Charge plays an important role in protein-protein interactions. In the case of excessively charged proteins, their electrostatic potentials contribute to the processes of recognition and binding with other proteins or ligands. We present an automated computational framework for determining the contribution of each charged amino acid to the electrostatic properties of proteins, at atomic resolution level. This framework involves computational alanine scans, calculation of Poisson-Boltzmann electrostatic potentials, calculation of electrostatic similarity distances (ESDs), hierarchical clustering analysis of ESDs, calculation of solvation free energies of association, and visualization of the spatial distributions of electrostatic potentials. The framework is useful to classify families of mutants with similar electrostatic properties and to compare them with the parent proteins in the complex. The alanine scan mutants introduce perturbations in the local electrostatic properties of the proteins and aim in delineating the contribution of each mutated amino acid in the spatial distribution of electrostatic potential, and in biological function when electrostatics is a dominant contributing factor in protein-protein interactions. The framework can be used to design new proteins with tailored electrostatic properties, such as immune system regulators, inhibitors, and vaccines, and in guiding experimental studies. We present an example for the interaction of the immune system protein C3d (the d-fragment of complement protein C3) with its receptor CR2, and we discuss our data in view of a binding site controversy. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 316-325, 2011