Conformational Reorganization Coupled to the Ionization of Internal Lys Residues in Proteins

Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction and catalysis. Because the protein interior is usually neither as polar nor as polarizable as water, these groups tend to have anomalous pKa values, and their ionization tends to be coupled to conformational reorganization. To elucidate mechanisms of energy transduction in proteins, it is necessary to understand the structural determinants of the pKa values of these buried groups, including the range and character of the conformational reorganization that the ionization of these buried groups can elicit. The L25K and L125K variants of staphylococcal nuclease (SNase) were used to characterize the diverse types of structural reorganization that can be promoted by the ionization of buried groups. NMR relaxation dispersion and ZZ-exchange experiments were used to identify the locations and measure the time scales and extent of pH-dependent conformational exchange in these two proteins. The buried Lys-25 and Lys-125 residues titrate with pKa of 6.3 and 6.2, respectively. The L25K protein fluctuates between the native state and an ensemble of locally unfolded states on the 400 μs to 7 ms time scale. On the 100 to 500 ms time scale the native state exchanges with a subglobally unfolded state in which the β-barrel is partially reorganized. The equilibrium between the native state and this alternative state is highly pH dependent; at pH values below the pKa of Lys-25 the state with the partially reorganized β-barrel is the dominant state. In contrast, the L125K protein only exhibited pH-independent fluctuation in the microsecond to millisecond time scale in the region near Lys-125. The study illustrates how diverse and how localized the coupling between conformational reorganization and ionization of buried groups can be. The pH-sensitive exchange between the fully native and subglobally or locally unfolded states in time scales well into hundreds of milliseconds will challenge all computational methods for structure-based calculations of pKa values.