A three-dimensional difference map of the N intermediate in the bacteriorhodopsin photocycle: part of the F helix tilts in the M to N transition.
The N intermediate of the bacteriorhodopsin photocycle was trapped for electron diffraction studies in glucose-embedded specimens of the site-directed mutant Phe219 --> Leu. At neutral pH, the N-bR difference Fourier transform infrared spectrum of this mutant is indistinguishable from published difference spectra obtained for wild-type bacteriorhodopsin at alkaline pH. An electron diffraction difference map of the N intermediate in projection shows large differences near the F and the G helix, which are very similar to the features seen in the M intermediates of the Asp96 --> Gly mutant [Subramaniam et al. (1993) EMBO J. 12, 1-8]. This similarity was anticipated on the basis of Fourier transform infrared data, which have shown that the M intermediate trapped in Asp96 mutants already has the protein structure of the N intermediate [Sasaki et al. (1992) J. Biol. Chem. 267, 20782-20786]. A preliminary three-dimensional difference map of the N intermediate, calculated from electron diffraction data of samples tilted at 25 degrees, clearly shows that the change on the F helix consists of an outward movement of the cytoplasmic end of the helix. In addition, the cytoplasmic side of the G helix moves or becomes more ordered. Comparison with published difference maps of the M intermediate indicates that the F helix tilt occurs in the M to N transition, but the G helix change represents an earlier step in the photocycle.
Vonck
J
J
Lawrence Berkeley National Laboratory, Life Sciences Division, Donner Laboratory, University of California, Berkeley 94720, USA.
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D004563:Electrochemistry; D006217:Halobacterium / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D015394:Molecular Structure; D010777:Photochemistry; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared
D001436:Bacteriorhodopsins