Determination of electrostatic potential around specific locations on the surface of actin by diffusion-enhanced...

In this study we have established systematic procedures for the measurement of electrostatic potentials around specific and localized portions of protein surfaces. Diffusion-enhanced fluorescence energy transfer was used for these determinations. Energy transfer from donor molecules to freely diffusing acceptors is sensitive to the electrostatic potential around the donor when the acceptors possess electric charges. To quantify this sensitivity phenomenon in a controllable environment, we observed energy transfer from excited terbium chelate donors of known electric charge to a series of acceptors of different charges but bearing the same chromophore group. The rate of energy transfer was calculated theoretically (considering the structural arrangement of charged groups around the chromophore center), as well as determined experimentally (by time-resolved detection of terbium luminescence), with the two values obtained by the different means in close agreement. Having established the validity of this procedure using a relatively simple system, we studied the electrostatic conditions around two specific sites on the surface of actin molecules. A negative potential was found at both sites; the potential at one location (around the myosin binding site) was nearly neutralized by the addition of myosin subfragment-1, while the potential at the other site (around the phalloidin binding site) was not significantly affected.