Modulating thermal folded stability and catalytic activity of a de novo metalloenzyme through chemical backbone modification.

The relationship between conformational heterogeneity and catalytic activity in enzymes is complex and poses important fundamental biological questions. Few experimental tools are available to rationally control conformations within a folded protein ensemble. Modification of the protein backbone through chemical synthesis has previously been used to alter enzyme conformational dynamics at a site essential for catalysis. Here, we explore the application of the approach to manipulate inherent conformational preferences in a dynamic helical region of an enzyme remote from its active site. Focusing on a conformationally heterogeneous helix in the de novo metal-dependent hydrolase MID1-zinc identified in prior work, a series of variants is generated with backbone-modified residues that site-specifically enhance or decrease local helical propensity. Impacts of these backbone modifications on structure, thermal stability, and esterase activity were assessed using biophysical methods. Results of these experiments reveal a complex interplay between thermal folded stability and catalytic activity in MID1-zinc. More broadly, the findings reported here validate a potentially general approach to produce enzymes with comparable oligomeric assemblies and secondary structure content but highly variable thermal stabilities and catalytic efficiencies.