Time-resolved detection of transient movement of helices F and G in
doubly spin-labeled bacteriorhodopsin.

Radzwill N, Gerwert K, Steinhoff HJ.

Lehrstuhl fur Biophysik, Ruhr-Universitat Bochum, D-44780 Bochum, Germany.

Photo-excited structural changes of the light-driven proton pump bacteriorhodopsin were
monitored using double-site-directed spin labeling combined with electron paramagnetic
resonance (EPR) spectroscopy. The inter-spin distances between nitroxides attached at residue
positions 100 and 226, 101 and 160, and 101 and 168 were determined for the BR initial state
and the trapped M photo-intermediate. Distance changes that occur during the photocycle were
followed with millisecond time resolution under physiological conditions at 293 K. The kinetic
analysis of the EPR data and comparison with the absorbance changes in the visible spectrum
reveal an outward movement of helix F during the late M intermediate and a subsequent
approach of helix G toward the proton channel. The displacements of the cytoplasmic moieties
of these helices amount to 0.1-0.2 nm. We propose that the resulting opening of the proton
channel decreases the pK of the proton donor D96 and facilitates proton transfer to the Schiff
base during the M-to-N transition.
Biophys J 2001 Jun;80(6):2856-66