The photocycle of bacteriorhodopsin (bR) is of great interest to the structure and function of bR. The proton-pumping and spectral tuning are related to the detail structure of bR intermediates, the determination of which relies on the discrimination of the individual intermediates and on the chemical shift assignments when solid state NMR method, the effective probe in protein structure determination, is used. In Gdn-HCl at pH10, the 15N NMR experiments revealed that 2 M states were observed, of which one is the normal M state, called Mo, and the other one is abnormal, called MN, dependent on whether they occur in NaCl medium. MN, trapped at -10 ~C, is more stable than Mo, prepared at temperature lower than -40 ~C. The two M's are sequential, with Mo relaxing to MN. At the same time the relaxation also produces NN state, which usually coexists with MN. From the signals of 14-13C and e-13C, Mo consists of two isomers and NN is different from the N state in NaCl due to the absence of the 14-13C signal at 115 ppm. The following bR photocycle is concluded in Gdn-HCl: bR568~~~>Mo~~~>MN~~~>NN~~~>bR568. In NaCl at pH10, the photocycle is different from that in Gdn-HCl. First Mo is comprised of several substates which are indicated by a broad 14-13C signal. The next difference is the absence of MN state in NaCl. The final one is that the 14-13C signal of N state occurs at 115 ppm, about 10 ppm upfield to that of NN. The concluded photocycle in NaCl is bR568~~~>Mo's~~~>N~~~>bR568. The further studies on L intermediate in NaCl add more information to our conclusion. The 15N NMR experiments showed that L relaxes to Mo's at a proper temperature. Therefore the photocycle is expanded to bR568~~~>L~~~>Mo's~~~>N~~~>bR568. The SB 15N chemical shifts are assigned for all the observed intermediates and the 14-13C and e-13C chemical shifts are also assigned for all but L state. The study of L in Gdn-HCl, the 13C chemical shift assignments for L in both media and the structure determination of all the intermediates are under way.
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