Protein-based therapeutics typically require high concentrations
of the active protein, which can lead to protein aggregation and high
solution viscosity. Such solution behaviors can limit the stability,
bioavailability, and manufacturability of protein-based therapeutics
and are directly influenced by the charge of a protein. Protein charge
is a system property affected by its environment, including the buffer
composition, pH, and temperature. Thus, the charge calculated by summing
the charges of each residue in a protein, as is commonly done in computational
methods, may significantly differ from the effective charge of the
protein as these calculations do not account for contributions from
bound ions. Here, we present an extension of the structure-based approach
termed site identification by ligand competitive saturation-biologics
(SILCS-Biologics) to predict the effective charge of proteins. The
SILCS-Biologics approach was applied on a range of protein targets
in different salt environments for which membrane-confined electrophoresis-determined
charges were previously reported. SILCS-Biologics maps the 3D distribution
and predicted occupancy of ions, buffer molecules, and excipient molecules
bound to the protein surface in a given salt environment. Using this
information, the effective charge of the protein is predicted such
that the concentrations of the ions and the presence of excipients
or buffers are accounted for. Additionally, SILCS-Biologics also produces
3D structures of the binding sites of ions on the proteins, which
enable further analyses such as the characterization of protein surface
charge distribution and dipole moments in different environments.
Notable is the capability of the method to account for competition
between salts, excipients, and buffers on the calculated electrostatic
properties in different protein formulations. Our study demonstrates
the ability of the SILCS-Biologics approach to predict the effective
charge of proteins and its applicability in uncovering protein–ion
interactions and their contributions to protein solubility and function.