title: pH-induced structural changes in bacteriorhodopsin studied by Fourier transform infrared spectroscopy.
abstract: Previous C13-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 pKa 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 (pKa = 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 pKa value (pKa1 = 9.3 +/- 0.3 and pKa2 = 11.5 +/- 0.2). Comparison of results obtained for bR and the D96N mutant of bR shows that the pKa approximately 11.5 characterizes not a direct titration of Asp-96 but a protein conformational change that makes Asp-96 accessible to the external medium.
authors: Institute of Biophysics, Hungarian Academy of Sciences, Szeged.
authors: S
authors: S
authors: Száraz
authors: D
authors: D
authors: Oesterhelt
authors: P
authors: P
authors: Ormos
mesh_terms: D001224:Aspartic Acid; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002247:Carbon Isotopes; D019617:Halobacterium salinarum / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation / Q000379:methods; D013329:Structure-Activity Relationship
chemical_list: D002247:Carbon Isotopes; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001436:Bacteriorhodopsins