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Journal of the Optical Society of America B

Journal of the Optical Society of America B


  • Vol. 17, Iss. 2 — Feb. 1, 2000
  • pp: 202–212

Phenomenological characterization of bacteriorhodopsin–D85N photocycle

Doğan A. Timuçin and John D. Downie  »View Author Affiliations

JOSA B, Vol. 17, Issue 2, pp. 202-212 (2000)

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An operational characterization of the molecular photocycle of a genetic variant of bacteriorhodopsin, BR–D85N, is presented. Steady-state bleach spectra and pump–probe absorbance data are obtained with thick hydrated films that contain BR–D85N embedded in a gelatin host. Simple two- and three-state models are used to analyze the photocycle dynamics and to extract relevant information such as pure-state absorption spectra, photochemical-transition quantum efficiencies, and thermal lifetimes of dominant states that appear in the photocycle, the knowledge of which should facilitate the analysis and the design of optical applications based on this photochromic medium. The remarkable characteristics of this material and their implications from the viewpoint of optical data storage and processing are discussed.

© 2000 Optical Society of America

OCIS Codes
(090.2900) Holography : Optical storage materials
(160.2900) Materials : Optical storage materials
(210.4810) Optical data storage : Optical storage-recording materials
(230.6120) Optical devices : Spatial light modulators
(260.5130) Physical optics : Photochemistry
(300.1030) Spectroscopy : Absorption

Doğan A. Timuçin and John D. Downie, "Phenomenological characterization of bacteriorhodopsin–D85N photocycle," J. Opt. Soc. Am. B 17, 202-212 (2000)

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  1. See, for instance, R. R. Birge, “Nature of the primary photochemical events in rhodopsin and bacteriorhodopsin,” Biochim. Biophys. Acta 1016, 293–327 (1990); R. R. Birge, “Photophysics and molecular electronic applications of the rhodopsins,” Annu. Rev. Phys. Chem. 41, 683–733 (1990); C. Bräuchle, N. Hampp, and D. Oesterhelt, “Optical applications of bacteriorhodopsin and its mutated variants,” Adv. Mater. ADVMEW 3, 420–428 (1991); D. Oesterhelt, C. Bräuchle, and N. Hampp, “Bacteriorhodopsin: a biological material for information processing,” Q. Rev. Biophys. QURBAW 24, 425–478 (1991); J. K. Lanyi, “Proton translocation mechanism and energetics in the light-driven pump bacteriorhodopsin,” Biochim. Biophys. Acta BBACAQ 1183, 241–261 (1993). [CrossRef] [PubMed]
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  3. See, for instance, T. Mogi, L. J. Stern, T. Marti, B. H. Chao, and H. G. Khorana, “Aspartic acid substitutions affect proton translocation by bacteriorhodopsin,” Proc. Natl. Acad. Sci. USA 85, 4148–4152 (1988); S. Subramaniam, T. Marti, and H. G. Khorana, “Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82→Ala and Asp-85→Glu: the blue form is inactive in proton translocation,” Proc. Natl. Acad. Sci. USA 87, 1013–1017 (1990); H. Otto, T. Marti, M. Holz, T. Mogi, L. J. Stern, F. Engel, H. G. Khorana, and M. P. Heyn, “Substitution of amino acids Asp-85, Asp-212, and Arg-82 in bacteriorhodopsin affects the proton release phase of the pump and the pK of the Schiff base,” Proc. Natl. Acad. Sci. USA PNASA6 87, 1018–1022 (1990). [CrossRef] [PubMed]
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  15. All absorption spectra shown in this paper were obtained with a Shimadzu UV-2501PC UV-VIS Recording Spectrophotometer.
  16. See, for instance, R. A. Mathies, C. H. Brito Cruz, W. T. Pollard, and C. V. Shank, “Direct observation of the femtosecond excited-state cis–trans isomerization in bacteriorhodopsin,” Science 240, 777–779 (1988); M. Rohr, W. Gärtner, G. Schweitzer, A. R. Holzwarth, and S. E. Braslavsky, “Quantum yields of the photochromic equilibrium between bacteriorhodopsin and its bathointermediate K. Femto- and nanosecond optoacoustic spectroscopy,” J. Phys. Chem. 96, 6055–6061 (1992); S. L. Logunov, L. Song, and M. A. El-Sayed, “pH dependence of the rate and quantum yield of the retinal photoisomerization in bacteriorhodopsin,” J. Phys. Chem. JPCHAX 98, 10674–10677 (1994); K. C. Hasson, F. Gai, and P. A. Anfinrud, “The photoisomerization of retinal in bacteriorhodopsin: experimental evidence for a three-state model,” Proc. Natl. Acad. Sci. USA PNASA6 93, 15124–15129 (1996); F. Gai, K. C. Hasson, J. C. McDonald, and P. A. Anfinrud, “Chemical dynamics in proteins: the photoisomerization of retinal in bacteriorhodopsin,” Science SCIEAS 279, 1886–1891 (1998). [CrossRef] [PubMed]
  17. Owing to the insensitivity of BR–D85N, a large pump fluence is required for bleaching the film to steady-state conditions (e.g., of the order of a few hundred J/cm2 for 633-nm excitation). Clearly, this energy can be delivered within a short time period by use of a powerful source; however, this presents the danger of denaturing the protein owing to excessive heat dissipation, 13 which would lead to a (highly undesirable) loss of photoactive material. In our experiments we therefore used relatively weak pump beams to ensure adiabatic bleaching of the material.
  18. Perhaps the most direct evidence for this would be the failure of the absorption spectra of the bleached film in forming a perfect isosbestic point, and close inspection of the 520–540-nm region in Figs. 2, 3, and 6 indeed reveals that the bleached spectra do not intersect the B-state spectrum at a single point, as they would for a truly two-state photocycle. This, however, can also be due to (1) a type of irreversibility (or fatigue) whereby erasure between exposure with different wavelengths does not return the film to the same initial state or (2) the thermal denaturation of the protein upon continuous high-power exposure. (Incidentally, the former is actually the case here, as discussed in the text.17) Therefore the lack of an isosbestic point does not by itself provide conclusive evidence for the inadequacy of the two-state model.
  19. Since ΦKB is roughly 0.1 at λ0=633 nm whereas ΦBL is nearly unity, this low value of ΦL0) indicates a strong photochemical backconversion K→B in the 13–cis cycle, which is consistent with the fact that the K state, with a spectrum centered around 640 nm, absorbs prominently in the red (Refs. 6789101112).
  20. In aqueous films with no chemical cross linking between the BR molecules and the host matrix, optically recorded information “fades” owing to molecular diffusion: BR molecules spatially arranged into different states by the recording beams subsequently migrate to attain uniform population densities throughout the film volume, causing the loss of recorded information in the process; see J. D. Downie, D. A. Timuçin, D. T. Smithey, and M. Crew, “Long holographic lifetimes in bacteriorhodopsin films,” Opt. Lett. 23, 730–732 (1998). This, of course, is of no consequence in PM (wild-type and D96N) BR films where M-state molecules quickly decay back to the B state before appreciable diffusion can take place. In the present case, however, since the sample under study is a large and uniformly bleached portion of the film (i.e., no spatial variation in the initial molecular population densities), diffusion effects cannot be responsible for the change in film absorbance observed over the time scales of interest here. [CrossRef]
  21. A. Papoulis, Probability, Random Variables, and Stochastic Processes, 3rd ed. (McGraw-Hill, New York, 1991).
  22. A. Popp, M. Wolperdinger, N. Hampp, C. Bräuchle, and D. Oesterhelt, “Photochemical conversion of the O-intermediate to 9–cis-retinal-containing products in bacteriorhodopsin films,” Biophys. J. 65, 1449–1459 (1993). [CrossRef] [PubMed]

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