Strategies for the thermodynamic characterization of linked binding/local folding reactions within the native state application to the LID domain of adenylate kinase from Escherichia coli.

Conformational fluctuations in proteins have emerged as an important aspect of biological function, having been linked to processes ranging from molecular recognition and catalysis to allostery and signal transduction. In spite of the realization of their importance, however, the connections between fluctuations and function have largely been empirical, even when they have been quantitative. Part of the problem in understanding the role of fluctuations in function is the fact that the mere existence of fluctuations complicates the interpretation of classic mutagenesis approaches. Namely, mutagenesis, which is typically targeted to an internal position (to elicit an effect), will change the fluctuations as well as the structure of the native state. Decoupling these effects is essential to an unambiguous understanding of the role of fluctuations in function. Here, we use a mutation strategy that targets surface-exposed sites in flexible parts of the molecule for mutation to glycine. Such mutations leave the ground-state structure unaffected. As a result, we can assess the nature of the fluctuations, develop a quantitative model relating fluctuations to function (in this case, molecular recognition), and unambiguously resolve the probabilities of the fluctuating states. We show that when this approach is applied to Escherichia coli adenylate kinase (AK), unique thermodynamic and structural insights are obtained, even when classic mutagenesis approaches targeted to the same region yield ambiguous results.