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PROTON TRANSLOCATION and CONFORMATIONAL CHANGES DURING THE BACTERIORHODOPSIN PHOTOCYCLE: TIME‐RESOLVED STUDIES WITH MEMBRANE‐BOUND OPTICAL PROBES and X‐RAY DIFFRACTION *
Author(s) -
Dencher Norbert A.,
Heberle Joachim,
Bark Carola,
Koch Mlchel H. J.,
Rapp Gert,
Oesterhelt Dieter,
Bartels Klaus,
Büldt Georg
Publication year - 1991
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1991.tb02107.x
Subject(s) - bacteriorhodopsin , chemistry , proton , kinetics , crystallography , halobacterium salinarum , diffraction , halobacteriaceae , neutron diffraction , membrane , analytical chemistry (journal) , crystal structure , optics , quantum mechanics , chromatography , biochemistry , physics
– For the first time, we monitored time‐resolved conformational changes inherent in bacterio‐rhodopsin's reaction cycle as well as the H + ‐release events directly at the surface of bacteriorhodopsin (bR) at room temperature. Signals of optical pH‐indicators in the aqueous bulk phase were compared with those of probes covalently linked to bR. The kinetics of H + ‐translocation and the correlation of the photocycle with the pumping cycle can only be determined with indicators bound to bR. The proton appears at the extracellular surface during the L 550 to M 412 transition. Upon short photo‐excitation, diffraction patterns were obtained from both guanidine hydrochloride‐treated wild type bR and an Asp96Asn mutant with millisecond time resolution by x‐ray synchrotron radiation. The measured time course and location of the structural changes confirm and extend our previous static neutron diffraction study at ‐180°C. The temporal correlation of photocycle intermediates and proton translocation steps with structural changes in the protein under similar environmental conditions strongly contribute to the understanding of the pumping mechanism.