Analysis of the factors that stabilize a designed two‐stranded antiparallel β‐sheet

Abstract

Autonomously folding β-hairpins (two-strand antiparallel β-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 β-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 β-hairpin stability by comparing 1 with ^DP→ 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 ^DPG segment decreases the entropic cost of β-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of ^DPG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed β-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.