ELECTROSTATIC INTERACTIONS AND ION BINDING IN TRYPSIN, BOVINE PANCREATIC TRYPSIN INHIBITOR, AND OTHER PROTEINS (STABILITY, PROTON)

The modified Tanford-Kirkwood electrostatic theory has been used to assess the stabilizing and destabilizing interactions among charged sites in several proteins. Various methods for considering solvent accessibility and ion binding effects are discussed. Application of the theory to the bovine pancreatic trypsin inhibitor (BPTI) defines the electrostatic origins of an observed conformational equilibrium as well as yielding pK values that correlate well with numerous ('13)C NMR observations. The net stability of the inhibitor is remarkable considering its high positive charge, and is shown to originate in selective exposure of positive charges which maximizes their neutralization by counterions. This strategy is also found to help stabilize the positive trypsin molecule. The electrostatic contribution to equilibrium constants for cation and proton binding to trypsin are discussed and related to its stability, conformation, and function. The extremely tight association of trypsin with the BPTI is considered and shown to be essentially nonelectrostatic in origin but is not disfavored electrostatically despite the high net positive charge, as a consequence of specific favorable interactions evaluated only by the complete multipolar treatment. The ovomucoid family of protease inhibitors was studied electrostatically and the magnitude of their interactions found to be minimal, as is the case of BPTI. This may relate to the presence of a rather high density of disulfide bonds functioning as tertiary structure supports in these proteins. The charge array is nevertheless organized to conserve stabilizing interactions and could thus possibly help facilitate folding. Electrostatic contributions to binding of azide ion to myoglobin as well as the fixation of the myoglobin A-helix to the rest of the molecule were reconsidered using the revised accessibility treatment, which was found to agree more closely with experiment. Consideration of the nature and variation in size of the overlapping electrostatic domains in proteins and their several types of stabilizing strategies lays the foundation for the understanding of the many biological phenomena that are based upon charge interactions.