Time-resolved ultraviolet resonance Raman of protein structural changes in the KL-intermediate of bacteriorhodopsin

Abstract

To obtain high quality time-resolved ultraviolet resonance Raman (UVRR) spectra of transient intermediates in the bacteriorhodopsin (BR) photocycle, we developed a new UVRR spectrometer. A home-made F = 3.5 prism prefilter was used in front of a 50 cm CCD detected spectrograph to give high throughput, wide tunability, and excellent stray light rejection along with low dispersion. Using this system, we obtained 239.5 nm excited time-resolved UVRR spectra of BR which revealed small but significant features associated with the formation of the KL-intermediate at 10 ns delays. This difference spectrum exhibits intensity decreases at 1624, 1561, 1012 and 763 cm-1 due to an altered environment of one or more Trp residues and a frequency shift of the Tyr ν8b band at 1602 cm-1. These signals show that the photoisomerization of retinal from all-trans to 13-cis induces significant changes in the structure and environment of aromatic residues that line the retinal binding pocket in only 10 ns. © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group.