The pH dependence of protein biophysical characteristics is often analyzed to gain an improved understanding of protein stability, enzyme activity, and protein-ligand-binding processes. Indeed, much of our understanding of the catalytic mechanisms of enzymes derives from studies of the pH dependence of catalytic activity, and the ability to redesign the pH-dependent properties of enzymes continues to be of high relevance for both industrial and medical applications of proteins. This chapter discusses current theoretical methods for calculating protein pK(a) values and illustrates how one can analyze protein pK(a) calculation results to study calculation accuracy, pH stability profiles, and enzymatic pH activity profiles. A description of how one can analyze the importance of individual titratable groups is presented along with details on methods for redesigning protein pK(a) values and enzymatic pH activity profiles. Finally, I discuss novel methods for fitting experimental nuclear magnetic resonance titration curves and enzymatic pH activity profiles that can be used to derive information on electrostatic interaction energies in proteins.
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