Surface charge of bacteriorhodopsin detected with covalently bound pH indicators at selected extracellular and cytoplasmic sites

We present a method that allows the detection of the surface charge density of bacteriorhodopsin (bR) at any selected protein surface site. The optical pH indicator fluorescein was covalently bound to the sulfhydryl groups of single cysteine residues, which were introduced at selected positions in bR by site-directed mutagenesis. On the extracellular side, the positions were in the BC loop (72) and in the DE loop (129-134). On the cytoplasmic side, one position in each loop was labeled: 35 (AB), 101 (CD), 160 (EF), and 231 (carboxy tail). The apparent pKs of fluorescein in these positions were determined for various salt concentrations. The local surface charge density was calculated from the 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 more negative on the cytoplasmic side (averaged over all positions, -2.5 +/- 0.2 elementary charges per bR) than on the extracellular side (average, -1.8 +/- 0.2 elementary charges per bR) with little variation along the surface. Since the experiments were performed with electrically neutral CHAPS/DMPC micelles, these values represent the charge present on bR itself.(ABSTRACT TRUNCATED AT 250 WORDS)