Understanding Cysteine Reactivity in Protein Environments with Electric Fields

The role cysteine residues play in proteins is mediated by their protonation state, whereby the thiolate form of the side chain is highly reactive while the thiol form is more inert. However, the pK(a) of cysteine residues is hard to predict as it can differ widely from its reference value in solution, an effect that is accentuated by local effects in the heterogeneous protein environment. Here, we present a new approach to the prediction of cysteine reactivity based on electric field calculations at the thiol/thiolate group. We validated our approach by predicting the protonation state of cysteine residues in different protein environments (in the active site, at the protein surface, and buried within the protein interior), including Cys-25 in papaya protease omega, which was proven problematic for the more traditional constant pH molecular dynamics (MD) technique. We predict pK(a) shifts consistent with experimental observations, and the decomposition of the electric fields into contributions from molecular fragments provides a direct handle to rationalize local pH and pK(a) effects in proteins without introducing parameters other than those of the force field used for MD simulations.