In silico concurrent multisite pH titration in proteins

The concurrent proton binding at multiple sites in macromolecules such as proteins and nucleic acids is an important yet challenging problem in biochemistry. We develop an efficient generalized Hamiltonian approach to attack this issue. Based on the previously developed generalized-ensemble methods, an effective potential energy is constructed which combines the contributions of all (relevant) protonation states of the molecule. The effective potential preserves important phase regions of all states and, thus, allows efficient sampling of these regions in one simulation. The need for intermediate states in alchemical free energy simulations is greatly reduced. Free energy differences between different protonation states can be determined accurately and enable one to construct the grand canonical partition function. Therefore, the complicated concurrent multisite proton titration process of protein molecules can be satisfactorily simulated. Application of this method to the simulation of the pKa of Glu49, Asp50, and C-terminus of bovine pancreatic trypsin inhibitor shows reasonably good agreement with published experimental work. This method provides an unprecedented vivid picture of how different protonation states change their relative population upon pH titration. We believe that the method will be very useful in deciphering the molecular mechanism of pH-dependent biomolecular processes in terms of a detailed atomistic description.