - t u r n (reverse turn s t a b i l i z e d by hydrogen-bonding between the carbonyl o f residue i and the N-H o f residue i+3) and a γ-turn (reverse turn s t a b i l i z e d by hydrogen bonding between the carbonyl of r e s i d u e i and the N-H o f residue i+2)* The i s o t r o p i c "^C chemical s h i f t s f o r the a l i p h a t i c carbons o f t h i s c r y s t a l l i n e peptide are e s s e n t i a l l y superimposable on the s h i f t s i n s o l u t i o n (Figure 1), thus confirming from NMR data that the peptide has the same conformation i n s o l u t i o n and i n the s o l i d * Only the presence of the asymmetric doublets f o r the α carbon, c a r b o n y l , and Pro resonances i n the s o l i d state spectrum y i e l d e d d i f ferences between the two spectra* E s p e c i a l l y noteworthy i s the appearance o f an unusually high f i e l d p r o l i n e C g resonance i n both spectra* This u p f i e l d p o s i t i o n i s due to an e c l i p s i n g o f the Pro carbonyl and g-methylene group which r e s u l t s from a very low trans' ψ angle n e c e s s i t a t e d by the p a r t i c i p a t i o n o f t h i s residue i n a γ-turn (19)* The c l o s e correspondence o f chemical s h i f t s i n the s o l u t i o n and s o l i d state s p e c t r a , e s p e c i a l l y the high f i e l d Pro Cg resonance, i n d i c a t e s that l o c a l intramolecular e f f e c t s predominate on determining chemical s h i f t s , and that spectra f o r s o l i d samples may be c o r r e l a t e d with peptide conformation* The peptide c y c l o ( G l v - P r o - G l y ) ο presents a quite d i f f e r e n t situation* The a n a l y s i s o f i t s NMR spectrum leads to the c o n c l u s i o n that i t adopts a Cp-symmetric conformation i n s o l u t i o n , at l e a s t on NMR timescales* The s o l u t i o n NMR spectrum (Figure 2B) shows the minimum number of resonances expected (one per c a r bon i n the repeating t r i p e p t i d e u n i t ) * By c o n t r a s t , i n the s o l i d s t a t e spectrum there i s c l e a r i n d i c a t i o n o f asymmetry s i n c e there are two Pro resonances i n Figure 2A* X-ray d i f f r a c t i o n analy s i s has revealed an asymmetric molecular conformation f o r the c r y s t a l l i n e peptide (17)» with two d i f f e r e n t types o f β-turns, only one o f which i s i n t r a m o l e c u l a r l y hydrogen-bonded* Analysis o f the s o l i d state i s o t r o p i c chemical s h i f t s i n terms o f l o c a l conformation y i e l d s a p i c t u r e of the molecule which i s c o n s i s t e n t with the X-ray data* A t h i r d c y c l i c peptide studied i s cvclo(D-Phe-Gly-Ala-GlyPro)* Figure 3 compares i t s s o l i d state and s o l u t i o n NMR spectra* Two aspects of t h i s peptide have been studied* F i r s t , the i s o t r o p i c chemical s h i f t p o s i t i o n s show that s t r u c t u r e I i s p r e f e r r e d i n the s o l i d state and both s t r u c t u r e s I and I I e x i s t i n s o l u t i o n * The existence of a γ - t u r n with p r o l i n e as the i+1 residue i n the s o l i d s t a t e conformation i s i n d i c a t e d by the high f i e l d p o s i t i o n o f the Pro resonance* By c o n t r a s t , the Pro resonance p o s i t i o n i n s o l u t i o n supports an e q u i l i b r i u m be tween s t r u c t u r e s I and I I , s i n c e i t occurs midway between an unperturbed Pro and one i n a γ -turn* Second, a s t r i k i n g f e a t u r e of the s o l i d s t a t e ^C spectrum of t h i s peptide i s i n the aromatic region where a broad i l l - d e f i n e d resonance i n the base l i n e i s a l l that i s seen f o r the δ and ε carbons while theY 1


NMR

SPECTROSCOPY


236

Figure 1. C NMR spectra of cyc\o(D-Phe-Pro-Gly-O-Ala-Pro). Chemical shifts for all spectra are relative to external Me Si at 38 MHz resonance frequency. Key: A, polycrystalline sample; and B, solution (CDCl ) sample. 13

k

3


Solid State NMR

of

Peptides

237


GIERASCH E T A L .

Figure 2. C NMR spectra of cyc\o(Gly-Pro-Gly) at 38 MHz resonance frequency. Key: A, polycrystalline sample; and B, solution [(CD) SO/H O] sample. 13

g

s

t


NMR

SPECTROSCOPY


238

Figure 3. C NMR spectra of cyc\o(D-Phe-Gly-Ala-Gly-Pro) at 63 MHz resonance frequency. Key: A, poly crystalline sample; and B, solution {CDClJMeOH) sample. 13



12.

GIERASCH E T A L .

Solid Slate NMR

of

Peptides

239

and ζ resonances appear as sharp l i n e s from the Phe residue* This w i l l be analyzed i n the next s e c t i o n i n terms o f dynamics* The s t u d i e s of these three c y c l i c peptides demonstrates the s e n s i t i v i t y o f the p r o l i n e chemical s h i f t s o f resonances to the geometry o f the p y r r o l i d i n e r i n g * The observed v a r i a t i o n i n the resonance p o s i t i o n of the Pro appears to be c o r r e l a t e d with the p r o l i n e Ψ angle* Such a c o r r e l a t i o n has been proposed (19) and used i n s o l u t i o n NMR o f p r o l i n e c o n t a i n i n g peptides (8.» 10. 20)* The s o l i d - s t a t e NMR spectra serve two purposes; since the "^C s h i f t s i n the s o l i d can be d i r e c t l y compared to Xray determined Ψ angles, and the s h i f t s provide a means o f assessing the p r e f e r r e d Ψ angle i n peptides whose s t r u c t u r e s have not been determined by X-ray d i f f r a c t i o n * Table I l i s t s the Pro Cg and Cy chemical s h i f t s f o r a v a r i e t y of peptides* For those examples with known X-ray d i f f r a c t i o n s t r u c t u r e s , the observed φ ,Ψ angles are c i t e d * Im portant c o n c l u s i o n s can be drawn from c o n s i d e r a t i o n o f these data* The c o r r e l a t i o n o f a small Δβγ with a low trans' Ψ angle leads to the c o n c l u s i o n that cvclo(D-Phe-Gly-Ala-GlyPro) takes up a conformation with the p r o l i n e p a r t i c i p a t i n g in a Ύ-turn i n the s o l i d state* This i s i n c o n t r a s t to what would be i n f e r r e d from examination o f the s o l u t i o n spectrum (where the intermediate values o f Δβγ (2*5 ppm) i s c o n s i s t e n t with conformational averaging* The data i n Table I and the spectrum o f s o l i d Ala-Pro (Figure 4) that shows two d i s t i n c t sets o f p r o l i n e resonances i n d i c a t e that t h i s dipept i d e e x i s t i n forms with c i s and trans Pro residues* According to resonance i n t e n s i t i e s , there i s a 2:1 r a t i o o f c i s and trans conformers* Spectra o f s o l i d peptides can be used to d e s c r i b e c o n f o r mational f e a t u r e s o f the peptides* Of p a r t i c u l a r u t i l i t y are the Pro Cg and Cy chemical s h i f t s , which can d e s c r i b e r i n g geometry or occurrence o f c i s or trans X-Pro bond confor mation* Dynamical A n a l y s i s of Lineshapes S o l i d - s t a t e NMR spectroscopy i s well suited f o r s t u d i e s o f i n t r a m o l e c u l a r dynamics because side chain motions can be analyzed independent o f o v e r a l l molecular r e o r i e n t a t i o n i n c r y s t a l l i n e samples* I t i s an a l t e r n a t i v e s p e c t r o s c o p i c s t r a t e g y to s o l u t i o n NMR because p a r t i a l motional averaging of the a n i s o t r o p i c spin i n t e r a c t i o n s occurs* D i p o l a r , chemical s h i f t , and quadrupolar i n t e r a c t i o n s can be used to d e s c r i b e the dynamics o f aromatic r i n g s of p r o t e i n s and peptides.


NMR

240

SPECTROSCOPY

TABLE I P r o l i n e C g and C f o r Peptides

Chemical S h i f t s

i n the S o l i d

a

State


Φ , Ψ Compound

Pro C β

Pro C γ

Dihedral Angles from X-ray

Δ3γ

Proline

32.9

25.3

7.6

N-Acetyl-Proline

29.6

24.7

4.9

Ala-Pro ,

31.9 34.4

26.3 22.9

5.6 11.5

Pro-Ala

33.2

26.8

6.4

t-Boc-Gly-Pro-Gly-OBz

31.7

25.9

5.8

H* Pro-Leu-Gly-NH

30.7

26.9

3.8

— ,152.9

cyclo(D-Phe-Pro-GlyD-Ala-Pro)

26.2 29.8

25.5 25.5

0.7 4.3

-82,59 -64,128

cyclo(Gly-Pro-Gly>2

29.0 30.8

25.3 25.3

3.7 5.5

27.3

26.6

0.7

trans cis

2

cyclo(D-Phe-Gly-Ala-GlyPro)

a

Maximum u n c e r t a i n t y ± 0.5 ppm;

t y p i c a l u n c e r t a i n t y ± 0.2

^ r e f e r e n c e 15 c

r e f e r e n c e 16

^ r e f e r e n c e 17


b

c

c

d

r -53,126 -66,-36

1

ppm.

d

Solid State NMR

GIERASCH E T A L .

12.

of

Peptides

241

The r i n g dynamics o f the two phenylalanine c o n t a i n i n g c y c l i c peptides have been studied* In s o l u t i o n , both o f these peptides show H and NMR s p e c t r a c o n s i s t e n t with r a p i d r e o r i e n t a t i o n o f the aromatic r i n g s about the C 3 -Cy bond a x i s * The aromatic regions o f the s o l i d s t a t e NMR s p e c t r a o f the two peptides are shown i n Figure 5» The spectrum of c y c l o (D-Phe-Pro-Gly-D-Ala-Pro) shows i n t e n s i t y from a l l s i x carbon s i t e s o f the r i n g * This i n d i c a t e s a long residence time i n an asymmetric environment, and that on NMR timescales the r i n g i s r i g i d * The spectrum o f the aromatic r i n g carbons o f c r y s t a l l i n e c v c l o (D-Phe-Gly-AlaGly-Pro) i s much d i f f e r e n t * While the γ and ζ carbon resonances are sharp l i n e s there i s o n l y a very broad nonde s c r i p t resonance f o r the other four carbons* I t s very unusual appearance i s r e t a i n e d at two f i e l d strengths and under a v a r i e t y of experimental c o n d i t i o n s * The most l i k e l y e x p l a n a t i o n i s that the phenyl r i n g i s i n intermediate exchange, probably f l i p p i n g at a r a t e around 1 0 0 sec" about the C3 -Cy bond a x i s * This rate was derived from the a p p r o x i mate chemical s h i f t d i f f e r e n c e f o r the asymmetric environments based on our experience with l i n e a r and c y c l i c peptides* A d d i t i o n a l evidence from other n u c l e i and other i n t e r a c t i o n s supports these c o n c l u s i o n s (£); t h e r e f o r e the peptide i s a l i k e l y candidate f o r v a r i a b l e temperature s t u d i e s *


1

1

There are l a r g e d i f f e r e n c e s i n the dynamics o f the phenylalanine r i n g s o f the two c y c l i c peptides studied* The dramatic d i f f e r e n c e i n the spectra shown i n Figure 5 can o n l y be due to i n t r a m o l e c u l a r dynamics because a l l the a l i p h a t i c and carbonyl s i t e s g i v e narrow l i n e s * The s i g n i f i c a n t d i f ferences i n phenylalanine r i n g dynamics may be a consequence o f the Phe residue preceding p r o l i n e i n one case and g l y c i n e i n the other* 1l

1

Conformational A n a l y s i s o f *N- ^C D i p o l a r Couplings The resonances from carbons d i r e c t l y bonded to n i t r o g e n are asymmetric doublets i n s o l i d s t a t e NMR s p e c t r a ( 2 J J * This unusual lineshape r e s u l t s from the f a i l u r e o f magic angle sample spinning to completely suppress the ^^C-^\ d i p o l a r c o u p l i n g s , the success of which depends on the spins o f i n t e r e s t being quantized along the a p p l i e d magnetic f i e l d * ^N with s p i n S=l, has a quadrupole moment whose c o u p l i n g with the n o n s p h e r i c a l l y symmetric e l e c t r i c f i e l d g r a d i e n t produces a quadrupole i n t e r a c t i o n with magnitude comparable to the ^N Zeeman i n t e r a c t i o n * Thus, the a x i s o f q u a n t i z a t i o n o f the Ν s p i n s i s not along the a p p l i e d magnetic f i e l d but i s quantized along a r e s u l t a n t of the quadrupole and Zeeman interactions* 1

1


NMR

242

SPECTROSCOPY

NH?-CH-C-N^3 CH

3

COO

0

L-Ala-L-Pro


Trans^ Ρλ *l

ι

I 180

Figure 4.

ι ι ι

I

ι

ι ι

160

ι

ι ι ι

60

ι

I

40 PPM

I

I

j

ι ι ι

20

ι 0

A C NMR spectrum of polycrystalline Ala-Pro at 38 MHz resonance frequency. 13

A Cyclic(D-Phe-Gly-Ala-Gly-Pro)

Β Cyclic(D-Phe-Pro-Gly-D-Ala-Pro)

; ι 150

I

I

jιιιιIιιιιIιιιι I 140 130 120 110 PPM

I

Figure 5. A romatic regions of NMR spectra of cyclic peptides containing phenyl alanine. Key: A, expansion of Figure 3; and B, expansion of Figure 1. 13


12.

GIERASCH E T A L .

Solid State NMR

of Peptides

When the presence o f both these i n t e r a c t i o n s i s taken i n t o account, i t i s found that the appropriate truncated d i p o l a r Hamiltonian must be expanded to i n c l u d e a d d i t i o n a l terms whose angular dependence corresponds to the C and D terms o f the c l a s s i c a l d i p o l a r Hamiltonian (22), These terms contain the Euler angles a and 3 which o r i e n t the i n t e r n u c l e a r v e c t o r i n the coordinate system, which determines the d i p o l a r c o u p l i n g f o r the s p e c i f i c *^N- ^C s i t e s o f i n t e r e s t . This i s the p r i n c i p l e a x i s system o f the e l e c t r i c f i e l d g r a d i e n t , whose o r i e n t a t i o n with respect to the a p p l i e d magnetic f i e l d determines the \ s p i n s t a t e s and hence t h e i r d i p o l a r c o u p l i n g s . The dependence o f the d i p o l a r couplings on the o r i e n t a t i o n o f the i n t e r n u c l e a r v e c t o r i n the p r i n c i p l e a x i s system o f the e l e c t r i c f i e l d gradient has inherent information about the molecular geometry o f the r e l e v a n t \ and sites. For example, i n a peptide bond, where two carbons are bound to the same n i t r o g e n , the d i p o l a r coupling o f each depends on the o r i e n t a t i o n o f i t s r e s p e c t i v e i n t e r n u c l e a r v e c t o r with respect to the common coordinate system o f the nitrogen e l e c t r i c f i e l d gradient p r i n c i p l e a x i s system. Thus, the NMR spectrum i s analyzed in terms o f the r e l a t i v e o r i e n t a t i o n o f the i n t e r n u c l e a r vectors. In p r i n c i p l e , t h i s can lead to an independent determination o f the conformation o f the peptide bond. As an example, the a n a l y s i s o f the d i p e p t i d e A l a - A l a i s desc r i b e d . Figure 6 contains the s o l i d - s t a t e NMR spectrum o f t h i s compound, with expansions f o r the carbonyl and alpha carbon resonances i l l u s t r a t i n g t h e i r asymmetric doublet c h a r a c t e r . For the peptide bond, the Z-axis o f the e l e c t r i c f i e l d gradient p r i n c i p l e a x i s system i s expected to be perpendicular to the plane o f the bond (21) and although the l o c a t i o n o f the Xa x i s i s unknown, by analogy with N-Acetyl V a l i n e i t i s expected to l i e along the N-H bond (24), These r e l a t i o n s h i p s are depicted i n Figure 7* The d i p o l a r i n t e r a c t i o n s and the spectra can be c a l c u l a t e d as described p r e v i o u s l y (4., 2 1 ) , Using the quadrupole coupling constant and asymmetry determined f o r N-acetyl v a l i n e (24), the t h e o r e t i c a l spectra are compared to the experimental spectra i n Figure 6, Both the s p l i t between the peaks o f the asymmetric doublet and the lineshape p r e d i c t e d from the theory agree well with the experimental spectrum. This close f i t confirms the appropriateness o f the conformational and spectroscopic parameters used i n the c a l c u l a t i o n . These r e s u l t s i n d i c a t e geometrical information i s a v a i l a b l e from lineshapes a n a l y s i s o f resonances from carbons bonded to n i t r o g e n . This a n a l y s i s i s straightforward f o r planar peptide bonds, and s i g n i f i c a n t s p e c t r a l d e v i a t i o n s are expected f o r nonplanar peptide bonds. D

D

1

1


243

1


NMR

244

E

NH -CH-C-N-CH-COO 3


CH,°

CH

SPECTROSCOPY

E

3

I ' ' ' I 180

160 PPM

Figure 6. NMR spectrum of poly crystalline Ala-Ala at 63 MHz resonance fre quency. Expansions are of peptide carbonyl, peptide Ca, and terminal Ca resonances from low to high field. Spectra were calculated as in Ref. 4. Theoretical spectra in applied field of 5.87 T . Parameters used: carbonyl carbon of peptide bond R n = 1.33 A; a = 126.0°; β° = 90.0°; e Q q / h = (-) 3.20 MHz; and = 0.31; Ca of peptide bond R = 1.445 A ; « = 112.0; β° = 90.0°; e Q q / h = (-) 3.20 MHz, and η = 0.31; C of ^-terminal L-ala R = 1.49 A; a = 0.0; β = 3.9 A; e Q q / h = 1.1 MHz, and = 0.26. 13

C

D

2

η

D

CN

a

2

D

CN

Ό

η


2


GIERASCH E T A L .

Figure 7.

Solid State NMR

of

Peptides

245

Drawing of peptide bond showing orientation of dipolar and quadrupolar vectors.


246

N M R SPECTROSCOPY


Acknowledgements We thank A, L* Rockwell f o r t e c h n i c a l a s s i s t a n c e * This work i s supported by grants GM-24266 (S*J*0*) and GM-27616 (L*M*G*) from the National I n s t i t u t e s o f Health and by grants from the American Chemical Society* M*H*F* i s supported by a C e l l and Molecular Biology T r a i n i n g Grant* J*G*H* i s sup ported by the NIGMS graduate research t r a i n i n g program (GM-07612) o f the Department o f Anesthesiology, U n i v e r s i t y o f Pennsylvania* S*J*0* i s a Fellow o f the A* P* Sloan Foundation (1980-1982)*

Literature Cited 1. Wuthrich, Κ., NMR in Biological Research: Peptides and Proteins", Elsevier, NY, 1976. 2. Frey, M. and Opella, S., JCS Chem. Comm. 1980, 474. 3. Pease, L.G., Frey, M.H., and Opella, S.J., J. Am. Chem. Soc. 1981, 103, 467. 4. Opella, S.J., Hexem, J.G., Frey, M.H., and Cross, Τ.A., Phil Trans R. Soc. Lond. A. 1981, 299, 665. 5. Gierasch,L.M., Opella, S. J., and Frey, M.H., Proc. of Seventh Amer. Peptide Symp. (Rich,D.H.and Gross, Ε., eds.) Pierce Chem. Co., Rockford, IL, in press. 6. Pines, A., Gibby, Μ., and Waugh, J., J. Chem. Phys., 1973, 51, 569. 7. Schaefer, J. and Stejskal, E.O.,J.Am.Chem.Soc.1976,98,1030. 8. Pease, L.G. and Watson, C., J. Am. Chem. Soc., 1978, 100, 1279. 9. Pease, L.G., Niu, C.-H., and Zimmermann, G.,J.Am.Chem. Soc., 1979, 101, 184. 10. Pease, L.G., in "Peptides: Structure and Biological Function, Proceedings of the Sixth American Peptide Symposium", E. Gross and J. Meienhofer, Eds., Pierce Chem. Co., Rockford, IL, 1979, 197. 11. Smith, J.A. and Pease, L.G., CRC Crit. Rev. in Biochem. 1980, 8, 315. 12. Balasubramanian, R., Lakshminarayanan, A.V., Sabesan, M.N., Tegoni, G., Kenkatesan, Κ., and Ramachandran, G.N., Int. J. Protein Research 1971, III, 25. 13. Ramachandran,G.N.and Sasiskharan, V., Adv. Prot. Chem. 1968, 23, 283. 14. Schimmel, P.R. and Flory, P.J., J. Mol. Biol. 1968, 34, 105. 15. Reed, L.L. and Johnson, P.L., J. Am. Chem. Soc. 1973, 95, 7523. 16. Karle, I.L., in "Perspectives in Peptide Symmetry", A. Eberle, R. Geiger, and T. Wieland, eds., S. Karger, Basel, 1981, 261. 17. Kostansek, E.C., Thiessen, W.E., Schomburg, D., and Lipscomb, W.N., J. Am. Chem. Soc. 1979, 101, 5811.



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GIERASCH ET AL.

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18. (b) Pease, L.G., Deber, C.M., and Blout, E.R.,J.Am.Chem. Soc. 1973, 95, 258. 19. Siemion, T.Z., Wieland, Τ., and Pook, K.H., Angew. Chem. 1975, 87, 712. 20. Madison, V., Atreyi, Μ., Deber, C.M., and Blout, E.R., J. Am. Chem. Soc. 1974, 96, 6725. 21. Hexem, J.G., Frey, Μ.Η., and Opella, S.J., J. Am. Chem. Soc., 1981, 103, 224. 22. Abragam, A. "The Principles of Nuclear Magnetism " 1961, Oxford Press. 23. Lucken, E.A.C., "Nuclear Quadrupole Coupling Constants", 1969, Academic Press, London. 24. Stark, R.E., Haberkorn, R.A., and Griffin, R.G. J. Chem. Phys. 1978, 68, 1996. RECEIVED

November 18, 1981.


NMR Spectroscopy - American Chemical Society

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