No abstract

Designed peptides that fold autonomously to specific conformations in aqueous solution are useful for elucidating protein secondary structural preferences. For example, autonomously folding model systems have been essential for establishing the relationship between ␣-helix length and ␣-helix stability, which would be impossible to probe with ␣-helices embedded in folded proteins. Here, we use designed peptides to examine the effect of strand length on antiparallel ␤-sheet stability. ␣-Helices become more stable as they grow longer. Our data show that a two-stranded ␤-sheet (''␤-hairpin'') becomes more stable when the strands are lengthened from five to seven residues, but that further strand lengthening to nine residues does not lead to further ␤-hairpin stabilization for several extension sequences examined. (In one case, all-threonine extension, there may be an additional stabilization on strand lengthening from seven to nine residues.) These results suggest that there may be an intrinsic limit to strand length for most sequences in antiparallel ␤-sheet secondary structure.M ost proteins must fold to a specific three-dimensional shape to perform their biological functions. There is great interest in identifying the factors that determine native conformations; however, despite considerable study, it is not yet possible to predict tertiary folding patterns on the basis of primary structure. A few secondary structures (especially ␣-helix and ␤-sheet) recur throughout known protein structures, and understanding the forces that control conformational preferences within the common secondary structures should contribute to our understanding of conformational preferences at tertiary and quaternary levels. The ␣-helix has been very carefully scrutinized because there are well-established design principles for creating synthetic peptides that adopt ␣-helical secondary structure in the absence of a specific tertiary context (1-7). Until recently, the lack of autonomously folding ␤-sheet model systems made it impossible to conduct analogous studies with this secondary structure (8). In the past several years, however, a number of short peptides (9-24 residues) that display double-or triple-stranded antiparallel ␤-sheet conformations in aqueous solution have been reported (9-11). These model systems provide thermodynamic (12-23) and kinetic (24) insights on ␤-sheet folding behavior. [Solvent-exposed ␤-sheets in specific tertiary contexts have provided a complementary approach for elucidation of ␤-sheet conformational preferences (25,26)]. Here, we show how small designed peptides can be used to assess an aspect of ␤-sheet stability that has not previously been addressed experimentally.␣-Helices become more stable as the length of the helix increases (5-7). This length-dependent effect on conformational stability arises because helix initiation is thermodynamically unfavorable but helix propagation is favorable, at least for some residues (1, 2). Analogous length-dependent stabilization is observed for double-helical nuclei...

The contributions of interstrand side chain-side chain contacts to beta-sheet stability have been examined with an autonomously folding beta-hairpin model system. RYVEV(D)PGOKILQ-NH2 ((D)P = D-proline, O = ornithine) has previously been shown to adopt a beta-hairpin conformation in aqueous solution, with a two-residue loop at D-Pro-Gly. In the present study, side chains that display interstrand NOEs (Tyr-2, Lys-9, and Leu-11) are mutated to alanine or serine, and the conformational impact of the mutations is assessed. In the beta-hairpin conformation Tyr-2 and Leu-11 are directly across from one another (non-hydrogen bonded pair). This "lateral" juxtaposition of two hydrophobic side chains appears to contribute to beta-hairpin conformational stability, which is consistent with results from other beta-sheet model studies and with statistical analyses of interstrand residue contacts in protein crystal structures. Interaction between the side chains of Tyr-2 and Lys-9 also stabilizes the beta-hairpin conformation. Tyr-2/Lys-9 is a "diagonal" interstrand juxtaposition because these residues are not directly across from one another in terms of the hydrogen bonding registry between the strands. This diagonal interaction arises from the right-handed twist that is commonly observed among beta-sheets. Evidence of diagonal side chain-side chain contacts has been observed in other autonomously folding beta-sheet model systems, but we are not aware of other efforts to determine whether a diagonal interaction contributes to beta-sheet stability.

We examine the relationship between covalent structure and conformational propensity among a series of β-amino acid tetramers. These experiments focus on the hairpin folding motif. Among conventional peptides, the minimum increment of β-sheet secondary structure is a "β-hairpin," in which two strands are connected via a short loop. The present studies are aimed at optimizing hairpin stability among β-peptides. Previous work from our laboratory has identified optimal substitution patterns for residues that form strands in an antiparallel β-peptide sheet (Krautha ¨user et al. J. Am. Chem. Soc. 1997, 119, 11719), and we have shown that a dinipecotic acid segment can promote sheet-type interactions between attached strand residues (Chung et al.