Biochemistry 2000 Feb 8;39(5):1120-1127

Time-Resolved Site-Directed Spin-Labeling Studies of
Bacteriorhodopsin: Loop-Specific Conformational Changes in M.

Mollaaghababa R, Steinhoff HJ, Hubbell WL, Khorana HG

Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,
Institut fur Biophysik, Ruhr-Universitat Bochum, 44780 Bochum, Germany, and Jules Stein Eye Institute and
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-7008.

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A spin-label at site 101 in the C-D loop of bacteriorhodopsin was previously found to detect a conformational change
during the M --> N transition [Steinhoff, 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 extended these time-resolved electron
paramagnetic resonance studies in purple membranes by analyzing conformational changes detected by a spin-label at
another site in the C-D loop (103), and at sites in the A-B loop (35), the 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 background that has
a prolonged M intermediate. We find that among the examined sites, only spin-labels in the C-D loop detect a
significant change in the local 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 conformational change detected by a spin-label at site 101. In this mutant, a conformational 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 demonstrate that the motion detected at
site 101 occurs in M, prior to Schiff base reprotonation.