Catching Excited States in the Act: Functional Unfolding in E. Coli Adenylate Kinase

Abstract

Excited states of proteins are important for many functions, however they are difficult to study due to their low population and transient nature. Using an entropy-enhancing, valine to glycine mutation scheme we are able to stabilize a locally unfolded (LU) excited state in the LID domain of E. coli adenylate kinase (AK) gaining access to varying populations of the LU state. With nuclear magnetic resonance experiments we are able to observe structural changes in the LID upon mutation consistent with local unfolding. More importantly conformational dynamics were measured from the both the folded and locally unfolded states giving access to the populations of both states and the kinetics of the folding/unfolding transition. Furthermore, the changes in population upon mutation are driven by an increase in the unfolding rate of the LID while enzyme kinetics are unaffected at physiological temperatures, evidence that LID unfolding is not rate limiting in the catalytic reaction. These experiments allow insight into the protein dynamics that drive function and exemplify the ensemble nature of the native state and the importance of conformational fluctuations in tuning biological activity.