Polyhedral assembly of a membrane protein in its three-dimensional crystal

Kouyama, Tsutomu; Yamamoto, Masaki; Kamiya, Nobuo; Iwasaki, Hitoshi; Ueki, Tatzuo; Sakurai, I.

doi:10.1016/0022-2836(94)90005-1

Cited by 37 publications

(30 citation statements)

References 17 publications

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“…To promote electrostatic adsorption of the vesicles, we first silanized the substrate surface with (3-aminopropyl)trimethoxysilane (APTMS) (15), followed by treatment with 0.1 M HCl, to obtain a positively charged surface. Both bR͞PC or bR͞OTG vesicle suspensions showed light-induced inward proton pumping as was found previously (13,16). Based on this observation, we postulate a preferential orientation of bR with the cytoplasmic side facing the substrate surface after vesicle fusion to form bR monolayer membranes, consistent with orientation-dependent transmembrane proton translocation (17).…”

Section: Resultssupporting

confidence: 87%

Bacteriorhodopsin (bR) as an electronic conduction medium: Current transport through bR-containing monolayers

Jin

Friedman²,

Sheves³

et al. 2006

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

157

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Studying electron transport (ET) through proteins is hampered by achieving reproducible experimental configurations, particularly electronic contacts to the proteins. The transmembrane protein bacteriorhodopsin (bR), a natural light-activated proton pump in purple membranes of Halobacterium salinarum, is well studied for biomolecular electronics because of its sturdiness over a wide range of conditions. To date, related studies of dry bR systems focused on photovoltage generation and photoconduction with multilayers, rather than on the ET ability of bR, which is understandable because ET across 5-nm-thick, apparently insulating membranes is not obvious. Here we show that electronic current passes through bR-containing artificial lipid bilayers in solid ''electrode-bilayer-electrode'' structures and that the current through the protein is more than four orders of magnitude higher than would be estimated for direct tunneling through 5-nm, water-free peptides. We find that ET occurs only if retinal or a close analogue is present in the protein. As long as the retinal can isomerize after light absorption, there is a photo-ET effect. The contribution of light-driven proton pumping to the steady-state photocurrents is negligible. Possible implications in view of the suggested early evolutionary origin of halobacteria are noted. molecular electronics ͉ vesicles ͉ bioelectronics B acteriorhodopsin (bR) is a protein-chromophore complex that serves as a light-driven proton pump in the purple membrane (PM) of Halobacterium salinarum (1). It has been shown that the protein is composed of seven transmembrane helices with a retinal chromophore covalently bound in the central region via a protonated Schiff base to a lysine residue (Fig. 1A). The PM is organized in a 2D hexagonal crystal lattice with a unit cell dimension of Ϸ6.2 nm. Electron crystallography has indicated that bR is organized into trimers in which lipids mediate intertrimer contact (2). Light absorption by bR initiates a multistep reaction cycle with several distinct spectroscopic intermediates: J 625 , K 590 , L 550 , M 412 , N 560 , and O 640 . More details on the molecular alterations that occur during the photocycle were recently obtained from x-ray diffraction studies (see ref. 3 for a recent review). The light-adapted form of bR contains only all-trans retinal, whereas the dark-adapted form contains a 1:1 mixture of 13-cis and all-trans (4). Because of its long-term stability against thermal, chemical, and photochemical degradation and its desirable photoelectric and photochromic properties, bR has attracted much interest as a material for biooptics and bioelectronics (5). Most of these efforts focused on multilayers and their photovoltage͞photocurrent generation (6-8) and photoconduction (9).In principle, PM patches (Ϸ5 nm thick, a few m in size; see Fig. 1B) can serve as a model protein material that is important for both planar junction fabrication and current transport measurements, because the 5-nm membrane is well beyond the thickness over which tu...

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Section: Resultssupporting

confidence: 87%

Bacteriorhodopsin (bR) as an electronic conduction medium: Current transport through bR-containing monolayers

Jin

Friedman²,

Sheves³

et al. 2006

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

157

View full text Add to dashboard Cite

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“…This activity agrees with literature data on such vesicles. [28] Similar inward proton-pumping activity was also detected in OTG vesicles as previously reported, [29] indicating the same orientation of bR in both kinds of vesicles.…”

Section: Full Papersupporting

confidence: 76%

“…The vesicles with high bR content were prepared by following the procedure of Kouyama and co-workers [29,60]. Briefly, after washing with 0.17 % Tween 20 in the presence of 0.16 M NaCl and 10 mM Hepes (pH 8.0) for 30 min, the PM (5 mg mL -1 ) was vesicularized by incubating at 32°C for five to seven days with 6.7 mM OTG in the presence of 1 M ammonium sulfate (AS) and 0.4 M NaCl at pH 6.4 (maintained by a phosphate buffer).…”

Section: Methodsmentioning

confidence: 99%

Bacteriorhodopsin‐Monolayer‐Based Planar Metal–Insulator–Metal Junctions via Biomimetic Vesicle Fusion: Preparation, Characterization, and Bio‐optoelectronic Characteristics

Jin

Friedman

Sheves

et al. 2007

Adv Funct Materials

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A reliable and reproducible method for preparing bacteriorhodopsin (bR)‐containing metal–biomolecule–monolayer‐metal planar junctions via vesicle fusion tactics and soft deposition of Au top electrodes is reported. Optimum monolayer and junction preparations, including contact effects, are discussed. The electron‐transport characteristics of bR‐containing membranes are studied systematically by incorporating native bR or artificial bR pigments derived from synthetic retinal analogues, into single solid‐supported lipid bilayers. Current–voltage (I–V) measurements at ambient conditions show that a single layer of such bR‐containing artificial lipid bilayers pass current in solid electrode/bilayer/solid electrode structures. The current is passed only if retinal or its analogue is present in the protein. Furthermore, the preparations show photoconductivity as long as the retinal can isomerize following light absorption. Optical characterization suggests that the junction photocurrents might be associated with a photochemically induced M‐like intermediate of bR. I–V measurements along with theoretical estimates reveal that electron transfer through the protein is over four orders of magnitude more efficient than what would be estimated for direct tunneling through 5 nm of water‐free peptides. Our results furthermore suggest that the light‐driven proton‐pumping activity of the sandwiched solid‐state bR monolayer contributes negligibly to the steady‐state light currents that are observed, and that the orientation of bR does not significantly affect the observed I–V characteristics.

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“…An interesting and unusual approach to membrane protein crystallization was proposed in 1998 [15,16]. The authors observed that purple membranes (two-dimensional hexagonal native crystals of bR) treated with the neutral detergent under certain conditions lead to the creation of spherical protein clusters (~50 nm in diameter).…”

Section: Crystallization From Vesiclesmentioning

confidence: 99%

“…The authors observed that purple membranes (two-dimensional hexagonal native crystals of bR) treated with the neutral detergent under certain conditions lead to the creation of spherical protein clusters (~50 nm in diameter). Using a standard vapor diffusion method for crystallization from bR vesicles with a high protein/lipid ratio, well diffracting hexagonal crystals were obtained [15][16][17]. This new crystal belongs to the space group P622 with unit cell dimensions of a = b = 104.7 Å and c = 114.1 Å.…”

Section: Crystallization From Vesiclesmentioning

confidence: 99%