Vol. 10, No. 5, September-October 1977 (18) J. J. Hermans, J . Colloid Interface Sci., 58, 286 (1977). (19) J. M.Burgers, Verh. K. Ned. Akad. Wet.,Afd.Nntuurk., Reeks 1, 16, No. 4,113 (1938). (20) S. C. Szu and J. J. Hermans, J . Polym. Sci., Polym. Phys. Ed., 11,1941 (1973). (21) E. L. Chang and R. M. Mazo, J . Chem. Phys., 64,1389 (1976). (22) T . S. Chow and J. J. Hermans, Kolloid-Z. 2. Polym., 250,404 (1972). (23) S. C. Szu and J. J. Hermans, J. Polym. Sci., Polym. Phys. Ed., 12, 1743 (1974). (24) R. B. Bird, H. R. Warner, Jr., and D. C. Evans, Ado. Polym. Sci., 8, 1 (1971). (25) C. F. Curtiss, R. B. Bird, and 0. Hassager, Ado. Chem. Phys., 35, 31 (1976). (26) H. Yamakawa, J. Chem. Phys., 53,436 (1970). (27) H. Yamakawa and J. Yamaki, J . Chem. Phys., 57,1542 (1972). (28) H. Yamakawa and G. Tanaka, J. Chem. Phys., 57,1537 (1972). (29) H. Fujita, J. Polym. Sci., Polym. Phys. Ed., 11,899 (1973). (30) S. Wakiya, J. Phys. SOC.Jpn., 22,1101 (1967). (31) S. Wakiya, J. Phys. SOC.Jpn., 31,1587 (1971). (32) P. G. Wolynes and J. M. Deutch, J . Chem. Phys., 65,2030 (1976); see also in this connection the fundamental study by P. G. Wolynes, Phys. Reo. A, 13,1235 (1976). (33) H. A. Kramers, J. Chrm. Phys., 14,415 (1946).

Oligopeptides Containing Proline and Glycine 1049 (34) W. H. Stockmayer, W. Gobush, Y. Chikahisa, and D. K. Carpenter, Faraday Discuss. Chem. SOC49,182 (1970). (35) Y. Chikahisa and W. H. Stockmayer, Rep. Prog. Polym. Phys., Jpn., 14, 79 (1971). (36) M. Doi and K. Okano, Polym. J., 5,216 (1973). (37) H. Yamakawa, G. Tanaka, and W. H. Stockmayer, J . Chem. Phys., 61, 4535 (1974). (38) B. U. Felderhof, J. M. Deutch, and U. M. Titulaer, J. Chem. Phys., 63,740 (1975). (39) W. H. Stockmayer, G. Wilemski, H. Yamakawa, and G. Tanaka, J. Chem. Phys., 63,1039 (1975). (40) P. Mazur and D. Bedeaux, Physica (Utrecht),76,235 (1974). (41) B. U. Felderhof, Physica (Utrecht),84,557,569 (1976). (42) S. C. Szu and J. J. Hermans, J. Fluid Mech., 66,385 (1974). (43) M. Bixon and R. Zwanzig, J . Stat. Phys., 3,245 (1971); K. S. J. Nordholm and R. Zwanzig, ibid., 11,143 (1974). (44) R. F. Rodriguez and N. G. Van Kampen, Physica (Utrecht), 85, 347 (1976). (45) D. Bedeaux and P. Mazur, Physica (Utrecht),78,505 (1974). (46) The result eq V.6 might seem to be time de,pendent, but this is'only ap) a function of the difparently so. In a stationary ensemble ( h ( t ) O ( s ) is ference t - s, say f ( t - s), and the integral may therefore be written Jo"dzz-'lzf(z), which is independent o f t .

Conformational Studies of Oligopeptides Containing Proline and Glycine1 Evelyn R. Stimson,2aS. Scott Zimmerman,2b a n d Harold A. Scheraga* Department of Chemistry, Cornell University, Ithaca, New York 14853. Received March, 29,1977

ABSTRACT: Nuclear magnetic resonance, infrared, and circular dichroism methods were used to study the conformational properties of N-acetyl-N'-ethylprolineamide(blocked Pro single residue), N-acetyl-N'-methylglycineamide (blocked Gly single residue), N-acetyl-N'-methylglycylprolineamide(blocked Gly-Pro), and N-acetyl-N'-methylprolylglycineamide (blocked Pro-Gly) in various solvents. It was found that all four peptide molecules exist in solution a t 29 "C as an ensemble of several conformations. In the blocked Pro single residue, the acetyl-Pro peptide bond was found to exist in both the cis and the trans forms, with hGotransdcis = 0.7 kcal/mol in CD2Cl2 and ~ 0 . kcal/mol 4 in DMSO a t 29 "C. In the trans form, the hydrogen-bonded conformation (C7eq,with $pro = 80") and nonhydrogenbonded conformations (with $pro being unidentified) coexist. The blocked Gly single residue was found to exist as a flexible molecule, with the C7 and other conformations present; no cis-trans isomerism was observed. Blocked GlyPro showed cis-trans isomerism about the Gly-Pro peptide bond with AGotrans-cis 0.7 kcal/mol in DMSO and -1.5 kcal/mol in CDZC12 at 29 "C. The Gly residue in blocked Gly-Pro was found to exist, to a large extent, in the fully extended (C5J structure, with the Pro residue in Gly-Pro assuming the C7eq conformation as well as other (nonhydrogen-bonded) conformations. No evidence for large amounts of /3 bend was detected in Gly-Pro. The temperature dependence of the NMR and CD data of blocked Gly-Pro in water was found to be similar to that of poly(G1y-Pro), indicating that the conformational properties of the polymer can be attributed to local interactions (Le., those within the dipeptide unit). The predominant conformations of blocked Pro-Gly were found to contain a 4 4 1 hydrogen bond; the molecules also showed restricted segmental motion of the Gly residue. These observations indicate the presence of /3 bends (along with other conformations). The experimentally determined properties of the four peptide molecules were compared with those determined from previous theoretical studies using empirical conformational energy calculations and, with some exceptions, were found to be in good agreement.

The hypothesis has been p r e ~ e n t e dthat ~ . ~ the conformation of a given sequence of amino acids in a protein is determined largely, but not exclusively, by intra-residue interactions. If this hypothesis is correct, a short sequence of amino acid residues in a globular protein should exist in a conformation similar to one or more of the stable structures of the same amino acid sequence in an oligomer in solution. This is the basis for using random copolymers for determining the tendency of single amino acid residues to adopt the a-helical conformation5 and for studying short peptides to determine their tendency toward formation of chain reversal (bend) conformations in pr0teins.6-l~In this paper, we examine oligopeptides (specifically, blocked single residues and blocked dipeptides) containing proline and glycine. The a-amino and a-carboxyl groups are blocked to eliminate charge effects and to simulate the environment of the polypeptide chain. Earlier studiesl4-l6 have treated unblocked oligopeptides of proline

and glycine, where the peptide molecules are ionizable. The proline and glycine residues are of interest in protein structure because they are found frequently in /%bend conformations, Pro-Gly having a high propensity for forming a bend but Gly-Pro having a very low 0ne.13J7J8 Proline is of special interest because cis conformations have been observed in peptide bonds preceding Pro in small mole~ules~~-~6J9-25 and in proteins.26 The purposes of this paper are to employ proton nuclear magnetic resonance (NMR), infrared (IR), and circular dichroism (CD) methods to: (a) determine the preferred conformations (including possible cis and trans peptide forms) of the N-acetyl-N'-ethylamide of Pro and the N-acety1-N'methylamides of Gly, Gly-Pro, and Pro-Gly; (b) compare the calculated structures of the N-acetyl-"-methylamides of Gly,27 Pro,27 G l y - P r ~ , and ~ ~ tP~~~o - G l ywith ~ ~ ,those ~ ~ observed experimentally; (c) determine the influence of solvent

1050 Stimson, Zimmerman, Scheraga

Macromolecules Table I NMR Data for Blocked Oligopeptides Chemcial shift, d ppm

Peptide

Solvent

Temp, 'C

NHG~~

Pro

CaH~iy

4.15 (d) 4.24 (d) 4.17 (d) CD2C12

GlY

DMSO-d6

Gly-Pro

CDzClz DMSO-d6

CDzClz

DMSO-d6

CDzClz

CH$O 1.79 (s) 1.94 (s) 1.94 (s)

4.39 (d) 4.23 (4) 4.41 (d) 4.19 (4)

-21

29

Pro-Gly

CaHpro

64 __

29

7.98 (t) 7.76 (t)

29

7.96 (t)

67

7.67 (t)

-23

6.7"

29

6.4"

29

8.20 (t)

64

8.02 (t)

-51 29

7.93 (t) 7.02"

2.07 ( s i 1.97 (s) 2.04 (s) 1.95 (s) 6.57 (t) 3.56 (d) 1.84 (s) 3.56 (d) 1.84 (s) Too insoluble for NMR experiments 3.88 (d) 1.86 (s) 4.37 (4) 4.22 (d) 3.75 (d) 3.84 (d) 1.85 (s) 4.26 (t)' -4.33 (q) -3.76 (d) 1.80 (s) 4.22 (d) 3.78 (q) 4.02 (4) 3.55 (4) 4.41 (d) 3.95 (d) 1.97 (s) 4.22 (4) 3.78 (q) 1.69 ( s i 4.28 (9) 3.57 (d) 2.00 ( s ) ~ 4.13 (t) 4.20 (t) 3.59 (d) 1.99 (s) 4.18 (ti 4.24 (ti 3.95 (q)

3.72 (q) 3.78 (p)

2.09 (s) 2.07 (s) 1.89 (s)

NHCH3" 7.64 (t) 7.94 (t) 7.78 (t) 7.50 (t) 7.19 (t) 6.93 (t) 1.10 (t) 7.65 (9) 7.43 (9) 7.74 (9) 7.50 (9) 6.7' 6.4" 7.78 (9) 7.52 (4) 7.69 (4) 7.43 (4) 7.59 (9) 7.02c

NHCH3' 0.97 (t) 0.99 (t) 0.98 1.07 (t) 1.10 (t) 1.07 (t) 2.56 (d) 2.56 (d) 2.54 (d) 2.60 (d) 2.55 (d)' -2.59 (di 2.50 (d) 2.58 (d) 2.76 (d) 2.70 (d) 2.55 (d) 2.48 (d) 2.58 (d) 2.67 (ti 2.69 (d) 2.73 (d)

0 In the proline single residue, this column refers to the NHCHzCH3 proton. In the proline single residue, this column refers t o the NHCHzCH3 protons. This resonance partially overlaps with the other resonance indicated. This broad acetyl resonance (90 MHz) was shown to contain an upfield component (separated by 0.04 ppm) in the resolution enhanced correlation spectra obtained with a 250-MHz NMR spectrometer.

of coupling patterns, area ratios, and decoupling experiments. Cis-trans isomerism about the X-Pro peptide bond in blocked Pro, blocked Gly-Pro, and blocked Pro-Gly was Methods detected and measured quantitatively by the presence of, and the relative areas under, the doubled proton resoA description of the syntheses and experimental methods n a n c e ~ , ~ particularly ~ - ~ ~ , ~ the ~ , Pro ~ ~ CaH and the Gly CaH is given in the Appendix. or the end group CH3CO resonances. Other doubled proton Conformational Energy Calculations. The nomenclaresonances also were used. For example, in Figure 1,the cisture, conventions, definitions, and methods of conformational trans ratio a t 29 "C was determined by measuring the relative energy calculations are those used earlier.27-30Rotations about areas under the doubled N H resonance, where the upfield the peptide bond between the N-terminal acetyl group component was assigned to the trans form in DMSO-d6 and ("residue" 1)and the first full residue (residue 2) are described the downfield component was assigned to the trans form in by w l ; rotations about the peptide bond following the first full CD2C12. No doubling of resonances, and hence no cis-trans residue are described by w2; those following the second residue isomerism, was detected in blocked Gly. Table I1 gives the by w3; and so on. Values of w near 0" and 180' are referred to percent cis and the free energy difference between cis and as cis and trans, respectively. trans, AGotrans-cis, for the peptide molecules in various solAn energy contour diagram was drawn for $ ~ l , , vs. $pro in vents at 29 'C. Table I1 also contains the values of AGotrms-cis blocked Pro-Gly by calculating the total conformational enas determined by conformational energy c a l c ~ l a t i o n s . ~ ~ - ~ ~ ergy at 5" intervals over the range 0' I $pro I 180' and -180' It is assumed, based on prior experimentallg and theoretiI# ~ I l 180' ~ and then, by interpolation, determining lines c a P results, that the proportion of cis isomer of the peptide of constant energy at 0.5-kcal/mol increments above the global group following the proline ring is vanishingly small. minimum. N-Acetyl-N'-ethylprolineamide.Cis-Trans Isomerism. Results a n d Discussion The coalescence of the CH&O resonance and of the CaH resonance as the temperature is raised from 29 to =64 "C in NMR spectra were determined for N-acetyl-N'-ethylprolineamide (blocked Pro), N-acetyl-N'-methylglycineamide DMSO-& (see Table I) indicates, according to the method of Shanan-Atidi and Bar-Eli,31 that the energy barrier between (blocked Gly), and the N-acetyl-N'-methylamidesof Gly-Pro the cis and trans forms in the Pro single residue is approxiand Pro-Gly, primarily in the deuterated solvents dimethyl mately 18 kcal/mol, in agreement with cis-trans barriers in sulfoxide (DMSO-&) and methylene chloride (CD&12), at small peptides estimated p r e v i o ~ s l y Although . ~ ~ ~ ~ ~the ~~~ various temperatures. Some representative NMR results are CH3CO and CaH resonances coalesce as the temperature is shown in Table I. raised from 29 to 64 'C, the NHCHpCH3 resonance is still The assignments of the resonances were made by comparing the spectra obtained here to published s p e ~ t r a ~ for ~ - ~ ~doubled , ~ ~ ,at~64~ "C because the initial difference (at 29 'C) in chemical shift between the two N H peaks is larger than that the same amino acid residues in similar solvents and by use

on the conformational properties of oligopeptides; and (d) determine the influence of local and long-range interactions on proline-containing oligo- and polypeptides.

Oligopeptides Containing Proline and Glycine 105 1

Vol. 10, No. 5, September-October 1977

rcL

Table I1 Cis-Trans Isomerism in Peptide Bonds Preceding Proline in Blocked Oligopeptides % cis AGOtrans-cis, kcdm01 Peptideo Solvent (exptUb Exptlb CalcdC DMSO a l 64'

Pro

A

I\ Gly-Pro DMSO a1 2 9 O C

Pro-Clyd

a t -21°C-

h i

-

90

l

.

L

i

80 Chemical

33

DMSO DzO MeOD CDpClp

25 19

DMSO CD2C12

22

Conformational studies of oligopeptides containing proline and glycine.

Vol. 10, No. 5, September-October 1977 (18) J. J. Hermans, J . Colloid Interface Sci., 58, 286 (1977). (19) J. M.Burgers, Verh. K. Ned. Akad. Wet.,Afd...
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