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 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 β‐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 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.