Application of Raman Spectroscopy to Retinal Proteins

Raman spectroscopic studies on photoreactive retinal proteins are comprehensively described, including the basic physics of Raman scattering and illustrative examples of the types of information on the structure and function of the retinal chromophore and its environment which can be obtained from the vibrational Raman spectra. In addition, practical advice and recipes are given which should enable the reader to plan and eventually perform a Raman experiment in a photolabile retinal protein. A dominant role is played by the resonance Raman (RR) experiment with visible laser excitation which selectively probes the retinal chromophore. Much discussion is devoted to bacteriorhodopsin (bR) and its photocycle as a paradigm for a light-induced reaction of a retinal protein. Various time-resolved techniques are described to study the temporal evolution of the bR chromophore by probing RR spectra of intermediate states. Vibrational Raman spectra are interpreted in terms of structure and structural changes of the chromophore. RR spectroscopic studies on halorhodopsin, sensory rhodopsin, and visual pigments are reported, as well as on modified proteins in which retinal analogues are incorporated, and on site-specific mutants. Results of ultraviolet RR experiments which selectively probe the aromatic side chains in the protein backbone are reported. In addition, a promising new technique of near-infrared Raman excitation is discussed. Finally, application of coherent anti-Stokes Raman spectroscopy (CARS) to retinal proteins is reported.