A novel ensemble-based scoring and search algorithm for protein redesign, and its application to modify the substrate specificity of the gramicidin synthetase A phenylalanine adenylation enzyme

摘要: Realization of novel molecular function requires the ability to alter complex formation. Enzymatic can be altered by changing enzyme-substrate interactions via modification an enzyme's active site. A redesigned enzyme may either perform a reaction on its native substrates or substrates. number computational approaches have been developed address combinatorial nature protein redesign problem. These typically search for global minimum energy conformation among exponential conformations. We present algorithm redesign, which combines statistical mechanics-derived ensemble-based approach computing binding constant with speed and completeness branch-and-bound pruning algorithm. In addition, we efficient deterministic approximation algorithm, capable approximating our scoring arbitrary precision. practice, decreases execution time mutation factor ten. To test method, examined Phe-specific adenylation domain non-ribosomal peptide synthetase gramicidin (GrsA-PheA). Ensemble scoring, using rotameric partition functions bound unbound states GrsA-PheA, is first used predict wildtype previously described mutant (selective leucine), second, switch specificity toward leucine, two site sequences computationally predicted searching through space possible mutations. The top in silico mutants were created wetlab dissociation / constants determined fluorescence quenching. tested mutations exhibit desired change from Phe Leu. Our flexibly models both ligand rotamer-based functions, has application prediction protein-ligand binding, computer-aided drug design.