Abstact

Protein unfolded states are heterogeneous but can manifest local and long-range order. Replacement of side chains through site-directed mutagenesis is a common method to manipulate the unfolded state and elucidate its role in the folding process. Modification of the protein backbone represents a less explored complementary approach with the potential to elicit dramatic changes in conformational preferences from minimal chemical alteration. Prior work has shown backbone modification can affect unfolded ensembles as well as intrinsically disordered sequences. Here, we show that it can be used to rationally tune structural characteristics of the unfolded state of a folded protein. Using the GCN4 leucine zipper as a host, canonical alpha-residues throughout the chain are individually replaced by beta(3) or C(alpha)-Me-alpha analogues. The former modification enhances conformational freedom, the latter restricts it, and both retain the side chain at the substitution site. Characterization by circular dichroism and X-ray crystallography shows that the variants adopt folded structures identical to the prototype. Thermal and thermodynamic stability vary in complex ways with the context and nature of backbone modification; however, a uniform relationship is observed between substitution type and the sensitivity of folding free energy to chemical denaturant. This finding suggests systematic changes in solvent-accessible surface area of the unfolded ensemble among isomeric proteins differing only in the position of a single CH(2) group. Collectively, these results demonstrate a platform for predictably tuning the properties of the unfolded state through minimal chemical modification, enabling new avenues for fundamental research on folding behavior of proteins as well as protein mimetics.