Functional roles of aspartic acid residues at the cytoplasmic surface of bacteriorhodopsin

The functions of the four aspartic acid residues in interhelical loops at t he cytoplasmic surface of bacteriorhodopsin, Asp-36, Asp-38, Asp-102, and A sp-104, were investigated by studying single and multiple aspartic acid to asparagine mutants. The same mutants were examined also with the additional D96N residue replacement. The kinetics of the M and N intermediates of the photochemical cycles of these recombinant proteins were affected only in a minor, although self-consistent, way. When residue 38 is an aspartate and anionic, it makes the internal proton exchange between the retinal Schiff b ase and Asp-96 about 3 times more rapid, and events associated with the rei somerization of retinal to all-trans about 3 times slower. Asp-36 has the o pposite effect on these processes, but to a smaller extent. Asp-102 and Asp -104 have even less or none of these effects. Of the four aspartates, only Asp-36 could play a direct role in proton uptake at the cytoplasmic surface . In the 13 bacterioopsin sequences now available, only this surface aspart ate is conserved.