In order to enforce vectorial proton transport in bacteriorhodopsin (bR), it is necessary that there be a change in molecular structure between deprotonation and reprotonation of the chromophore --- i.e., there must be at least two different M-intermediates in the functional photocycle. We have recently published the results that two consecutive M intermediates, M,, and M., were trapped which resemble the putative M I and M2 states. SSNMR studies on the retinal chromophore in these two states indicate that the two deprotonated states resemble each other in the chemical shifts of SB 15N, [12-13C]ret, [14-13C]ret, etc.. Two subisomers, M01 and M,,2, in M,, were barely resolved through the small splitting in the chemical shift of [ 14-13C]ret. The later M state,Mn, coexists with the N state under all the conditions tested so far and their differences other than protonation state have not yet been detected in terms of chemical shifts. The improved resolution of a 500 or a 750 MHz spectrometer (compared to 317 MHz) is crucial for resolving the spectral components of the close intermediates, a prerequisite for examining their structure. Further experiments will determine the unique spectral and structural characteristics of the intermediates and their relationship in the bR photocycle.
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