Fourier-transform Raman spectroscopy applied to photobiological systems.

Abstract

Fluorescence and initiation of photoreactions are problems frequently encountered with resonance Raman spectroscopy of photobiological systems. These problems can be circumvented with Fourier-transform Raman spectroscopy by using the 1064-nm wavelength of a continuous wave neodymium-yttrium/aluminum-garnet laser as the probing beam. This wavelength is far from the absorption band of most pigments. Yet, the spectra of the investigated systemsbacteriorhodopsin, rhodopsin, and phycocyanin-show that these systems are still dominated by the chromophore, or that preresonant Raman scattering is still prevalent. Only for rhodopsin were contributions of the protein and the membrane discernible. The spectra of phycocyanin differ considerably from those obtained by excitation into the UV-absorption band. The results show the usefulness of this method and its wide applicability. In addition, analysis of the relative preresonant scattering cross sections may provide a detailed insight into the scattering mechanism.Resonance Raman spectroscopy is a powerful technique for selectively studying chromophores in biological systems. By tuning the probing laser beam to the electronic transition of the chromophore(s) within the biological matrix (protein, lipids, etc.), the resonance-enhancement effect increases the scattering from the chromophore by several orders of magnitude. Thus, the spectrum contains only bands caused by the chromophore. The technique has contributed considerably to our understanding of the mechanism of retinal proteins and to the investigation of chromophore-protein interaction in proteins containing heme, bilin, chlorophyll, and carotenoid chromophores (1-6). There are only a few, but in some cases severe, drawbacks to this method, which can make its application difficult. Because the probing beam excites the chromophore into a higher electronic state, strong fluorescence may override the weaker spontaneous Raman scattering, making it undetectable against the fluorescence background. In active photobiological systems, the strong probing beam also initiates photoreaction(s), and special techniques must be applied to control the state of the system under investigation. This last point may become especially severe for systems in which the photoreaction is not reversible (i.e., systems that "bleach," such as vertebrate visual pigments) or in which the back-reaction is very slow (such as dark adaptation of bacteriorhodopsin or reversion of phytochrome). By pumping the sample through a capillary jet, the accumulation of the bleached state or of long-lived intermediates can be avoided, but large amounts of material are usually required.For a long time, analyzing the Raman-scattered light by a Fourier-transform (FT) spectrophotometer, or FT-Raman spectroscopy, was thought to have disadvantages as compared with conventional Raman spectroscopy (7-9). However, about 2 years ago, it was realized that by using a near-infrared laser, such as a neodymium-yttrium/aluminum-garnet (Nd:YAG) laser, as the probing beam, t...

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

“…Wild-type spectra are for all-trans (light-adapted) and 13-cis (from a dark-adapted sample that contains 33% all-trans) chromophores. These are essentially the same as reported before (39) and shown as controls only. For the mutants spectra of dark-adapted samples are shown.…”

“…HPLC of the extracted retinal gave 40 and 55% 13-cis content for dark-adapted D85N and D85N͞D96N, respectively, but could give no information on the configuration of the C 15 AN bond (37). Recently FT-Raman (39,40) became the method of choice for determining the configuration of retinal because (i) it does not perturb the protein, (ii) unlike resonance Raman it utilizes 1.064 m excitation that has virtually no effect on the isomeric state, and (iii) it can distinguish between 13-cis,15-anti and 13-cis,15-syn. When the protonation state of the Schiff base is unchanged, the amplitudes of several vibrational modes determine the configuration of the bonds between C 13 and C 15 unambiguously.…”

Infrared spectroscopy of phytochrome and model pigments

“…Thus it appears that, for bilin pigments, the two types of resonance Raman spectra cannot be directly compared. However, the former spectrum agrees reasonably well with the Fourier-transform Raman spectrum obtained under pre-resonance conditions (Sawatzki et al, 1990).…”

“…1 a), has been studied (Margulies and Stockburger, 1979;Margulies and Toporowicz, 1984), but this pigment differs from phytochrome by having a fully unsaturated chromophore system. Another model pigment is phycocyanin, a photosynthetic antenna pigment bearing a chromophore which is very similar to the one of phytochrome, and which has been investigated by resonance Raman (Szalontai et al, 1988;Szalontai et al, 1989;Margulies and Toporowicz, 1988), pre-resonance Raman (Sawatzki et al, 1990) and resonant coherent anti-Stokes Raman spectroscopy (CARRS) (Schneider et al, 1988a, b).…”

Photochemical Reaction Cycle and Proton Transfers inNeurospora Rhodopsin

“…10 shows FT-Raman spectra of the dark-adapted states of BR and NR, containing bands nearly entirely from the chromophore (53). As judged from the relative amplitude of the 1187 cm Ϫ1 band in the fingerprint region and a shoulder in the ethylenic stretch band, both proteins are mixtures of all-trans and 13-cis retinal species (54).…”