Light-Induced Reaction of Halorhodopsin Prepared under Low Salt Conditions1

Ogurusu, Tarou; Maeda, Akio; Sasaki, Natsuhi; Yoshizawa, Tôru

doi:10.1093/oxfordjournals.jbchem.a133591

Cited by 39 publications

(21 citation statements)

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“…7). We have observed this phenomenon in intact membranes (8), and it has also been reported by Ogurusu et al (21). The changes at 275 nm may be caused by perturbation in the charge densities around aromatic residues or retinal (or both) or by a change in the interaction of these chromophores.…”

Section: Spectroscopy Rapid Transient Absorbance Changes Weresupporting

confidence: 82%

Purification of photochemically active halorhodopsin.

Taylor¹,

Bogomolni²,

Weber³

1983

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

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We have developed a procedure for the purification of halorhodopsin in a photochemically active state. Solubilization of membranes from a bacteriorhodopsin-negative Halobacterium strain with octyl glucoside was followed by chromatography on hydroxylapatite and octyl-Sepharose gels. All steps were carried out in high-ionic-strength solutions. The procedure resulted in 270-fold enrichment with a 35% yield. The eluted pigment had an absorption maximum at 575 nm and an A280/A575 ratio of 2. On removal of the detergent by dialysis, the purified halorhodopsin was chemically bleached, regenerated with [3H]-retinal, and reduced with cyanoborohydride. Such samples showed one main and one satellite band after staining or fluorography of NaDodSO4/polyacrylamide gels. The apparent molecular weight of the main band was 25,000. Purified halorhodopsin underwent a photocycle after excitation with pulsed laser light and showed a 9-nm blue shift (at neutral pH) on removal of chloride ion. The pigment also underwent a photoreversible shift at alkaline pH to a form absorbing maximally at 410 nm. All three reactions closely resembled those of membrane-bound halorhodopsin.Three retinal-containing pigments are synthesized by Halobacterium halobium. These membrane-bound pigments are bacteriorhodopsin (bR), halorhodopsin (hR), and the slow rhodopsin-like pigment (s-rhodopsin; sR); they have similar UV and visible absorption spectra, with maxima at 568 nm (bR), 578 nm (hR), and 587 nm (sR), and all three undergo characteristic photochemical cycles after excitation by actinic light (1-4). Whereas bR, the light-driven proton pump, converts light energy into a protonmotive force that can be harnessed by bacterial cells for ATP synthesis (for review see ref. 1), hR is probably involved in electrogenic transport of Cl-into the cells (5). The physiological significance of this light-driven process is not known with certainty. Some evidence suggests that sR participates in the phototactic responses of halobacterial cells (3).Many structural and functional studies require purification of the photoactive chromoproteins. This task is relatively simple for bR, because it is the only protein of the purple membrane that can be fractionated on sucrose gradients (6). In our available halobacteria strains, hR is not so organized and has to be purified by detergent solubilization and subsequent fractionation.Using a strain deficient in bR [determined by flash spectroscopy as described (4, 7) were suspended in 150 ml of 4 M NaCl and lysed by dialysis in the presence of 10 mg of DNase at 4°C for 18 hr against 6 liters of 50 mM Hepes (pH 7.0). The lysate was cleared of large debris (containing little or no hR) by centrifugation (6,000 x g, 15 min) and the membranes were then sedimented (260,000 x g, 1 hr). The bluish pellet was suspended in 50 mM Hepes buffer (pH 7.0). The sedimentation and resuspension of membranes was repeated twice in Hepes buffer and then once in 4 M NaCl. Finally, the membranes were stored in 4 M NaCl at 4°C at 10-20 mg/ml.Pro...

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Section: Spectroscopy Rapid Transient Absorbance Changes Weresupporting

confidence: 82%

Purification of photochemically active halorhodopsin.

Taylor¹,

Bogomolni²,

Weber³

1983

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

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“…It was concluded from this (Lanyi, 1984b) that the retinal configuration in HRSZO.C1-is cis (perhaps 13-cis as in the photointermediates of bacteriorhodopsin), rather than all-trans as in the parent molecule (Smith et al, 1984;Alshuth et al, 1985;Maeda et al, 1985). Extraction and analysis of the retinal has demonstrated the 13-cis isomeric form in illuminated halorhodopsin when the pigment was trapped in a stable form during the illumination, either as the deprotonated product (Ogurusu et al, 1981) or as the oxime.2 Flash-induced difference spectra, based on experiments similar to those in Figure 1, are shown in Figure 2. The absorption changes are given as extinction changes, calculated from the magnitudes of the flash-induced absorption changes at the indicated wavelengths and the amount of pigment that photocycled.…”

Section: Resultsmentioning

confidence: 97%

Flash spectroscopic studies of the kinetics of the halorhodopsin photocycle

Lanyi¹,

Vodyanoy²

1986

Biochemistry

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The photoreactions of halorhodopsin are complicated by the fact that the parent pigment and its photoproducts interact with chloride. Thus, in any photoreaction scheme at least four species have to be accounted for: HR565 and HR578 Cl-, as well as HR640 and HR520 Cl-. A photocycle scheme proposed earlier places the two main photointermediates of halorhodopsin, HR520 Cl- and HR640, into a single photocycle, with a chloride-dependent equilibrium between them [Oesterhelt, D., Hegemann, P., & Tittor, J. (1985) EMBO J. 4, 2351-2356]. This scheme, with the additional feature of direct photoproduction of HR640 from HR565, was tested in this work by using numerical solutions of the appropriate differential equations to simulate flash-induced absorption changes at 500 nm (production of HR520 Cl-) and at 660 nm (production of HR640). The time scale of the simulation was ms following the flash. Comparison of the simulated curves with experimental traces yielded a unique set of three rate constants. The proposed photocycle scheme and these rate constants predict well the shapes and amplitudes of flash traces at various chloride concentrations. It appears from the photocycle scheme, and the numerical values of rate constants, that chloride is bound with high affinity to the parent halorhodopsin molecule, but with much lower affinity to its main photointermediate. This may be the consequence of the fact that in the parent halorhodopsin in the retinal configuration is all-trans, but in the two photointermediates it is 13-cis.

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“…For example, removal of Cl − from pHR causes a 20-nm red shift of the visible absorption band, while sHR shows a 10-nm blue shift upon removal of Cl − . 30,31 The former absorption change is a normal reaction that could be induced upon removal of a negative charge in the active site, as indicated by previous spectroscopic studies of BR in which a 30-nm red shift was observed upon protonation of Asp85 by acidification with sulfuric acid. 32 The red-shifted species of BR generated at acidic pH, usually called the acid blue form, has been reported to mimic the O intermediate occurring in the last step of its proton pumping cycle.…”

Section: Introductionmentioning

confidence: 86%