Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pK_a Measurements

Abstract

The pKa 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 pKa 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 pKa 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 N-acetyl-L-histidine methylamide. In the unliganded and unfolded protein, the pKa 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 pKa value is depressed by 1.03 ± 0.25 units, which is ~0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa 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.

Electrostatic Interactions in Protein Structure, Folding, Binding, and Condensation

“…These p K a ’s are significantly perturbed from model-compound values, but, when used in eq , yield a pH dependence of the folding free energy consistent with direct measurement. p K a ’s have now been determined for the unfolded state of at least one other marginally stable protein and for several intrinsically disordered proteins (or fragments). − Using a new NMR methodology, it is even possible to determine p K a ’s for a sparsely populated folding intermediate and deduce its pH-dependent stability relative to the folded state . Again, in all these cases, perturbed p K a ’s implicate charge–charge interactions.…”