Molecular determinants of pKa values of histidine residues in staphylococcal nuclease

Elucidating the molecular determinants of pKa values is essential for understanding the character of electrostatic contributions to the stability and function of proteins. The studies described in this dissertation focus on two poorly understood factors that can affect the pK a values of surface ionizable groups: short-range polar and Coulomb interactions, and local structural flexibility.;In another set of experiments I examined the roles of local conformational flexibility in modulating the pKa values of histidines. The approached involved substitution with Gly residues at positions surrounding His-121 and His-124 to promote local flexibility. In some of the Gly-containing variants the pKa values of His-121 and His-124 approached the normal value of 6.3 without exhibiting any evidence of major structural changes. This is fully consistent with the notion that the p Ka values of surface groups reflect the flexibility of their microenvironments.;The pKa values measured by NMR spectroscopy were used to challenge a variety of continuum methods for pK a calculations based on Poisson-Boltzmann electrostatics. Our results show that when static structures are used in the calculations, arbitrarily high dielectric constant are not sufficient to reproduce the effects of short-range interactions and local conformational flexibility in a self consistent manner. They suggest that calculations with static structures can never be accurate, and that explicit treatment of local conformational dynamics will be necessary to improve pKa calculations.;The approach involved measurement of the pKa values of histidine residues in staphylococcal nuclease using NMR spectroscopy. In one set of experiments, I examined the roles of short-range polar or Coulomb interactions in the network surrounding His-121 and His-124. The pKa values of His-121 and His-124 in the parent protein are 5.3 and 5.7, respectively. In variants where the network surrounding these groups was disrupted, the pKa values range from 4.03 to 6.46 for His-121, and 5.04 to 5.99 for His-124. The data suggest that hydrogen bonds and hydration of charged groups are maximized in the network, whereas Coulomb interactions are not. The tautomeric state of the histidines was the same in all variants, suggesting that the network is quite rigid and incapable of reorganizing. This was consistent with what was observed in the crystal structure of a variant in which a key interaction between His-121 and Asp-75 was disrupted---the network appears to be incapable of reorganizing in response to perturbations. The conformational properties of the network appear to be governed by hydrogen bonds.