Backbone Conformational Fluctuations as Determinants of pKa Values of Surface Residues in Proteins

Abstract

The pKa values of surface ionizable residues in proteins are usually similar to the pKa values of ionizable residues in water. This is still difficult to reproduce using structure-based electrostatics calculations with static structures, which tend to exaggerate the shifts in pKa values because the magnitude of Coulomb effects is exaggerated and the balance between Coulomb and dehydration effects is not reproduced correctly. These problems can be minimized by using artificially high protein dielectric constants, but sometimes the discrepancy persists even when the protein interior is treated with the dielectric constant of water. This suggests that pKa values are affected by the conformational dynamics of the protein, which are not reflected in the crystal structure. Molecular dynamics or Monte Carlo simulations can be used to attempt to reproduce these dynamic effects implicitly, but there are no useful data for testing this approach directly. To examine the role of fluctuations of the backbone in determining pKa values, NMR spectroscopy was used to measure the pKa values of all 20 Asp and Glu residues in variants of staphylococcal nuclease with Gly substituted at select locations. These substitutions were intended to enhance backbone fluctuations without affecting the overall protein structure. Some Gly substitutions detectably shifted the pKa values of some carboxylic groups. Crystal structures of these variants showed no significant changes relative to the structure of the reference protein. Calculations with continuum methods using these crystal structures do not reproduce the measured shifts in pKa values. NMR spectroscopy studies suggest Gly substitutions affect the dynamics of the protein. Efforts are underway to better characterize these changes. Our results suggest that the high apparent polarizability of proteins might be due to subtle structural fluctuations that are difficult to reproduce computationally.