Application of Raman Spectroscopy to Retinal Proteins

Abstract

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 chromophoreand 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 selelctively probes the retinal chromophore. Much discussion is devoted to bacteriorhodopsin (bR) and its photocycle as a paradigm for a light-inducedreaction of a retinal protein. Various time-resolved techniques are described to study the temporal evolution of the bR chromophore by probing RR spectra of intermediatestates. VibrationalRaman 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 modifiedproteins in which retinal analoguesare incorporated,and on sitespecific 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.

Functional characterization of sensory rhodopsin II from Halobacterium salinarum expressed in Escherichia coli

“…The high frequency of this band correlates well to the absorption maximum of 490 nm in the visible range [25]. The side band at 1582 cm À1 may be attributed to a higher energetic C‚C mode (presumably C 13 ‚C 14 stretch [26]). The fingerprint region ($1100-1300 cm À1 ) where predominantly the C-C stretches absorb is very sensitive to the conformation of the retinal.…”

“…The sensitivity of this band towards deuteration of the Schiff base nitrogen argues for the C‚N-H bond in syn-configuration in the 13-cis isomer [28] which is characteristic for the thermally isomerized 13-cis state. Finally, the strong band at 1009 cm À1 can be assigned to the in-plane rocking mode of the two methyl groups of the retinal chain and the weak bands at 882 and 826 cm À1 are due to HOOP (hydrogen-outof-plane) modes [26].…”

A local electrostatic change is the cause of the large-scale protein conformation shift in bacteriorhodopsin

“…The C 14 OC 15 stretch band at 1168 cm Ϫ1 is prominent in all-trans but weak in 13-cis, while the opposite is true for the C 10 OC 11 band at 1182 cm Ϫ1 . For reasons of steric constraint in the binding pocket, the C 14 hydrogen-out-of-plane band at 800 cm Ϫ1 is strong in 13-cis,15-syn retinal but has negligible amplitude in all-trans or 13-cis,15-anti (41)(42)(43). Fig.…”

Time‐resolved resonance Raman spectroscopy of sensory rhodopsin II in the micro‐ and millisecond time range using gated cw excitation

“…This technique has substantially contributed to a better understanding of the relationships between structure, dynamics, and function, specifically of biological photoreceptors. 1,2 In time-resolved RR experiments, the spectrum is probed after a delay time following initiation of the photoinduced processes by a pump laser. On the one hand, time-resolution of such pump-probe RR experiments may be established through the excitation with pulsed pump and probe lasers.…”

“…2 Processes on the micro-and millisecond time scale, however, can be studied with continuous wave (cw) lasers, which allow milder excitation conditions such that the risk of unwanted photoinduced processes is reduced. 1 In this case, time-resolution is achieved by moving the sample with a well-defined velocity subsequently through the pump and the probe laser beams such that their spatial separation defines the delay time between the pump and the probe event.…”