PH-INDUCED STRUCTURAL-CHANGES IN BACTERIORHODOPSIN STUDIED BY FOURIER-TRANSFORM INFRARED-SPECTROSCOPY

Previous C-13-NMR studies showed that two of the four internal aspartic acid residues (Asp-96 and Asp-115) of bacteriorhodopsin (bR) are protonated up to pH = 10, but no accurate pK(a), of these residues has been determined. In this work, infrared spectroscopy with the attenuated total reflection technique was used to characterize pH-dependent structural changes of ground-state, dark-adapted wild-type bacteriorhodopsin and its mutant (D96N) with aspartic acid-96 replaced by asparagine. Data indicated deprotonation of Asp-96 at high pH (pK(a) = 11.4 +/- 0.1), but no Asp-115 titration was observed. The analysis of the whole spectral region characteristic to complex conformational changes in the protein showed a more complicated titration with an additional pK(a) value (pK(a1) = 9.3 +/- 0.3 and pK(a2) = 11.5 +/- 0.2). Comparison of results obtained for bR and the D96N mutant of bR shows that the pK(a) approximate to 11.5 characterizes not a direct titration of Asp-96 but a protein conformational change that makes Asp-96 accessible to the external medium.