Incorporating β-Turns and a Turn Mimetic out of Context in Loop 1 of the WW Domain Affords Cooperatively Folded β-Sheets

Abstract

To probe the conformational requirements of loop 1 in the Pin1 WW domain, the residues at the i + 2 and i + 3 positions of a β-turn within this loop were replaced by dPro-Gly and Asn-Gly, which are known to prefer the conformations required at the i + 1 and i + 2 positions of type II‘ and type I‘ β-turns. Conformational specificity or lack thereof was further examined by incorporating into the i + 2 and i + 3 positions a non-α-amino acid-based β-turn mimetic (4-(2‘-aminoethyl)-6-dibenzofuran propionic acid residue, 1), which was designed to replace the i + 1 and i + 2 positions of β-turns. All these Pin WW variants are monomeric and folded as discerned by analytical ultracentrifugation, NMR, and CD. They exhibit cooperative two-state transitions and display thermodynamic stability within 0.5 kcal/mol of the wild-type WW domain, demonstrating that the acquisition of native structure and stability does not require a specific sequence and, by extension, conformation within loop 1. However, it could be that these loop 1 mutations alter the kinetics of antiparallel β-sheet folding, which will be addressed by subsequent kinetic studies.