Chemical Frustration in the Protein Folding Landscape: Grand Canonical Ensemble Simulations of Cytochrome c

A grand canonical formalism is developed to combine discrete simulations for chemically distinct species in equilibrium. Each simulation is based on a perturbed funneled landscape. The formalism is illustrated using the alkaline-induced transitions of cytochrome c as observed by FTIR spectroscopy and with various other experimental approaches. The grand canonical simulation method accounts for the acid/base chemistry of deprotonation, the inorganic chemistry of heme ligation and misligation, and the minimally frustrated folding energy landscape, thus elucidating the physics of protein folding involved with an acid/base titration of a protein. The formalism combines simulations for each of the relevant chemical species, varying by protonation and ligation states. In contrast to models based on perfectly funneled energy landscapes that contain only contacts found in the native structure, this study introduces chemical frustration from deprotonation and misligation that gives rise to many intermediates at alkaline pH. While the nature of these intermediates cannot be easily inferred from available experimental data, this study provides specific structural details of these intermediates, thus extending our understanding of how cytochrome c changes with an increase in pH. The results demonstrate the importance of chemical frustration for understanding biomolecular energy landscapes.