Using multiconformation continuum electrostatics model for pKa and proton transfer pathway calculations in protein

Residue protonation state changes and proton transfer reactions in protein are basic and ubiquitous reactions in biological chemistry. The Multiconformation Continuum Electrostatics (MCCE) program is applied here to calculate the pKa of residues in the specially engineered protein staphylococcal nuclease (SNase) and further modified to investigate the unidirectional proton transfer mechanism in the proton pump protein bacteriorhodopsin. The pKas are obtained by Monte Carlo sampling of coupled side chain protonations and positions as a function of pH in MCCE. The pKas of 96 acids and bases introduced into buried sites in the SNase protein were calculated and the results compared with experimental values. It''s found that the pKas of the introduced residues have a clear dependence on the protein dielectric constant epsilon in the continuum electrostatics analysis, while native ionizable residues do not. The native residues have electrostatic interactions with other residues in the protein favoring ionization, which are larger than the desolvation penalty favoring the neutral state, while the introduced residues have a larger desolvation penalty and negligible interactions with residues in the protein. For these residues changing protein dielectric constant epsilon has a large influence on the calculated pKa. An epsilon of 8-10 and a Lennard-Jones scaling of .25 is best here. A hydrogen-bonded network of residues and water in protein can be also obtained with Monte Carlo sampling of coupled side chain protonations and positions at a fixed pH. The proton transfer pathways can then be analyzed from the hydrogen-bonded network and the free energy barriers of the pathways can be further estimated. Bacteriorhodopsin has three key sites that bind and release protons: D96 near the cytoplasmic side, the central (D85, D212 and the Schiff Base) and exit (E194 and E204) clusters. Its proton transfer networks have been analyzed in crystal structures of bacteriorhodopsin trapped in the ground and M states. Internal pathways between D85 and the exit cluster are seen in the bR but not M state structures. In contrast, in the M state but not the bR structures the exit cluster is connected to the extracellular side. The free energy is found for the non-equilibrium protonation states for single element hops between D85 and the exit cluster. The proton transfer from R82 to a nearby water is the highest energy intermediate, ≈10 kcal/mol above the reactant state.