stabilizing helices - PNAS

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model for nucleation of a-helix: Role of water molecules in stabilizing helices ... detect stable conformations (for example, a-helices) in short oligopeptides isolated from ... It has been rather widely assumed that short oligopeptides will not have a ..... hydration of polar and nonpolar groups is an essential con- formationalĀ ...
Proc. Nati. Acad. Sci. USA Vol. 87, pp. 871-875, February 1990

Biophysics

Design of crystalline helices of short oligopeptides as a possible model for nucleation of a-helix: Role of water molecules in stabilizing helices (x-ray diffraction/amphipathic hefices/protein folding)

R. PARTHASARATHY*, SANJEEV CHATURVEDI,

AND

KUANTEE Go

Center for Crystallographic Research and Department of Biophysics, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263

Communicated by Herbert A. Hauptman, October 30, 1989 (received for review August 7, 1989)

We have designed, synthesized, crystallized, ABSTRACT and performed x-ray analysis of several hydrophobic tripeptides that show an extended near a-helical structure in the crystalline state. All of the tripeptides that show this remarkably stable helix crystallize with two or three water molecules; they all have glycine at the N terminus and have increasing hydrophobicity as one moves from the N to C terminus. Even though three residues in the oligomer are not sufficient to complete a turn, one of the water molecules acts as an added residue and links up adjacent tripeptide segments along the helix axis so that in the crystal, the helix appears effectively as one long continuous helix. Two of these tripeptides are stabilized by two water molecules that enable the peptides to complete a turn of the helix and extend the helical structure throughout the crystal by linking translationally related peptides by hydrogen bonds. In two other peptides, these roles are played by three rather than two water molecules. Though these tripeptides have different crystal symmetry, they all show the basic pattern of hydrated helix and packing, indicating the strong conformational preference for a stable structure even for these tripeptides. Such conformationally stable hydrated structures for short specific related sequences illustrate their possible importance in nucleating protein folding and in the role water molecules play in such events.

oligopeptides isolated from helix-containing regions of proteins gave negative results (8, 9). It has been rather widely assumed that short oligopeptides will not have a preferred and well-defined conformation. For example, it has been shown that a given sequence in a peptide does not always assume the same conformation in different proteins (10). Also, the Zimm-Bragg equation (11) predicts that peptides as short as 20 residues should not show measurable a-helix formation in water regardless of the temperature and amino acid sequence (12). However, Lerner and his coworkers (13) have found that a highly immunogenic nonapeptide from influenza virus shows structure that is measurable by NMR. Further, a-helices of short peptides of particular sequences have been found to be stable and their stability in the solution state has been attributed to be due to salt bridges (14, 15); a similar conclusion has been drawn for the stability of a-helices in the crystalline state (16). Though many empirical correlations have been put forward for amino acid preferences for specific locations at the ends of helices (17-19) and have been used in designing a-helical peptides (20, 21), ". . . there are few examples of short, noncyclic peptides that adopt stable'conformations in solution unless they are bound to a biological receptor" (3). Based on our earlier work of two short peptides that are crystalline helices (22), we have now designed, synthesized, and crystallized (at pH 7.0) three more tripeptides and carried out x-ray diffraction studies of two of these that show stable helical conformation in the crystalline state. Taken together, we now have four clear examples of tripeptides-namely, Gly-L-Ala-L-Phe (GAF) (22), Gly-Gly-L-Val (GGV) (22, 23), Gly-L-Ala-L-Val (GAV) (Fig. 1), and Gly-L-Ala-L-Leu (GAL) (Fig. 2)-that are helical in the crystalline state and a fifth one, Gly-L-Ala-L-Ile (GAI) (whose x-ray structure determination is not yet completed), that shows all of the characteristics of the other four peptides.

How proteins start to fold and what clues the primary structure itself gives to the folding process have long been a mystery. Though it was demonstrated as early as 1960 by Anfinsen, White, and Sela (for a review, see ref. 1) that polypeptide chains can refold in vitro, the mechanism of the folding process and how different amino acid sequences direct chains into unique conformations have not been elucidated. It is being increasingly realized (for recent short reviews on protein folding, see refs. 2 and 3) that not only do the amino acid sequences determine the final folded conformation, but they also influence the folding process to reach the final folded state rapidly and with near perfect fidelity. One of the key elements of the folding process, as originally suggested by Anfinsen (4) and later experimentally observed by Anfinsen and coworkers (5) and by many others (6, 7), is the presence of nucleating sites in the polypeptide. These nucleating sites are short fragments of the polypeptide chains that, during the folding process, can flicker in and out of the conformation that they assume in the final fold; if these fragments have well-defined conformations, they seed effectively the folding process. Anfinsen and coworkers detected the native-like folding of fragments of the polypeptide that have highly unfavorable equilibrium constants using a conformation-sensitive antibody technique. Early attempts to detect stable conformations (for example, a-helices) in short

The crystal structures GAV and GAL are reported here and those of GAF and GGV have been discussed by us earlier (22). GAV (C1OH19N304-3H20) and GAL (C11H21N3043H20), Mr = 299 and 313, respectively, were synthesized by solution-phase peptide synthesis. Blocked tripeptides BocGly-Ala-Val-OBzl and Boc-Gly-Ala-Leu-OBzl (where Boc = butoxycarbonyl and OBzl = benzyloxy) were made from the respective X-OBzl C-terminal residue, which was coupled with Boc-Ala and Boc-Gly by N,N'-dicyclohexylcarbodiimide/HOBt method consecutively (24). Finally, the Bzl and Boc blocking groups were removed, respectively, by hydrogenation and acid treatment (trifluoroacetic acid/CH2Cl2, 1:1). The pH of the deblocked tripeptides was adjusted to 7,

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Ā§1734 solely to indicate this fact.

Abbreviations: GAF, Gly-L-Ala-L-Phe; GGV, Gly-Gly-L-Val; GAV, Gly-L-Ala-L-Val; GAL, Gly-L-Ala-L-Leu; GAI, Gly-L-Ala-L-Ile. *To whom reprint requests should be addressed. 871

MATERIALS AND METHODS

Proc. Natl. Acad. Sci. USA 87 (1990)

Biophysics: Parthasarathy et al.

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a

b

c

FIG. 1. GAV. (a) Stereoview of the helical turn stabilized by OWL. Note that OW1 is in the plane of the COO- group. (b) Stereoview of the crystalline helix formed by the tripeptide GAV. The side chains have been removed for clarity. The tripeptide molecules related by cell translations are linked by the water molecule OW1, giving rise to the appearance of an extended helix in the crystal. OW2 and OW3 add further stability to the helix through hydrogen bonds involving N1 ... OW2 ... OW3 ... N1 of adjacent molecule. (c) Same as b but contains side chains and hydrogen atoms.

and they were Iyophilized. The purity of the peptides was checked and confirmed by running them through a reversephase HPLC using Delta-Pak analytical C18 column (3.9 mm x 30 cm) from Waters; a linear gradient of 0-70% acetonitrile in 0.01% CH3COONH4 was used for elution. Crystals of GAV and GAL were grown by the vapor diffusion method from water/methanol solutions and sealed in capillary tubes with mother solutions. Three-dimensional x-ray diffraction data to the limit of the Cu sphere (1903 reflections, 1298 2 3oa for GAV, and 2045 reflections, 1318 2 3o, for GAL) were measured by the w-20 scan method. The scan widths and aperture widths were calculated using the relations (1.0 + 0.14 tanO)0 and (3.0 + 1.2 tanO)mm-1. The maximum time spent on any reflection was 100 s and the background count time was half the scan time. The variation in intensity of three standard reflections monitored every hour of x-ray exposure was