Vibrational Spectra of Rhodopsin and Bacteriorhodopsin

Kitagawa, Teizo; Maeda, Akio

doi:10.1111/j.1751-1097.1989.tb02918.x

Cited by 35 publications

(19 citation statements)

References 151 publications

(154 reference statements)

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“…These values are similar to those of the corresponding bands of L at 1400 and 1301 cm-I (Diller et al, 1987;Maeda, Sasaki, PfefferE, Shichida, and Yoshizawa, unpublished observations). Since H-bonding strength is related to the frequency of the N-H in-plane bending vibration (Maeda et al, 1985;Baasov et al, 1987;Kitagawa & Maeda, 1989), these results mean that the strength of H bonding for the interaction of the Schiff base with the protein of N is almost same as that of L.…”

Section: Discussionmentioning

confidence: 99%

Fourier transform infrared study of the N intermediate of bacteriorhodopsin

Pfefferlé¹,

Maeda²,

Sasaki³

et al. 1991

Biochemistry

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137

173

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Visible absorption spectroscopic experiments show that the N intermediate is the main photoproduct of a highly hydrated film of the light-adapted bacteriorhodopsin (70% water by weight) at pH 10 and 274 K. The difference Fourier transform infrared spectrum between the N intermediate and unphotolyzed light-adapted bacteriorhodopsin was recorded under these conditions. A small amount of the M intermediate present did not affect this spectrum significantly. The difference spectrum exhibited a positive band at 1755 cm-1 (probably due to Asp-85) and a negative band at 1742 cm-1 (due to Asp-96), neither of which was observed for the M intermediate. The spectrum of the N intermediate at pH 7 was nearly identical with that at pH 10. Spectra at pH 10 also were measured with isotope-substituted samples. A vibrational band at 1692 cm-1 due to the peptide bond disappeared, and a band at 1558 cm-1 emerged upon formation of the N intermediate. The spectrum also displayed bands containing the N-H and C15-H in-plane bending vibrational modes at 1394 and 1303 cm-1. These frequencies are similar to those of the L intermediate while the intensities of these bands are larger than those in the L intermediate, suggesting that the Schiff bases of both the L and N intermediates have a strong hydrogen-bonding interaction with the protein and that the C12-H to C15-H region of the chromophore is less twisted in the N intermediate than in the L intermediate.

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Section: Discussionmentioning

confidence: 99%

Fourier transform infrared study of the N intermediate of bacteriorhodopsin

Pfefferlé¹,

Maeda²,

Sasaki³

et al. 1991

Biochemistry

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137

173

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“…The difference between the C=N stretching vibration in H 2 0 and 2H 2 0 (BV H _ D ) thus must be dependent on the intrinsic frequency of the N-H in-plane bending vibration, which increases in frequency with increasing H-bonding strength of the Schiff base N-H. This value was calculated from the C=N stretching vibration frequencies in the resonance Raman spectra as 17 em:' for BR,48 24 cm-l for L,48 and 24 em:' for N. 4 9 The value of 17 ern:' is much smaller than 31 em:' for rhodopsin." being consistent with an argument for weak H-bonding on the basis of a solid-state ISN NMR study for BR.…”

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confidence: 99%

Application of FTIR Spectroscopy to the Structural Study on the Function of Bacteriorhodopsin

Maeda

1995

Israel Journal of Chemistry

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139

214

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Difference FTIR spectroscopy of the photointermediates of bacteriorhodopsin is informative for changes in H-bonding, the protonation states, and the bond orientation of functional residues such as C=O, N-H, and O-H of the chromophore, protein residues, peptide bonds, and internal water molecules. The vibrational bands of the chromophore are found at frequencies similar to those observed by resonance Raman spectra. Moreover, FTIR gives clear results on the N-H in-plane bending vibration and C 14 -C 15 stretching vibrational modes, both of which are useful for the analysis of the structure of the chromophore. The protonation states and H-bonding changes of intramembrane protonated aspartic acid residues can be revealed only by FTIR spectroscopy, in combination with proper isotope labeling and site-directed mutagenesis. The results with Asp-96 and Asp-85 in the L, M, and N photointermediates were especially useful for understanding the proton transfer mechanism. Besides amino acid groups in the protein, peptide C=O vibrations were assigned to a specific bond by the use of site-directed isotope labeling. Water molecules which undergo structural changes upon photoreaction were attributed specifically to those interacting with particular protein residues by mutational studies. On the basis of these FTIR studies, the role of the L intermediate is emphasized as the key intermediate that creates the conditions for the proton transfer reaction from the Schiff base to Asp-85 and subsequent proton uptake reactions in the cytoplasmic domain.

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“…Use of isotopically labeled retinal in resonanceRaman and FTIR experiments has shown that the absorbance bands observed between 1211 cm-' and 1164 cm-' are due to C-C stretching vibrations of the chromophore (for a recent review, see ref. 21). To account for the overlap of absorbance *175 bands of the ground state and the intermediates, the global fit analysis was also applied to the region between 1211 cm-' and 1164 cm-' by using the absorbance data for 24 wavenumbers simultaneously.…”

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Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy.

Gerwert¹,

Souvignier²,

Hess³

1990

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

275

203

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Absorbance changes in the infrared and visible spectral range were measured in parallel during the photocycle oflight-adapted bacteriorhodopsin, which is accompanied by a vectorial proton transfer. A global fit analysis yielded the same rate constants for the chromophore reactions, for protonation changes of protein side groups, and for the backbone motion. From this result we conclude that all reactions in various parts of the protein are synchronized to each other and that no independent cycles exist for different parts. The carbonyl vibration of Asp-85, indicating its protonation, appears with the same rate constant as the Schiff base deprotonation. The carbonyl vibration of Asp-96 disappears, indicating most likely its deprotonation, with the same rate constant as for the Schiff base reprotonation. This result supports the proposed mechanism in which the protonated Schiff base, a deprotonated aspartic acid (Asp-85) on the proton-release pathway, and a protonated aspartic acid (Asp-96) on the proton-uptake pathway act as internal catalytic proton-binding sites.Recently, the structure of two membrane proteins, the photosynthetic reaction center and bacteriorhodopsin (bR), have been determined at near-atomic and molecular resolution, respectively (1, 2). In order to understand the structurefunction relationship on an atomic level, time-resolving methods have to be applied to yield insight into the intramolecular reactions. Here, the light-driven proton pump bR (3) is investigated by time-resolved Fourier-transform infrared (FTIR) difference spectroscopy (4, 5).The proton-transfer reactions of bR are initiated by a light-induced isomerization reaction of the chromophore retinal. This reaction is followed by protonation changes of the protonated Schiff base binding site between chromophore and protein and internal aspartic acids of the protein (6, 7). Based on these results, it has been proposed that the interplay between the protonated Schiff base and a deprotonated and a protonated internal aspartic acid describes the principal features of the pump mechanism (5, 6). By using mutant proteins in which internal aspartic acids were altered, these two residues were identified as Asp-85 and Asp-96 by static FTIR difference spectroscopy (8, 9). Substitution of these two residues results in defective proton pumping by the mutated proteins (10, 11).To determine simultaneously the light-induced kinetics in various parts of the protein, including the protonation changes of Asp-85 and Asp-96 relative to the Schiff base, we performed time-resolved FTIR experiments and, in parallel, measured absorbance changes in the visible spectral range. MATERIALS AND METHODSPurple membrane was isolated as described (12). Wet, highly concentrated purple membrane pellets in distilled water were squeezed between two CaF2 windows, separated by a 2.5-,um spacer in a homemade sample chamber. The final OD was -0.5 and the pH -6. No salt was added. IR spectra were recorded on a Bruker IFS 88 instrument with the modifications and addition...

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Vibrational Spectra of Rhodopsin and Bacteriorhodopsin

Cited by 35 publications

References 151 publications

Fourier transform infrared study of the N intermediate of bacteriorhodopsin

Fourier transform infrared study of the N intermediate of bacteriorhodopsin

Application of FTIR Spectroscopy to the Structural Study on the Function of Bacteriorhodopsin

Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy.

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