Investigations of Local Unfolding Like Conformational Excursions in the Native State of Adenylate Kinase


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Elucidating the interplay between protein structure and dynamics is a prerequisite to an understanding of both function and adaptation in proteins. It has been difficult to experimentally separate these effects because it is challenging to rationally design mutations that will selectively effect dynamics. Here we adopt a mutation approach that is based on a thermal adaptation strategy observed in nature, and we use it to study the binding interaction of Escherichia coli adenylate kinase (AK). We rationally design several single-site, surface-exposed glycine mutations to selectively perturb the excited state conformational repertoire, leaving the ground-state X-ray crystallographic structure unaffected. The results not only demonstrate that the conformational ensemble of AK is significantly populated by a locally unfolded state that is depopulated upon binding, but also that the excited-state conformational ensemble can be manipulated through mutation, independent of perturbations of the ground-state structures. The implications of these results are twofold. First, they indicate that it is possible to rationally design dynamic allosteric mutations, which do not propagate through a pathway of structural distortions connecting the mutated and the functional sites. Secondly, the results reveal a general strategy for thermal adaptation that allows enzymes to modulate binding affinity by controlling the amount of local unfolding in the native-state ensemble. These findings open new avenues for rational modulation of protein function and fundamentally illuminate the role of local unfolding in function and adaptation.



Enzymology, Biophysics, Local unfolding, Adenylate Kinase, Protein Dynamics, Binding Affinity