Electrostatic Energetics of <i>Bacillus subtilis</i> Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine p<i>K</i>_a Measurements

The p<i>K</i>_a values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine p<i>K</i>_a values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have p<i>K</i>_a values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound <i>N</i>-acetyl-l-histidine methylamide. In the unliganded and unfolded protein, the p<i>K</i>_a values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent p<i>K</i>_a value is depressed by 1.03 ± 0.25 units, which is ∼0.5 units more than in either U or F. The depressed p<i>K</i>_a values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the p<i>K</i>_a differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function