Transient and linear dichroism studies on bacteriorhodopsin: Determination of the orientation of the 568 nm all-trans retinal chromophore

Heyn, Maarten P.; Cherry, Richard J.; Müller, Ulrike

doi:10.1016/0022-2836(77)90060-2

Cited by 186 publications

(121 citation statements)

References 33 publications

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“…The polyene chain was tilted slightly towards the extracellular side of the membrane and the polyene plane was arranged approximately perpendicular to the membrane plane. This orientation is in agreement with polarized visible [16,17], FTIR [18,19], resonance Raman [20] and neutron diffraction [21] measurements. It was assumed that the C9-and C12-methyl groups of retinal were pointed towards the cytoplasmic side of the membrane and the NH bond towards the extracellular medium in agreement with recent evidence (R. Mathies, personal communication; M. Heyn, personal communication).…”

Section: Methodssupporting

confidence: 89%

Conserved amino acids in F‐helix of bacteriorhodopsin form part of a retinal binding pocket

et al. 1989

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A 3-dimensional model for the retinal binding pocket in the light-driven proton pump, bacteriorhodopsin, is proposed on the basis of spectroscopic sfudies of bacteriorhodopsin mutants. In this model Trp-182, Pro-186 and Trp-189 surround the polyene chain while Tyr-185 is positioned close to the retinylidene Schiff base. This model is supported by sequence homologies in the F-helices of bacteriorhodopsin and the related retinal proteins, halorhodopsin and rhodopsins.

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Section: Methodssupporting

confidence: 89%

Conserved amino acids in F‐helix of bacteriorhodopsin form part of a retinal binding pocket

et al. 1989

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“…(22) (ro = 0.087 + 0.008, mean (r) = 270 us). The exponential decay time, r, of 8.3 As is ascribed to the rotation of BR monomers (19,20). The slower decay components of the signal, which were fitted by the distribution of exponentials, reflect a second, slower (4T-) exponential decay function with reduced (=50%) amplitude expected for the BR monomer (19) as well as the tumbling of the whole vesicles with different sizes within the aqueous phase.…”

Section: Resultsmentioning

confidence: 91%

“…The aggregation state of the BR population incorporated into the vesicular membranes was assayed by measuring the rotational diffusion of the protein within the lipid bilayer (18)(19)(20) and the excitonic coupling of the chromophores in the visible circular dichroism (CD) spectra (21). Rotational Diffusion.…”

Section: Resultsmentioning

confidence: 99%

Monomeric and aggregated bacteriorhodopsin: Single-turnover proton transport stoichiometry and photochemistry

Grzesiek

Dencher

1988

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

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The question of the basic functional transport unit of bacteriorhodopsin (BR) has been addressed by comparing the proton pumping stoichiometry as well as the photocycle kinetics of monomeric and aggregated BR in phospholipid vesicles. When time-resolved laser spectroscopy was used in combination with the optical pH-indicator pyranine, single-turnover experiments revealed approximately 0.5-0.8 and 0.8-1.2 protons vectorially translocated per photocycling monomeric and aggregated BR molecule, respectively. Since both these values are akin and very similar to the pumping stoichiometry of crystalline BR molecules in the purple membrane, the BR monomer has been proven to be the essential transport unit. The natural arrangement of the photopigments in a crystalline array of immobilized trimers is not required for efficient vectorial proton translocation.The light-energy-converting membrane protein bacteriorhodopsin (BR), which has a covalently bound retinal as chromophore (1), has gained special attention due to its relative simplicity. However, the molecular mechanism by which, during the complex photochemical cycle, one proton per BR molecule (2-6) is vectorially translocated across the membrane is far from understood. Interpretation of measured functional data is hampered by the extraordinary organization of the photopigment in the cytoplasmic membrane of the cell. As the only protein of the so-called purple membrane (PM), it is arranged in a two-dimensional hexagonal lattice of protein trimers (7,8) which are separated from neighboring trimer clusters by only one shell of lipids (9). To interpret various experimental results with the aim of constructing a working model for BR's proton-transport mechanism, the basic functional unit of this light-driven proton pump has to be defined; the functional unit could be the monomer, the trimer, or even the lattice.BR monomers do exhibit vectorial H+-transport activity (10-13). However, the important question whether or not monomeric BR has the same efficiency as the aggregate is controversial (12)(13)(14) To accelerate the relaxation of a pH gradient and a membrane potential generated after a single flash or after light adaptation, the vesicles were incubated with 1 ,M of the ion channel gramicidin above the phase transition temperature of the lipids for 10 min and thereafter sonicated for 20 s to improve the incorporation. The sample was then divided into two 1-ml aliquots, one was incubated additionally with 50 ,M pyranine in the extravesicular medium, and both were degassed at 0.1 torr (13 Pa) for 3 min. After pH adjustment (pH 7.0) at the temperature of the flash-spectroscopic experiments, the measurements were carried out at once. The buffer capacity of the samples containing pyranine was determined by titration with KOH and HCl in the range of pH 6.7-7.3. Flash Spectroscopy. The flash spectrometer (excitation wavelength Aex = 580 ± 20 nm; 1 mJ; flash duration 5-10 ns) equipped with a logarithmic time base has been described elsewhere (6). Before the flash and ...

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“…The optical density of the films of Ϸ2,000 membrane layers (thickness Ϸ 10 m) is Ϸ2 at the 565-nm visible absorption maximum. In these films, the retinal chromophores are oriented at Ϸ28°with respect to the plane (35)(36)(37), and the twodimensional orientation in the plane is random.…”

Section: Methodsmentioning

confidence: 99%

Resonant optical rectification in bacteriorhodopsin

Groma

Colonna

Lambry

et al. 2004

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

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The relative role of retinal isomerization and microscopic polarization in the phototransduction process of bacteriorhodopsin is still an open question. It is known that both processes occur on an ultrafast time scale. The retinal trans3cis photoisomerization takes place on the time scale of a few hundred femtoseconds. On the other hand, it has been proposed that the primary lightinduced event is a sudden polarization of the retinal environment, although there is no direct experimental evidence for femtosecond charge displacements, because photovoltaic techniques cannot be used to detect charge movements faster than picoseconds. Making use of the known high second-order susceptibility (2) of retinal in proteins, we have used a nonlinear technique, interferometric detection of coherent infrared emission, to study macroscopically oriented bacteriorhodopsin-containing purple membranes. We report and characterize impulsive macroscopic polarization of these films by optical rectification of an 11-fs visible light pulse in resonance with the optical transition. This finding provides direct evidence for charge separation as a precursor event for subsequent functional processes. A simple two-level model incorporating the resonant second-order optical properties of retinal, which are known to be a requirement for functioning of bacteriorhodopsin, is used to describe the observations. In addition to the electronic response, long-lived infrared emission at specific frequencies was observed, reflecting charge movements associated with vibrational motions. The simultaneous and phase-sensitive observation of both the electronic and vibrational signals opens the way to study the transduction of the initial polarization into structural dynamics.R etinal proteins play an essential role in a broad range of light-driven biological processes, including vision (1), energy transduction (2), and circadian control (3). All these functions involve both the conversion of light energy into charge separation and retinal isomerization, but the interplay of these processes is the subject of intense debate. The retinal protein of which the initial photochemistry is most extensively studied is the photosynthetic protein bacteriorhodopsin (bR). This protein colors the purple membrane of halobacteria and acts as a light-driven proton pump by means of a multistep process termed the photocycle (2). In the traditional model for the initial transduction step in this cycle is directly light-driven trans3cis isomerization of retinal (4-6); this model has been recently extended by including excited-state skeletal stretching (5, 7). However, experiments with modified bR containing nonisomerizable retinal analogs challenged this model by showing that the initial photo-induced events are not associated with retinal isomerization (8-10). In fact, in an early alternative hypothesis (11), the essential process was proposed to be dielectric relaxation of the protein as a response to sudden polarization upon retinal excitation (11-13). Recent molecular dynamic...

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Transient and linear dichroism studies on bacteriorhodopsin: Determination of the orientation of the 568 nm all-trans retinal chromophore

Cited by 186 publications

References 33 publications

Conserved amino acids in F‐helix of bacteriorhodopsin form part of a retinal binding pocket

Conserved amino acids in F‐helix of bacteriorhodopsin form part of a retinal binding pocket

Monomeric and aggregated bacteriorhodopsin: Single-turnover proton transport stoichiometry and photochemistry

Resonant optical rectification in bacteriorhodopsin

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