Effect of Allosteric Mutations on Conformational Equilibrium of DHFR

Abstract

Allosteric mutations, distal from the binding site of DHFR from B. stearothermophilus, were previously found to alter the ligand binding specificity. In this work, we investigate how the structural conformational equilibrium of DHFR is affected when one of these mutations is introduced to the enzyme. H38N and Y127A mutations are introduced to previously developed, fluorescently labeled, conformationally-sensitive Bs DHFR (C73A/S131CMDCC DHFR). Stopped-flow kinetic analysis is currently used to study the kinetics of protein conformational motions associated with methotrexate (MTX) binding. This approach allows us to examine the conformational changes associated with MTX binding for the mutant and compares them to the data for the wild type enzyme. The data will also reveal information about the conformational equilibrium preceding ligand binding. Understanding of the effect of these distal mutations on the conformational equilibrium will provide insights on the role of allosteric regions in ligand binding specificity and inform design and repurposing of selective DHFR drug compounds