Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale.

Terner, James; Campion, Alan; El‐Sayed, Mostafa A.

doi:10.1073/pnas.74.12.5212

Cited by 51 publications

(35 citation statements)

References 29 publications

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“…This approach was quite successful for the visual pigments rhodopsin and isorhodopsin (10). However, analogous studies of BR and its intermediates have resulted in ambiguous conclusions (21)(22)(23)(24). The ambiguity arises because the all-trans and 13-cis protonated Schiff base spectra are not sufficiently different when compared with t~picaZ protein-induced frequency and intensity changes.…”

Section: Chromophore Structure In Bacteriorhodopsinmentioning

confidence: 93%

Resonance Raman Determination of Retinal Chromophore Structure in Bacteriorhodopsin

Mathies

1984

Spectroscopy of Biological Molecules

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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.

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Section: Chromophore Structure In Bacteriorhodopsinmentioning

confidence: 93%

Resonance Raman Determination of Retinal Chromophore Structure in Bacteriorhodopsin

Mathies

1984

Spectroscopy of Biological Molecules

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“…Apparatus The resonance Raman spectrometer used is a modified version of that described previously (8,9). Laser light from a Spectra Physics model 165 argon ion laser or a model 375 dye laser (Spectra-Physics Inc., Laser Products Div., Mountain View, Calif.) was focused vertically across the sample.…”

Section: Samplesmentioning

confidence: 99%

“…The beginning resonance Raman work on bacteriorhodopsin studied the two intermediates, bR570 and bM412, which had the largest steady-state concentrations while under laser illumination (4-7). Some of the more recent resonance Raman work on bacteriorhodopsin has been what is called time-resolved or kinetic resonance Raman spectroscopy (8)(9)(10)(11). These methods are designed to obtain resonance Raman spectra of transient FIGURE I Photochemical cycle of bacteriorhodopsin as suggested by the work described in reference 3. Half times are given for room temperature.…”

Section: Introductionmentioning

confidence: 99%

“…The molecular flow technique was previously used to obtain the spectra of unphotolyzed rhodopsin and retinal isomers (12)(13)(14). At present, essentially complete spectra of bR570, bM412 and the dark-adapted bR DA form have been published (8,11,15), and features of bK590 and bL550 (7,10,15) as well as bM412 and bR570 (4)(5)(6) have been reported and discussed.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Time-resolved resonance Raman characterization of the bL550 intermediate and the two dark-adapted bRDA/560 forms of bacteriorhodopsin

Terner¹,

Hsieh²,

El‐Sayed³

1979

Biophysical Journal

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The resonance Raman spectrum of the second intermediate in the bacteriorhodopsin cycle, bL550, is obtained by a simple flow technique. The Schiff base linkage in this intermediate appears to be protonated, contrary to previous suggestion. The fingerprint region of the spectrum of bL550 does not closely match those of any presently available model Schiff bases of retinal isomers, though some comparisons can be made. The resonance Raman spectrum of dark-adapted bacteriorhodopsin is obtained and decomposed by computer subtraction of the spectrum of bR570. The remaining spectrum does not match the spectra of any model compounds presently in the literature. The spectra of bL550 and dark-adapted bRDA/560 from purple membrane in H2O are compared to those in D2O. It is found that changes in the spectrum occur in the 1,600 - 1,650 cm-1 region as well as in the 800 - 1,000 cm-1 region, but apparently not in the fingerprint region (1,100 - 1,400 cm-1). The possibilities of conformational changes of the retinal chromophore in the light adaptation process as well as the photosynthetic cycle are discussed.

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“…To overcome this problem infrared difference spectroscopy is used, because only modes that'change intensity or position during the photocycle will appear in the difference spectrum. Kinetic IR difference spectroscopy (8) cm'l, C=-C stretching vibrations (ethylenic stretch) between 1,500 and 1,620 cm-l, C-C stretches and C-C-H bends between 1,100 and 1,400 cm-' (fingerprint region), methyl rocks around 1,000 cm-1, and hydrogen out-of-plane vibrations between 750 and 1,050 cm-' (18,19,25 (5,(13)(14)(15)(16)(17)(18)(19)(20)(26)(27)(28)(29). For K, the peaks below 1,550 cm_1 agree with those found by resonance Raman (16,19,20,30); in particular the overall data for K presented in this communication are in good agreement with those obtained by Braiman and Mathies (20).…”

mentioning

confidence: 99%

Fourier transform infrared difference spectroscopy of bacteriorhodopsin and its photoproducts.

Bagley¹,

Dollinger²,

Eisenstein³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

174

138

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Fourier transform infrared difference spectroscopy has been used to obtain the vibrational modes. in the chro-*mophore and apoprotein 'that change in intensity or position between light-adapted bacteriorhodopsin and the K and M-intermediates in its photocycle and between dark-adapted and lightadapted bacteriorhodopsin. Our infrared' measurements provide independent verification of resonance Raman results that in lightadapted bacteriorhodopsin the protein-chromophore linkage is a protonated Schiff base and in the M state the Schiff base is un-,protonated. Although we cannot unambiguously identify the Schiff base stretching frequency in the K state, the most'likely interpretation of deuterium shifts of the chromophore hydrogen out-ofplane vibrations is that the Schiff base in K is protonated. The intensity of the hydrogen out-of-plane vibrations in the K state compared with the intensities of.those in light-adapted and'darkadapted bacteriorhodopsin shows that the conformation of the chromophore in K is considerably distorted. In addition, we find evidence that the conformation of the protein changes during the photocycle.Bacteriorhodopsin (bR) is the light-energy transducing protein found in the purple membrane (PM) of the extreme halophile Halobacterium halobium (1-4). The chromophore in bacteriorhodopsin is a single molecule of retinal, covalently bound to the £-amino group of a lysine (Lys-216) via a Schiffbase linkage (Fig 1). Upon absorption of light, the light-adapted form of bR (bR ) undergoes a photocycle, bRLA +--* K --L --M -O 0 bRLA, during which protons are pumped from the inside of the cell to the extracellular medium. The resulting proton gradient is used by the cell to generate chemical energy in the form ofATP and drive other energy-requiring processes. In the dark, bRLA thermally converts to the dark-adapted form of bR (bRDA).The mechanism of this light driven proton pump has been studied by using visible and ultraviolet, resonance Raman (5), and infrared (IR) (6-8) spectroscopies and chemical extraction techniques. These investigations strongly suggest that during the photocycle changes occur in both the isomeric state of the chromophore and the state ofprotonation of the Schiff'base. In particular, chemical extraction experiments have provided evidence that the chromophore in bRLA is in an all-trans configuration, that in the L and M states it is in a 13-cis configuration, and that in bRDA the chromophore exists in two isomeric forms, all-trans and 13-cis, in a ratio of approximately 1: 1 (9-11).Evidence for the conformation of the chromophore in situ comes primarily from comparisons between the resonance Raman vibrational spectra in both 'H20 and 2H20 of native bR, bR in which analogs ofretinal have been incorporated, and retinal 'Schiff bases. Analysis of the results from such work is dif-CH3 CH3

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Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale.

Cited by 51 publications

References 29 publications

Resonance Raman Determination of Retinal Chromophore Structure in Bacteriorhodopsin

Resonance Raman Determination of Retinal Chromophore Structure in Bacteriorhodopsin

Time-resolved resonance Raman characterization of the bL550 intermediate and the two dark-adapted bRDA/560 forms of bacteriorhodopsin

Fourier transform infrared difference spectroscopy of bacteriorhodopsin and its photoproducts.

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