DIRECT MOLECULAR-LEVEL MEASUREMENTS OF THE ELECTROSTATIC PROPERTIES OF A PROTEIN SURFACE

In this work, we used direct measurements with the surface force appar atus to determine the pH-dependent electrostatic charge density of a s ingle binding face of streptavidin. Mean field calculations have been used with considerable success to model electrostatic potential fields near protein surfaces, but these models and their inherent assumption s have not been tested directly at the molecular level, Using the forc e apparatus and immobilized, oriented monolayers of streptavidin, we m easured a pi of 5-5.5 for the biotin-binding face of the protein. This differs from the pi of 6.3 for the soluble protein and confirms that we probed the local electrostatic features of the macromolecule. With finite difference solutions of the linearized Poisson-Boltzmann equati on, we then calculated the pH-dependent charge densities adjacent to t he same face of the protein. These calculated values agreed quantitati vely with those obtained by direct force measurements. Although our st udy focuses on the pH-dependence of surface electrostatics, this direc t approach to probing the electrostatic features of proteins is applic able to investigations of any perturbations that alter the charge dist ribution of the surfaces of immobilized molecules.