The crystal structure of the L1 intermediate of halorhodopsin at 1.9 angstroms resolution.
The mutant T203V of the light driven chloride pump halorhodopsin from Halobacterium salinarum was crystallized and the X-ray structure was solved at 1.6 angstroms resolution. The T203V structure turned out to be nearly identical to the wild type protein with a root mean square deviation of 0.43 angstroms for the carbon alpha atoms of the protein backbone. Two chloride binding (CB) sites were demonstrated by a substitution of chloride with bromide and an analysis of anomalous difference Fourier maps. The CB1 site was found at the same position as in the wild type structure. In addition, a second chloride binding site CB2 was identified around Q105 due to higher resolution in the mutant crystal. As T203V showed a 10 times slower decay of its photocycle intermediate L, this intermediate could be trapped with an occupancy of 60% upon illumination at room temperature and subsequent cooling to 120 degrees K. Fourier transform infrared spectroscopy clearly identified the crystal to be trapped in the L1 intermediate state and the X-ray structure was solved to 1.9 angstroms resolution. In this intermediate, the chloride moved by 0.3 angstroms within binding site CB1 as indicated by peaks in difference Fourier density maps. The chloride in the second binding site CB2 remained unchanged. Thus, intraproteinous chloride translocation from the extracellular to the cytoplasmic part of the protein must occur in reaction steps following the L1 intermediate in the catalytic cycle of halorhodopsin.
Gmelin
Walter
W
Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Martinsried, Germany.
Zeth
Kornelius
K
Efremov
Ruslan
R
Heberle
Joachim
J
Tittor
Jörg
J
Oesterhelt
Dieter
D
D019943:Amino Acid Substitution; D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry; D018360:Crystallography, X-Ray; D019617:Halobacterium salinarum / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D025602:Halorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry
D002712:Chlorides; D025602:Halorhodopsins