Experimental pK_a Value Determination of All Ionizable Groups of a Hyperstable Protein

Electrostatic interactions significantly contribute to the stability and function of proteins. The stabilizing or destabilizing effect of local charge is reflected in the perturbation of the pK_a value of an ionizable group from the intrinsic pK_a value. Herein, the charge network of a hyperstable dimeric protein (ribbon-helix-helix (rhh) protein from plasmid pRN1 from Sulfolobus islandicus) is studied through experimental determination of the pK_a values of all ionizable groups. Transitions were monitored by multiple NMR signals per ionizable group between pH 0 and 12.5, prior to a global analysis, which accounted for the effects of neighboring residues. It is found that for several residues involved in salt bridges (four Asp and one Lys) the pK_a values are shifted in favor of the charged state. Furthermore, the pK_a values of residues C40 and Y47, both located in the hydrophobic dimer interface, are shifted beyond 13.7. The necessary energy for such a shift is about two-thirds of the total stability of the protein, which confirms the importance of the hydrophobic core to the overall stability of the rhh protein.