Determinants of miniprotein stability: can anything replace a buried H-bonded Trp sidechain?

A novel 20-residue fold, designated the `Trp-cage' motif, hasbeen shown to be 98+% folded in both water and 30 vol-%trifluoroethanol solution. Folding is cooperative andhydrophobically driven, resulting in the burial of the Trpsidechain and a stable H-bond from the Trp-εNH to a sequenceremote backbone carbonyl. In the present study the effects ofreplacing the Trp with His, Phe and both isomers of β-naphthylalanine are examined. The results suggest that thehydrophobic cluster is a specific interaction of proline ringswith the indole ring which can be partially mimicked by anaphthalene ring. The His and Phe mutants are completelyunfolded in aqueous medium. The naphthylalanine mutants forma stable hydrophobic cluster in 30% trifluoroethanol, but areless stable in water than the native structure.