Increasing protein stability by inferring substitution effects from high-throughput experiments.

ABSTRACT: We apply a computational model, global multi-mutant analysis (GMMA), to inform on effects of most amino acid substitutions from a randomly mutated gene library. Using a high mutation frequency, the method can determine mutations that increase the stability of even very stable proteins for which conventional selection systems have reached their limit. As a demonstration of this, we screened a mutant library of a highly stable and computationally redesigned model protein using an in vivo genetic sensor for folding and assigned a stability effect to 374 of 912 possible single amino acid substitutions. Combining the top 9 substitutions increased the unfolding energy 47 to 69 kJ/mol in a single engineering step. Crystal structures of stabilized variants showed small perturbations in heli

SUBMITTER: Norrild RK 

PROVIDER: S-EPMC9701609 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature