The Role of Dynamics in Protein Evolution

Abstract

re-equilibration of a chemical system following an instantaneous increase in temperature induced by a laser pulse tuned to an infrared water band. The reequilibration results in changes in the concentration of the species involved, and the transient changes are characterized using spectroscopic probes. To investigate the conformational changes associated with the binding of oxamate we studied the LDH from wild type cells as well as those from various single tryptophan mutants. These mutants were created by first replacing all tryptophans with tyrosine in wild type bsLDH to create a tryptophan-less template, followed by reintroduction of a single tryptophan at strategic sites in the protein. We probed the fluorescence emission of NADH in wild type and mutant bsLDH to report on the time evolution of the changes within the NADH environment over 100ms to 3ms time scale. Transients collected were then correlated to those resulting from a probe of tryptophan emissions. The results were then analyzed based on a plausible kinetic model. A comprehensive picture of the dynamics of ligand binding and Michaelis complex formation in bsLDH is obtained from the various structural reporters.