Local Effect of Glycine Substitution in a Model Helical Peptide

The amino acid glycine strongly destabilizes a-helical structure in proteins as well as in model helical peptides. We have investigated the role of a single glycine substitution in a helical host peptide system. Quantitatively, a single glycine leucine substitution has about one-third of the effect on the stability of helix as does triple substitution of these residues in the middle of the helix. The single glycine perturbs the distribution of helix in the peptide. NMR experiments detect a strong local drop in helix structure at the residues flanking the site of substitution, in addition to overall loss in helicity of the peptide seen at all positions in the chain. Introduction The &-helix’ is the single most abundant secondary structure in globular proteins? The factors that stabilize a-helical structure are of fundamental importance in understanding how structure is acquired,’ both in isolated fragments of proteins and in intact protein chains. In some, but not all$ cases a-helices serve as early intermediates in the folding of globular proteins. Polypeptides of different composition3 and peptides of different sequence5-10 exhibit varying degrees of helicity, so that each side chain in a helix appears to influence the helical structure present. Alanine and leucine are well-known to stabilize a-helical structure, for example, whereas proline and glycine destabilize h e l i c e ~ . ~ J ~ But how or why a particular side chain influences the stability of (1) Pauling. L.; Corey, R. B.; Branson, H. R. Proc. Nutl. Acad. Sci. U S A . 1951, 37, 205. (2) Creighton, T. E. Proteins; W . H. Freeman: New York, 1984. (3) Sueki, M.; Lee, S.; Powers, S. P.; Denton, J. B.; Konishi, Y.; Scheraga, H. A. Mucromolecules 1984, 17, 148. (4) (a) Roder, H.; Elove, G. A,; Englander, S. W . Narure 1988,335,700. (b) Udgaonkar, J. B.; Baldwin, R. L. Nature 1988, 335, 694. (5) (a) Brown, J. E.; Klee, W . A. Biochemistry 1971, 10,470. (b) Bierzynski, A.; Kim, P. s.; Baldwin, R. L. h o c . Narl. Acud. Sci. U.S.A. 1982, 79,2470. (c) Kim, P. S.; Bierzynski, A,; Baldwin, R. L. J . Mol. Biol. 1982, 162, 187. (d) Shoemaker, K. R.; Kim. P. S.; Brems, D. N.; Marqusee, S.; York, E. J.; Chaiken, 1. M.; Stewart, J. M.; Baldwin, R. L. Proc. Nurl. Acud. Sci. U.S.A. 1985,82, 2549. (e) Shoemaker, K. R.; Kim, P. S.; York, E. J.; Stewart, J. M.: Baldwin, R. L. Nature 1987,326, 563. (f) Strehlow, K. G.; Baldwin, R. L. Biochemistry 1989.28,2130. (g) Shoemaker, K. R.; Fairman, R.; Schultz, D. A.; Robertson, A.; York, E. J.; Stewart, J. M.; Baldwin, R. L. Biopolymers 1990, 29, I . (6) (a) Marqusce, S.; Baldwin, R. L. Proc. Narl. Acad. Sci. U.S.A. 1987, 84, 8988. (b) Marqusee, S.; Robbins, V.; Baldwin, R. L. Proc. Nutl. Acud. Sci. U S A . 1987,86,5286. (c) Padmanabhan, S.; Marqusee, S.; Ridgeway, T.; Lane, T. M.; Baldwin, R. L. Nature 1990, 344, 268. (7) (a) Merutka, G.; Stellwagen, E. Biochemistry 1989, 28, 352. (b) Merutka, G.; Stellwagen, E. Biochemistry 1990, 29, 894. (c) Merutka, G.; Lipton, W.: Shalongo, W.; Park, S.-H.; Stellwagen, E. Biochemistry 1990, 29, 7511. (8) (a) Lyu, P. C.; Marky, L. A.; Kallenbach, N. R. J . Am. Chem. Soc. 1989, 111,2733. (b) L p , P. C.; Marky, L. A.; Kallenbach, N. R. Peptides, Proceedings of the Eleventh American Peptide Symposium; Rivier, J. E., Marshall, G. R., Eds.; ESCOM: Leiden, 1990; p 632. (9) ONeil, K. T.; DeGrado, W. F. Science 1990, 250, 646. (IO) Lyu, P. C.; Liff, M. 1.; Marky, L. A.; Kallenbach, N. R. Science 1990, 250, 669. a-helix is not clear; it seems likely that no single mechanism will account for the role of all side chains. The intrinsic strength of hydrogen bonds in the helix must depend on the nature of the donor and acceptor side chains. Inspection of helix wheels” shows that longer range interactions among side chains along the helix are possible, particularly if the spacing of groups is appropriate. Thus charges or nonpolar side chains spaced at intervals of i, i + 4 lie on the same face of the helix and can presumably influence each other. On the other hand differences among chemically similar side chains such as leucine, isoleucine, and ~ a l i n e ” . ’ ~ ~ J ~ in identical environments imply that there are short-range effects of single substitution as well. The conformational restriction imposed by cy-helix formation has been shown to be a significant factor in the helix propensities of chemically similar side chains.I2 We have investigated10 a series of peptides that we refer to by the standard one-letter abbreviations for the three “guest” amino acids which are introduced into the central positions in “host” chains with the sequence succinyl-Tyr-Ser-Glu4-Lys4-XXX-Glu4-Lys4-NH2 CD spectroscopy of ten substituted chains allows us to assign an order of relative helix stabilizing effect to the different guest amino acids in the series:I0 Ala > Leu > Met > Gln > Ile > Val > Ser > Thr > Asn > Gly. This order is consistent with that determined in a series of host-guest coiled-coil peptides by O’Neil and Degrad^,^ but not with host-guest experiments on polyamino acids with alkylated glutamic acid as host side chain^.^ NMR analysis of members of the series indicates that each is partially helical, with the helix favoring the N terminus of the chain.” This analysis is based on distance criteria and coupling constants and shows that the chemical shift of the C a protons in the host blocks of these peptides affords a useful measure of the helix probability. The question that concerns us here is how a single substitution stabilizes a-helix relative to the triple substitutions used previously ( 1 I ) Schiffer, M.; Edmundsen, A. B. Biophys. J . 1967, 7, 121. (12) (a) Hermans, J.; Yun, R.-H.; Anderson, A. G. Private communication. (b) Piela, L.; Nemethy, G.; Scheraga, H. A. Biopolymers 1987,26, 1273. (13) Liff, M. I.; Lyu, P. C.; Kallenbach, N. R. J . Am. Chem. SOC. 1991, 113. 1014. 0002-7863/91/1513-3568$02.50/0