Simplified methods for pKa and acid pH-dependent stability estimation in proteins: removing dielectric and counterion boundaries.
Much computational research aimed at understanding ionizable group interactions in proteins has focused on numerical solutions of the Poisson-Boltzmann (PB) equation, incorporating protein exclusion zones for solvent and counterions in a continuum model. Poor agreement with measured pKas and pH-dependent stabilities for a (protein, solvent) relative dielectric boundary of (4,80) has lead to the adoption of an intermediate (20,80) boundary. It is now shown that a simple Debye-Huckel (DH) calculation, removing both the low dielectric and counterion exclusion regions associated with protein, is equally effective in general pKa calculations. However, a broad-based discrepancy to measured pH-dependent stabilities is maintained in the absence of ionizable group interactions in the unfolded state. A simple model is introduced for these interactions, with a significantly improved match to experiment that suggests a potential utility in predicting and analyzing the acid pH-dependence of protein stability. The methods are applied to the relative pH-dependent stabilities of the pore-forming domains of colicins A and N. The results relate generally to the well-known preponderance of surface ionizable groups with solvent-mediated interactions. Although numerical PB solutions do not currently have a significant advantage for overall pKa estimations, development based on consideration of microscopic solvation energetics in tandem with the continuum model could combine the large deltapKas of a subset of ionizable groups with the overall robustness of the DH model.
Institute of Food Research, Reading Laboratory, Earley Gate, Reading, United Kingdom. james.warwicker@bbsrc.ac.uk
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D001426:Bacterial Proteins; D003087:Colicins / Q000032:analysis; D003198:Computer Simulation; D006863:Hydrogen-Ion Concentration; D015233:Models, Statistical; D009010:Monte Carlo Method; D009113:Muramidase / Q000032:analysis; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000032:analysis; D012259:Ribonuclease, Pancreatic / Q000032:analysis; D012260:Ribonucleases / Q000032:analysis; D013696:Temperature
D001426:Bacterial Proteins; D003087:Colicins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic; D009113:Muramidase