Biophys. J., Vol 78, Issue 3. Copyright © 2000 by Biophysical Society

Unraveling Photoexcited Conformational Changes of
Bacteriorhodopsin by Time Resolved EPR Spectroscopy

Thomas Rink, Matthias Pfeiffer, Dieter Oesterhelt, Klaus Gerwert and Heinz-Jurgen Steinhoff

By means of time resolved electron paramagnetic resonance (EPR) spectroscopy the photoexcited structural changes
of site-directed spin labeled bacteriorhodopsin are studied. A complete set of cysteine mutants of the C-D loop,
positions 100 to 107, and of the E-F loop including the first alpha-helical turns of helices E and F, positions 154 to
171, was modified with a methanethiosulfonate spin label. The EPR spectral changes occurring during the photocycle
are consistent with a small movement of helix C and an outward tilt of helix F. These helix movements are
accompanied by a rearrangement of the E-F loop and of the C-terminal turn of helix E. The kinetic analysis of the
transient EPR data and the absorbance changes in the visible spectrum reveals the conformational change to occur
during the lifetime of the M intermediate. Prominent rearrangements of nitroxide side chains in the vicinity of D96 may
indicate the preparation of the reprotonation of the Schiff base. All structural changes reverse with the recovery of the
BR initial state.