Backbone engineering in the hydrophobic core of villin headpiece

Changing the backbone connectivity of proteins can impart useful new traits while maintaining essential structural and functional features. In design of artificial proteomimetic agents, backbone modification is usually isolated to sites that are solvent-exposed in the folded state, as similar changes at buried residues can alter the fold. Recent work has shown that core backbone modification without structural perturbation is possible; however, the modifications in that study were consistently destabilizing and made in a prototype of exceptionally high conformational stability. Here, we report efforts to broaden the scope and improve the efficacy of core backbone engineering by applying it to the C-terminal subdomain of villin headpiece. A series of variants are prepared in which different artificial residue types are incorporated at core positions throughout the sequence, including a crucial aromatic triad. Impacts on folding energetics are quantified by biophysical methods, and high-resolution structures of several variants determined by NMR. We go on to construct a variant with ∼40% of its core modified that adopts a fold identical to the prototype while showing enhanced thermodynamic stability.