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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...

Autonomously folding ␤-hairpins (two-strand antiparallel ␤-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a ␤-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in ␤-hairpin stability by comparing 1 with D P→ N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the D PG segment decreases the entropic cost of ␤-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of D PG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed ␤-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.

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