Resonance Raman Determination of Retinal Chromophore Structure in Bacteriorhodopsin

Abstract

Resonance Raman scattering is a versatile technique for studying the mechanism of proton-pumping in bacteriorhodopsin. This method is particularly valuable when spectra of isotopic derivatives of retinal are used to assign the vibrational features.Isotopic substitution provides a basis for the interpretation of the spectra and the in situ determination of chromophore structure. To accomplish this it is important to obtain reliable spectra and to understand the bases behind the spectral interpretations. First, current techniques for obtaining highquality spectra of bacteriorhodopsin and its intermediates are summarized, and their relative advantages and disadvantages are discussed. Then the vibrational structure of-all-trans and 13-cis retinal chromophores is outlined in light of the recent normal coordinate analyses on these isomers. With this foundation, the spectra of light-adapted bacteriorhodopsin, dark-adapted bacteriorhodopsin, K610, M412, and 0640 are presented. The "isotopic fingerprint" method is used to examine the geometric configuration of retinal in each intermediate. Finally, a model summarizing current knowledge about retinal structural changes during proton pumping in bacteriorhodopsin is presented.

Methyl Substituents at the 11 or 12 Position of Retinal Profoundly and Differentially Affect Photochemistry and Signalling Activity of Rhodopsin

“…The downshift in frequency of the C = C stretch vibration around 1550 cm − 1 accompanying the rhodopsin to Batho transition ( Figure 2, upper trace) reflects this red-shift of the electronic absorbance band. 8,47 There is no clear shift in the C = C band pattern of the 11-methyl difference spectrum ( Figure 2, middle trace), which would agree with only minor changes in spectral properties between 11-methylrhodopsin and its primary photoproduct. So far, we could not obtain reproducible spectral data by UV/vis cryospectroscopy to confirm this interpretation.…”

“…We assign this signal tentatively to the 12-methyl wag vibration. 8,47 This may have obtained stronger IR-activity as a result of the out-ofplane movement of the 12-methyl group upon isomerization, and closer interaction with the protein environment, in particular Trp265. 9,11,63 The C = C signal around 1550 cm − 1 shows a bilobal shape similar to that of the native state, suggesting that, like the native Batho, the 12-methyl photoproduct has a red-shifted absorbance band.…”

“…The peaks in the region 1800-1550 cm − 1 represent changes in the protein, while below 1550 cm − 1 the major changes relate to the chromophore. 30,36,[46][47][48] For the native state, most of the bands in the difference spectrum can be attributed to specific vibrations in the retinylidene backbone. Most prominent are C = C and C = N stretches in the range 1500-1600 cm − 1 , C-C stretches in the range 1100-1300 cm − 1 , and wag vibrations below 1050 cm − 1 .…”

A Resonance Raman Study Of the C=C Stretch Modes in Bovine and Octopus Visual Pigments with Isotopically Labeled Retinal Chromophores

“…(e) The ethylenic modes of both bovine and octopus isorhodopsins are delocalized. It confirms that the spectra of bovine isorhodopsin is analogous to that of 9-cis-protonated Schiff base retinal (16). It also confirms the similar shifting pattern upon isotopic substitution.…”

Applications

“…46 Optical micrograph of a cholesterol-bilirubin gallstone. Since Raman spectroscopic studies on them have been reviewed extensively, only the references are cited: hydroporphyrins and chlorophylls(84,85), flavins(86)(87)(88), iron-sulfur proteins(89,90), metaltyrosinate proteins(91), visual pigments and bacterial rhodopsin(92)(93)(94), photosynthesis(95), viruses and nucelproteins(96,97), membranes(98,99) enzyme-substrate reactions(100)(101)(102), and medical applications(103). cholesterol-bilirubin gallstone by using the MOLE and FT-IR.Figure 4-46 is a micrograph showing its layered structure.…”