Local Conformational Fluctuations of the Backbone Affect the p Ka Values of Ionizable Groups in Proteins

Abstract

The pKa values of all 20 Asp and Glu residues in staphylococcal nuclease (SNase) have been measured previously using NMR spectroscopy. Some of these pKa values are shifted relative to the normal values for Asp and Glu in water, but most are not. This does not agree with structure-based pKa calculations, according to which most of the pKa values are depressed because of strong Coulomb interactions with basic residues. This discrepancy persists even in calculations that treat the protein interior with the dielectric constant of water, suggesting that the crystal structure does not fully represent the ensemble average in solution. This idea is supported by calculations showing that the acid unfolding properties of SNase are reproduced better when the protein is treated as an ensemble of structures rather than as a static structure. We have tested the hypothesis that subtle, local conformational fluctuations contribute significantly to the pKa values of ionizable groups. This was examined experimentally by measuring pKa values of Asp, Glu, and His residues with NMR spectroscopy in variants of SNase with Gly substitutions at strategic locations, designed to enhance the flexibility of the backbone. Such substitutions should affect pKa values without affecting the protein structure. Consistent with our hypothesis, several Gly substitutions were found to shift the pKa values of ionizable groups without affecting the structure noticeably. Efforts are underway to determine what the effects of Gly substitutions on structure and fluctuations are. Our results suggest that the high apparent polarizability of proteins might be due to local or subglobal unfolding. We will present a computational model designed to capture the effects of local unfolding on pKa values in proteins.