Structural transition of bacteriorhodopsin is preceded by deprotonation of Schiff base: microsecond time-resolved x-ray diffraction study of purple membrane.
The structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of 7 angstroms by a time-resolved x-ray diffraction experiment utilizing synchrotron x rays from an undulator of SPring-8. The x-ray diffraction measurement system, used in coupling with a pulsed YAG laser, enabled us to record a diffraction pattern from purple membrane film at a time-resolution of 6 micros over the time domain of 5 micros to 500 ms. In the time domain, the functionally most important M-intermediate appears. A series of time-resolved x-ray diffraction data after photo-excitation showed clear intensity changes caused by the conformational changes of helix G in the M-intermediate. The population of the reaction intermediate was prominently observed at approximately 5 ms after a photo-stimulus. In contrast, absorption measurement indicated the deprotonation of the Schiff base predominantly occurred at approximately 300 micros after a photo-stimulus. These results showed that the conformational changes characterizing structurally the M-intermediate predominantly occur at a later stage of the deprotonation of the Schiff base. Thus, the M-intermediate can be divided into two metastable stages with different physical characteristics.
Oka
Toshihiko
T
Department of Physics, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, Japan. oka@phys.keio.ac.jp
Inoue
Katsuaki
K
Kataoka
Mikio
M
Yagi
Naoto
N
D001436:Bacteriorhodopsins / Q000737:chemistry*; D001703:Biophysics / Q000379:methods*; D005583:Fourier Analysis; D006217:Halobacterium / Q000378:metabolism; D007834:Lasers; D008027:Light; D009502:Neutrons; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011522:Protons; D018717:Purple Membrane / Q000378:metabolism*; D012545:Schiff Bases; D013997:Time Factors; D014961:X-Ray Diffraction
D011522:Protons; D012545:Schiff Bases; D001436:Bacteriorhodopsins