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• Two-Photon Absorption of Bacteriorhodopsin: Formation of a R...

  Two-Photon Absorption of Bacteriorhodopsin: Formation of a Red-Shifted Thermally Stable Photoproduct F620 Biophysical Journal, Volume 89, Issue 2, 1 August 2005, Pages 1175-1182 Thorsten Fischer and Norbert A. Hampp Abstract By means of high-intensity 532nm laser pulses, a photochemical conversion of the initial B state of bacteriorhodopsin (BR) to a stable photoproduct absorbing maximally at ≈620nm in BR suspensions and at ≈610nm in BR films is induced. This state, which we named F, is photochemically further converted to a group of three products with maximal absorptions in the wavelength range from 340nm to 380nm, which show identical spectral properties to the so-called P state reported in the literature. The photoconversion from B to F is most likely a resonant two-photon absorption induced step. The formation of F and P leads to a distinguished photo-induced permanent optical anisotropy in BR films. The spectral dependence of the photo-induced anisotropy and the anisotropy orientations at the educt (B) and product (F) wavelengths are strong indicators that F is formed in a direct photochemical step from B The chemical nature of the P products probably is that of a retro-retinal containing BR, but the structural characteristics of the F state are still unclear. The photo-induced permanent anisotropy induced by short laser pulses in BR films helps to better understand the photochemical pathways related to this transition, and it is interesting in view of potential applications as this feature is the molecular basis for permanent optical data storage using BR films. Abstract | Full Text | PDF (189 kb)

• Photochemistry in Dried Polymer Films Incorporating the Deio...

  Photochemistry in Dried Polymer Films Incorporating the Deionized Blue Membrane Form of Bacteriorhodopsin Biophysical Journal, Volume 75, Issue 4, 1 October 1998, Pages 1619-1634 Jack R. Tallent, Jeffrey A. Stuart, Q. Wang Song, Edward J. Schmidt, Charles H. Martin and Robert R. Birge Abstract The preparation and photochemical properties of dried deionized blue membrane (dIbR; ≈600nm, ϵ≈54, 760cm M, f≈1.1) in polyvinyl alcohol films are studied. Reversible photoconversion from dIbR to the pink membrane (dIbR; ≈485nm) is shown to occur in these films under conditions of strong 647-nm laser irradiation. The pink membrane analog, dIbR, has a molar extinction coefficient of ∼39,000cm M (f≈1.2). The ratio of pink→blue and blue→pink quantum efficiencies is 33±5. We observe an additional blue-shifted species (dIbR, ≈455nm) with a very low oscillator strength (f≈0.6, ϵ≈26,000cm M). This species is the product of fast thermal decay of dIbR. Molecular modeling indicates that charge/charge and charge/dipole interactions introduced by the protonation of ASP are responsible for lowering the excited-state all-→9- barrier to ∼6kcal mol while increasing the corresponding all-→13- barrier to ∼4kcal mol. Photochemical formation of both 9- and 13- photoproducts are now competitive, as is observed experimentally. We suggest that dIbR may be a 9-, 10-s-distorted species that partially divides the chromophore into two localized conjugated segments with a concomitant blue shift and decreased oscillator strength of the absorption band. Abstract | Full Text | PDF (857 kb)

• The M Intermediate of Pharaonis Phoborhodopsin Is Photoactiv...

  The M Intermediate of Pharaonis Phoborhodopsin Is Photoactive Biophysical Journal, Volume 78, Issue 6, 1 June 2000, Pages 3150-3159 Sergei P. Balashov, Masato Sumi and Naoki Kamo Abstract The retinal protein phoborhodopsin (pR) (also called sensory rhodopsin II) is a specialized photoreceptor pigment used for negative phototaxis in halobacteria. Upon absorption of light, the pigment is transformed into a short-wavelength intermediate, M, that most likely is the signaling state (or its precursor) that triggers the motility response of the cell. The M intermediate thermally decays into the initial pigment, completing the cycle of transformations. In this study we attempted to determine whether M can be converted into the initial state by light. The M intermediate was trapped by the illumination of a water glycerol suspension of phoborhodopsin from called phoborhodopsin (pR) with yellow light (>450nm) at −50°C. The M intermediate absorbing at 390nm is stable in the dark at this temperature. We found, however, that M is converted into the initial (or spectrally similar) state with an absorption maximum at 501nm upon illumination with 380-nm light at −60°C. The reversible transformations pR ⇔ M are accompanied by the perturbation of tryptophan(s) and probably tyrosine(s) residues, as reflected by changes in the UV absorption band. Illumination at lower temperature (−160°C) reveals two intermediates in the photoconversion of M, which we termed M′ (or M′) and pR′ (or pR′). A third photoproduct, pR′, is formed at −110°C during thermal transformations of M′ and pR′. The absorption spectrum of M′ (maximum at 404nm) consists of distinct vibronic bands at 362, 382, 404, and 420nm that are different from the vibronic bands of M at 348, 368, 390, and 415nm. pR′ has an absorption band that is shifted to shorter wavelengths by 5nm compared to the initial pR, whereas pR′ is redshifted by at least 3nm. As in bacteriorhodopsin, photoexcitation of the M intermediate of pR and, presumably, photoisomerization of the chromophore during the M → M′ transition result in a dramatic increase in the proton affinity of the Schiff base, followed by its reprotonation during the M′ → pR′ transition. Because the latter reaction occurs at very low temperature, the proton is most likely taken from the counterion (Asp) rather than from the bulk. The phototransformation of M reveals a certain heterogeneity of the pigment, which probably reflects different populations of M or its photoproduct M′. Photoconversion of the M intermediate provides a possible pathway for photoreception in halobacteria and a useful tool for studying the mechanisms of signal transduction by phoborhodopsin (sensory rhodopsin II). Abstract | Full Text | PDF (183 kb)

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Abstract

The quantum yield of proton release by bacteriorhodopsin was measured from volume changes after excitation of purple membrane fragments by short flashes. At low ionic strengths, about 0.25 mol of protons is released per einstein absorbed. This agrees well with quantum yields reported recently for the conversion of bacteriorhodopsin into a metastable state (M) that absorbs near 412 nm. However, the quantum yield of proton release increases gradually with increasing ionic strength; it plateaus with a value of 0.43 +/- 0.03 at ionic strengths above 200 mM. Changing the ionic strength has no detectable effect on the quantum yield of formation of the M spectral state. It thus appears that as many as two protons can be released and rebound in each photochemical cycle at high ionic strengths. The quantum yield of proton release is essentially independent of pH over the range 6.0–8.75. The quantum yield decreases with increasing flash strength, apparently due to photoreversal of the initial photochemical reaction.