SURFACE-CHARGE OF BACTERIORHODOPSIN DETECTED WITH COVALENTLY BOUND PHINDICATORS AT SELECTED EXTRACELLULAR AND CYTOPLASMIC SITES

We present a method that allows the detection of the surface charge de nsity of bacteriorhodopsin (bR) at any selected protein surface site. The optical pH indicator fluorescein was covalently bound to the sulfh ydryl groups of single cysteine residues, which were introduced at sel ected positions in bR by site-directed mutagenesis. On the extracellul ar side, the positions were in the BC loop (72) and in the DE loop (12 9-134). On the cytoplasmic side, one position in each loop was labeled : 35 (AB), 101 (CD), 160 (EF), and 231 (carboxy tail). The apparent pK s of fluorescein in these positions were determined for various salt c oncentrations. The local surface charge density was calculated from th e dependence of the apparent pK of the dye on the ionic strength using the Gouy-Chapman equation. The surface charge density at pH 6.6 is mo re negative on the cytoplasmic side (averaged over all positions, -2.5 +/- 0.2 elementary charges per bR) than on the extracellular side (av erage, -1.8 +/- 0.2 elementary charges per bR) with little variation a long the surface. Since the experiments were performed with electrical ly neutral CHAPS/DMPC micelles, these values represent the charge pres ent on bR itself. The validity of our approach is supported by the out come of the following two control experiments: (1) when the positively charged surface residue Arg-134 was replaced by cysteine, the surface charge as detected by the indicator at that site became more negative by one elementary charge; (2) removal of the negatively charged carbo xy tail on the cytoplasmic side reduced the negative charge density, a s sensed by a pH indicator on the same side, but not on the opposite s ide. The contribution of the functionally important internal residues Arg-82 and Asp-96 to the surface charge density was determined using t he double mutants G72C/R82A and V101C/D96A with the indicator dye atta ched to cysteine in position 72 and position 101, respectively. From t he comparison of the charge density data obtained for the double mutan ts to the results for the single mutants G7 2C and V101C, it is conclu ded that Arg-82 contributes about 0.55-0.6 positive charge and Asp-96 0.6-0.65 negative charge to the surface charge on the membrane side to which they are closest.