| Electrostatic effects in proteins govern many biological processes. For this reason, structure-based calculations of electrostatic energies and pKa values are necessary to bridge the gap between structure and function. To be useful, these calculations must be validated against experimental data, and they must embody the correct physics. Toward this end, we addressed three problems where insight from experiment was necessary to improve structure-based calculations: (1) why are the p Ka values of surface ionizable groups so similar to those of model compounds in water, (2) how does temperature affect electrostatic interactions in a protein and (3) what determines internal hydration?;To examine the reasons that pKa values of surface groups are minimally perturbed, the pKa values of all 20 Asp and Glu residues in staphylococcal nuclease (SNase) were measured using 13C-NMR spectroscopy. Fourteen titrated with pKa values shifted by 1.1 units or less from those of model compounds. Six titrated with pKa shifts of 1.5 units or larger; hydrogen bonding contributes more to these p Ka shifts than Coulomb effects. Structure-based calculations fail to reproduce these pKa values owing to exaggerated Coulomb interactions. Relaxation of the structure may improve agreement between calculation and experiment, but no evidence of a major pH-sensitive conformational rearrangement was detected.;The effects of temperature on pKa values in proteins are virtually unknown. To examine this issue, the temperature dependence of histidine pKa values in SNase was measured with 1H-NMR spectroscopy. A standard method for continuum electrostatics was modified to enable explicit treatment of temperature effects. In general, the properties of residues that exhibit abnormal enthalpies of ionization cannot yet be reproduced computationally.;It is becoming widely recognized that internal water molecules in proteins are essential for function. To learn how to hydrate the protein interior artificially, the DOWSER algorithm, developed by Li Zhang and Jan Hermans, was calibrated with 70 crystal structures of SNase, 16 of which show water molecules at an internal pocket with polar and ionizable groups.;These studies contribute novel and useful insight to guide further development of computational methods for accurate treatment of electrostatic energies in proteins. |
