Bacteriorhodopsin (bR) as an electronic conduction medium: Current transport through bR-containing monolayers Yongdong Jin*, Noga Friedman*, Mordechai Sheves*†, Tao He‡, and David Cahen†‡ Departments of *Organic Chemistry and ‡Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel Edited by Mostafa A. El-Sayed, Georgia Institute of Technology, Atlanta, GA, and approved April 19, 2006 (received for review December 28, 2005) Studying electron transport (ET) through proteins is hampered by surements. Monolayers of PM patches are problematic because of achieving reproducible experimental configurations, particularly the practical difficulty in capturing and holding such patches electronic contacts to the proteins. The transmembrane protein between two electrodes and to prepare monolayers with sufficiently bacteriorhodopsin (bR), a natural light-activated proton pump in high coverage. Conducting probe atomic force microscopy (AFM) purple membranes of Halobacterium salinarum, is well studied for of a single PM patch is complicated because of the small contact biomolecular electronics because of its sturdiness over a wide area (leading to very low currents; see below) and the problem of range of conditions. To date, related studies of dry bR systems making contact reproducibly. To date, only a few reports about focused on photovoltage generation and photoconduction with current flow through PM in dry systems, namely for PM multilayers multilayers, rather than on the ET ability of bR, which is under- (9) and as patches (10), have appeared. The underlying origins or standable because ET across 5-nm-thick, apparently insulating mechanisms have not been addressed. membranes is not obvious. Here we show that electronic current We find that reconstituting bR in lipid bilayers on a solid, passes through bR-containing artificial lipid bilayers in solid ‘‘elec- electrically conducting support provides a reliable basis for repro- trode–bilayer–electrode’’ structures and that the current through ducible electronic transport measurements. We prepared such the protein is more than four orders of magnitude higher than samples by vesicle formation and subsequent fusion. To form planar would be estimated for direct tunneling through 5-nm, water-free metal–protein–metal junctions we used the ‘‘lift-off, float-on’’ peptides. We find that ET occurs only if retinal or a close analogue (LOFO) technique (11) as a ‘‘soft,’’ nondestructive way to deposit is present in the protein. As long as the retinal can isomerize after gold contacts (60 nm thick, 2.10Ϫ3 cm2) on the monolayer. The light absorption, there is a photo-ET effect. The contribution of resulting structures are sufficiently robust to allow repeated and light-driven proton pumping to the steady-state photocurrents is reproducible electron transport studies at ambient condition and negligible. Possible implications in view of the suggested early room temperature. We used monolayers of native, apo-membranes evolutionary origin of halobacteria are noted. as well as membranes with artificial bR pigments derived from synthetic retinal analogs. Structures of native all-trans retinal and BIOPHYSICS ͉ ͉ molecular electronics vesicles bioelectronics the ‘‘locked’’ analogues used in this study are shown in Scheme 1. Based on our results with the modified bR samples, we conclude acteriorhodopsin (bR) is a protein–chromophore complex that that transmembrane electron transport occurs essentially only via Bserves as a light-driven proton pump in the purple membrane bR and not via the lipid bilayer and requires the presence of retinal (PM) of Halobacterium salinarum (1). It has been shown that the or a similar ␲-electron system in the protein. protein is composed of seven transmembrane helices with a retinal chromophore covalently bound in the central region via a proton- Results and Discussion ated Schiff base to a lysine residue (Fig. 1A). The PM is organized A suspension of PM fragments containing wild-type bR was CHEMISTRY in a 2D hexagonal crystal lattice with a unit cell dimension of Ϸ6.2 prepared (12) and reconstituted with exogenous egg phosphatidyl- nm. Electron crystallography has indicated that bR is organized choline (PC) into vesicles by a modification of the method of Racker into trimers in which lipids mediate intertrimer contact (2). Light (13). To further check protein-amount-dependent current–voltage absorption by bR initiates a multistep reaction cycle with several (I–V) characteristics, another vesicular bR suspension was prepared distinct spectroscopic intermediates: J625,K590,L550,M412,N560, and by using octylthioglucoside (OTG) as a detergent (14). Monolayers O640. More details on the molecular alterations that occur during of the native bR-containing membranes were then prepared by the photocycle were recently obtained from x-ray diffraction studies adsorbing the vesicles on an Al substrate (an Ϸ50-nm-thick film of (see ref. 3 for a recent review). The light-adapted form of bR Al evaporated on quartz) with a layer of natural aluminum oxide contains only all-trans retinal, whereas the dark-adapted form on its surface (represented here as AlOx). The attached vesicles contains a 1:1 mixture of 13-cis and all-trans (4). Because of its then open and fuse, forming solid-supported lipid bilayers. To long-term stability against thermal, chemical, and photochemical promote electrostatic adsorption of the vesicles, we first silanized degradation and its desirable photoelectric and photochromic the substrate surface with (3-aminopropyl)trimethoxysilane properties, bR has attracted much interest as a material for bioop- (APTMS) (15), followed by treatment with 0.1 M HCl, to obtain a tics and bioelectronics (5). Most of these efforts focused on ͞ ͞ ͞ positively charged surface. Both bR PC or bR OTG vesicle sus- multilayers and their photovoltage photocurrent generation (6–8) pensions showed light-induced inward proton pumping as was and photoconduction (9). In principle, PM patches (Ϸ5nmthick,afew␮m in size; see Fig. 1B) can serve as a model protein material that is important for both Conflict of interest statement: No conflicts declared. planar junction fabrication and current transport measurements, This paper was submitted directly (Track II) to the PNAS office. because the 5-nm membrane is well beyond the thickness over Abbreviations: bR, bacteriorhodopsin; PM, purple membrane; AFM, atomic force micros- which tunneling through this kind of medium can be expected to be copy; LOFO, lift-off, float-on; PC, phosphatidylcholine; OTG, octylthioglucoside; APTMS, efficient. However, systematic studies of the electron transport (3-aminopropyl)trimethoxysilane; I–V, current–voltage; BO, bacterioopsin. characteristics of bR-containing monolayers (or, for that matter, of †To whom correspondence may be addressed. E-mail: [email protected] or single bR or PM units) were hampered by the difficulty to find a [email protected]. reliable, reproducible experimental system that allows such mea- © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0511234103 PNAS ͉ June 6, 2006 ͉ vol. 103 ͉ no. 23 ͉ 8601–8606 Downloaded by guest on September 23, 2021 Fig. 1. Scheme of bR chemical tailoring and of the metal–protein–metal junction preparation. (A) Schematic representation of the 3D structure of bR. The seven ␣-helical domains form a transmembrane pore. The retinylidene residue is linked to the protein moiety via a protonated Schiff base linkage to Lys-216. (B) Representative AFM image (12 ϫ 12 ␮m) of native bR patches prepared by 5 min of adsorption on an Al͞AlOx substrate derivatized with APTMS. (C) Schematic of bR-containing vesicle. (D) Schematic of a Au͞(single-bR-layer)͞(APTMS-on-AlOx)͞Al junction and measuring scheme. found previously (13, 16). Based on this observation, we postulate tion analysis shows the highest average height feature to be Ϸ5.2 a preferential orientation of bR with the cytoplasmic side facing the nm, which agrees well with the thickness of a single PM patch, substrate surface after vesicle fusion to form bR monolayer mem- indicating the formation of a bR monolayer. The few white dots branes, consistent with orientation-dependent transmembrane pro- in Fig. 2A are left-over flattened vesicles on top of the film. ton translocation (17). Denser bR monolayer-containing membranes (Fig. 2B) result Fig. 2A shows a representative AFM image of a substrate, after Ϸ20-min adsorption of the vesicles on the substrate. Fig. 2B covered by bR-containing fused vesicle membranes, prepared by also allows a fortuitous measurement of the membrane͞ 10-min adsorption on the substrate. Because it is difficult to get monolayer thickness (5.1 nm between markers) because of a an ideal monolayer (100% coverage), there are always some crack in the membrane as a result of excessive drying. Longer Ͼ sample-free cracks or pinholes (typically tens of nanometers), adsorption times ( 30 min) led to multilayer formation. Mono- between fused vesicle membranes. These cracks and pinholes are layers of membranes with modified bR were prepared by using small enough for the Au pad (0.5-mm diameter) to bridge them the same procedures. All samples for electrical transport mea- (for transport measurement) but large enough for our AFM surements were prepared by 20 min of adsorption and checked measurements to make thickness measurements possible. Sec- by AFM to assure monolayer quality. It is worth mentioning that, unlike surface-supported pure lipid bilayers,