STRUCTURAL CHARACTERIZATION OF THE L-TO-M TRANSITION OF THE BACTERIORHODOPSIN PHOTOCYCLE

Structural intermediates occurring in the photocycle of wild-type bact eriorhodopsin are trapped by illuminating hydrated, glucose-embedded p urple membrane at 170K, 220K, 230K, and 240K, We characterize light-in duced changes in protein conformation by electron diffraction differen ce Fourier maps, and relate these to previous work on photocycle inter mediates by infrared (FTIR) spectroscopy. Samples illuminated at 170K are confirmed by FTIR spectroscopy to be in the L state; a difference Fourier projection map shows no structural change within the 0.35-nm r esolution limit of our data. Difference maps obtained with samples ill uminated at 220K, 230K, and 240K, respectively, reveal a progressively larger structural response in helix F when the protein is still in th e M state, as judged by the FTIR spectra, Consistent with previous str uctural studies, an adjustment in the position or in the degree of ord ering of helix G accompanies this motion. The model of the photocycle emerging from this and previous studies is that bacteriorhodopsin expe riences minimal change in protein structure until a proton is transfer red from the Schiff base to Asp(85). The M intermediate then undergoes a conformational evolution that opens a hydrated ''half-channel,'' al lowing the subsequent reprotonation of the Schiff base by Asp(96).