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• Characterization of the Azide-Dependent Bacteriorhodopsin-Li...

  Characterization of the Azide-Dependent Bacteriorhodopsin-Like Photocycle of Salinarum Halorhodopsin Biophysical Journal, Volume 82, Issue 4, 1 April 2002, Pages 1687-1695 Melinda Lakatos, Géza I. Groma, Constanta Ganea, Janos K. Lanyi and György Váró Abstract The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150mM binding constant in the absence of chloride and with 250mM binding constant in the presence of 1M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13- retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all- retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the “spectrally silent” intermediates M, M, and HR′, HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M to M transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed. Abstract | Full Text | PDF (164 kb)

• Charge Motions During the Photocycle of pharaonis Halorhodop...

  Charge Motions During the Photocycle of pharaonis Halorhodopsin Biophysical Journal, Volume 78, Issue 2, 1 February 2000, Pages 959-966 Krisztina Ludmann, Grazyna Ibron, Janos K. Lanyi and György Váró Abstract Oriented gel samples were prepared from halorhodopsin-containing membranes from and their photoelectric responses to laser flash excitation were measured at different chloride concentrations. The fast component of the current signal displayed a characteristic dependency on chloride concentration, and could be interpreted as a sum of two signals that correspond to the responses at high-chloride and no-chloride, but high-sulfate, concentration. The chloride concentration-dependent transition between the two signals followed the titration curve determined earlier from spectroscopic titration. The voltage signal was very similar to that reported by another group (Kalaidzidis, I. V., Y. L. Kalaidzidis, and A. D. Kaulen. 1998. 427:59–63). The absorption kinetics, measured at four wavelengths, fit the kinetic model we had proposed earlier. The calculated time-dependent concentrations of the intermediates were used to fit the voltage signal. Although no negative electric signal was observed at high chloride concentration, the calculated electrogenicity of the K intermediate was negative, and very similar to that of bacteriorhodopsin. The late photocycle intermediates (O, HR′, and HR) had almost equal electrogenicities, explaining why no chloride-dependent time constant was identified earlier by Kalaidzidis et al. The calculated electrogenicities, and the spectroscopic information for the chloride release and uptake steps of the photocycle, suggest a mechanism for the chloride-translocation process in this pump. Abstract | Full Text | PDF (178 kb)

• Halide Dependence of the Halorhodopsin Photocycle as Measure...

  Halide Dependence of the Halorhodopsin Photocycle as Measured by Time-Resolved Infrared Spectra Biophysical Journal, Volume 80, Issue 3, 1 March 2001, Pages 1452-1465 M. Shane Hutson, Sergey V. Shilov, Richard Krebs and Mark S. Braiman Abstract Time-resolved Fourier transform infrared (FTIR) difference spectra of the halorhodopsin (hR) photocycle have been collected from 3s to 100ms in saturating concentrations of KCl or KBr. Kinetic analysis of these data revealed two decay processes, with time constants of ≃150s and ≃16ms in the presence of either halide, with describing the return to the starting (hR) state. Comparison to previous low-temperature FTIR spectra of hR intermediates confirms that characteristic hK and hL spectral features are both present before the decay, in a state previously defined as hK (Dioumaev, A., and M. Braiman. 1997. 66:755–763). However, the relative sizes of these features depend on which halide is present. In Br, the hL features are clearly more dominant than in Cl. Therefore, the state present before is probably best described as an hK/hL equilibrium, instead of a single hK state. Different halides affect the relative amounts of hK and hL present, i.e., Cl produces a much more significant back-reaction from hL to hK than does Br. The halide dependence of this back-reaction could therefore explain the halide selectivity of the halorhodopsin anion pump. Abstract | Full Text | PDF (292 kb)

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Abstract

The light-driven chloride pump, halorhodopsin, is a mixture containing all-trans and 13-cis retinal chromophores under both light and dark-adapted conditions and can exist in chloride-free and chloride-binding forms. To describe the photochemical cycle of the all-trans, chloride-binding state that is associated with the transport, and thereby initiate study of the chloride translocation mechanism, one must first dissect the contributions of these species to the measured spectral changes. We resolved the multiple photochemical reactions by determining flash-induced difference spectra and photocycle kinetics in halorhodopsin-containing membranes prepared from Halobacterium salinarium, with light- and dark-adapted samples at various chloride concentrations. The high expression of cloned halorhodopsin made it possible to do these measurements with unfractionated cell envelope membranes in which the chromophore is photostable not only in the presence of NaCl but also in the Na2SO4 solution used for reference. Careful examination of the flash-induced changes at selected wavelengths allowed separating the spectral changes into components and assigning them to the individual photocycles. According to the results, a substantial revision of the photocycle model for H. salinarium halorhodopsin, and its dependence on chloride, is required. The cycle of the all-trans chloride-binding form is described by the scheme, HR-hv-->K<==>L1<==>L2<==>N-->HR, where HR, K, L, and N designate halorhodopsin and its photointermediates. Unlike the earlier models, this is very similar to the photoreaction of bacteriorhodopsin when deprotonation of the Schiff base is prevented (e.g., at low pH or in the D85N mutant). Also unlike in the earlier models, no step in this photocycle was noticeably affected when the chloride concentration was varied between 20 mM and 2 M in an attempt to identify a chloride-binding reaction.