Time-resolved site-directed spin-labeling studies of bacteriorhodopsin: Loop-specific conformational changes in M

A spin-label at site 101 in the C-D loop of bacteriorhodopsin was previousl y found to detect a conformational change during the M --> N transition [St einhoff, H. -J., Mollaaghababa, R., Altenbach, C., Hideg, K., Krebs, M. P., Khorana, H. G,, and Hubbell, W. L, (1994) Science 266, 105-107]. We have e xtended these time-resolved electron paramagnetic resonance studies in purp le membranes by analyzing conformational changes detected by a spin-label a t another site in the C-D loop (103), and at sites in the A-B loop (35), th e D-E loop (130), and the E-F loop (160). In addition, we have investigated the motion detected by a spin-label at site 101 in a D96A mutant backgroun d that has a prolonged M intermediate. We find that among the examined site s, only spin-labels in the C-D loop detect a significant change in the loca l environment after the rise of M. Although the D96A mutation dramatically prolongs the lifetime of the M intermediate, it does not perturb either the structure of bacteriorhodopsin or the nature of the light-activated confor mational change detected by a spin-label at site 101. In this mutant, a con formational change is detected during the lifetime of M, when no change in the 410 nm absorbance is observed. These results provide direct structural evidence for the heterogeneity of the M population in real time, and demons trate that the motion detected at site 101 occurs in M, prior to Schiff bas e reprotonation.