Analysis of Core Packing in a Cooperatively Folded Miniature Protein: The Ultrafast Folding Villin Headpiece Helical Subdomain

Abstract

The helical subdomain of the villin headpiece is the smallest naturally occurring cooperatively folded protein. Its small size, simple three-helix topology, and very rapid folding have made it an extremely popular model system for computational and theoretical studies of protein folding. The domain has a well-packed hydrophobic core comprised in part of an unusual set of three closely packed phenylalanine residues, F47, F51, and F58 (denoted using the numbering of the larger headpiece protein). Aromatic-aromatic interactions have been thought to play a critical role in specifying the subdomain fold and have been proposed to play a general role in stabilizing small proteins. The modest stability of the subdomain has hindered studies of core packing since multiple mutations can lead to constructs which fail to fold, and even single mutants can result in poorly folded variants. Using a previously characterized hyperstable mutant of the domain, generated by targeting surface residues, a complete set of single, double, and triple core Phe to Leu mutants were characterized. A highly conserved surface Trp which is part of a Trp-Pro interaction was also examined. All mutants are well-folded as judged by CD and NMR, and all exhibit sigmoidal urea and thermally induced unfolding transitions, thus proving that aromatic-aromatic, aromatic-proline, or aromatic-hydrophobic interactions are not required for specifying the subdomain fold. Double mutant cycle analysis demonstrates that F47 and F51 make the strongest pairwise interaction. Mutations which lack F58 are the most destabilized, although even the triple mutant is folded. Interestingly, mutation of the central Phe, F51, has the smallest effect on stability even though it makes contact with both F47 and F58 and appears to form the strongest pairwise interaction.