Is the rigid-body assumption reasonable?: Insights into the effects of dynamics on the electrostatic analysis of barnase–barstar

Electrostatically-driven association of proteins is important to many biological functions, and understanding which amino acid residues contribute to these interactions is crucial to protein design. Theoretical calculations that are used to elucidate the role of electrostatics in association are typically based on a single experimentally determined protein structure, while an underlying rigid-body assumption is adopted. The goal of this study was to investigate the role of conformational fluctuations on electrostatic interaction energies, as applied to the electrostatic analysis of barnase–barstar. For our calculations, we apply theoretical alanine-scan mutagenesis to introduce charge perturbations by replacing every ionizable residue with alanine, one at a time. Electrostatic clustering and free energy calculations based on the Poisson–Boltzmann method are used to evaluate the effects of each perturbation. Molecular dynamics simulations are performed for the barnase–barstar parent complex and seven experimental alanine mutations from the literature, in order to introduce relaxation before and after mutation. We discuss the effects of dynamics, in the form of pre- and post-mutation relaxation, on electrostatic clustering and free energies of association in light of experimental data. We also examine the utility of nine electrostatic similarity methods for clustering of barnase alanine-scan mutants. Our calculations suggest that the rigid-body assumption is reasonable for electrostatic calculations of barnase–barstar.