Protein thermostabilization with Protposer: Pushing the stability limits and folding reversibility of a highly-stabilized apoflavodoxin

Enhancing the stability of highly stable proteins represents an interesting challenge in protein design. We have used the computational tool Protposer to rapidly achieve large additional stabilization of apoflavodoxin, a protein strongly thermostabilized over the years through protein engineering based on educated guesses. By rationally combining top-ranked mutations onto a previously stabilized variant (6 M), we have generated a series of new mutants and characterized their stability by thermal and chemical denaturation. Relative to the starting variant, the T_m of 10 M apoflavodoxin is nearly 9 °C higher, while the simplified 3 M and 4 M mutants, showing improved refolding properties, display increases of 6/7.5 °C, respectively. The thermostabilizing effects of individual mutations are close to additive and translate into a large increase in conformational stability at 25.0 °C. Comparison of the x-ray structures of progressively stabilized WT, 6 M and 10 M flavodoxins reveals a concomitant mild trend toward shorter hydrogen bonds, reduced internal cavity volumes and narrower tunnels. Overall, these conformational changes are minor, and a functional assay confirms the mutants also preserve their catalytic activity. These findings demonstrate that even highly stable proteins can be further stabilized through rational design using a simple computational tool that automatically analyses PDB files and identifies stabilizing mutations.