A neutron diffraction study of spectroscopic states for the light-energized proton pump bacteriorhodopsin (BR) is presented. The photocycle states BR-568 and M were generated at temperatures above 40C and were measured after trapping at -1800C. In the BR-568 to M-state transition, which is known to be a key step in transmembrane proton pumping, reversible structural changes of the protein were detected. These structural alterations occur in the neighborhood of the cyclohexene ring and at the Schiff's base end of the chromophore retinal. They are interpreted as a 1-2°tilt of three or four of the transmembrane a-helices or as positional changes of four or five amino acids. The structural changes observed are inherent in the transport mechanism of bacteriorhodopsin.Membrane protein conformational changes are expected to be involved in transport mechanisms. For the light-driven proton pump bacteriorhodopsin (BR), however, the all-trans to 13-cis isomerization of the chromophore retinal, during the first steps of the photocycle, is the only structural alteration unambiguously determined to date (1, 2). A variety of spectroscopic and diffraction experiments have been performed to detect changes also in the protein moiety of BR connected with the transition from the BR-568 ground state to the photocycle intermediate M. These efforts resulted in contradictory ideas about the extent of the conformational changes occurring. They -range from lattice disorder and dramatic changes in the tertiary conformation to only subtle alterations in the structure at high resolution (for review, see ref.3). To settle this controversy, we have conducted a neutron diffraction study on different functional states of BR. The advantages of neutron diffraction for the study of biological membranes derive mainly from the natural contrast between protein and protein-associated water as well as between protein and lipid, which is higher for neutrons than for x-rays. Furthermore, since the coherent neutron scattering length of H (-3.74 fm) and 2H (6.67 fm) is different not only in magnitude but also in sign, there are rich possibilities of 2H labeling. By performing measurements in H20 and 2H20 on the BR-568 ground state and the M intermediate, it should be possible not only to detect structural differences in the protein but also to observe any redistribution of associated water and exchangeable hydrogens related to the transition.The results obtained by neutron diffraction unambiguously show that in the BR-568 to M transition, which is known to be a key step in transmembrane proton pumping, reversible structural changes of the protein in the vicinity of the chromophore retinal occur. These light-induced conformational changes are proposed to trigger the vectorial H+ translocation processes in the active center of BR.
METHODSSample Preparation. Purple membranes were isolated from Halobacterium halobium (strain ET 1001). Neutron diffraction experiments require '100 mg of BR to perform the experiments in a reasonable time of ""2 days for t...
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