Electron transfer (ET) through proteins, a fundamental element of many biochemical reactions, is studied intensively in aqueous solutions. Over the past decade, attempts were made to integrate proteins into solid-state junctions in order to study their electronic conductance properties. Most such studies to date were conducted with one or very few molecules in the junction, using scanning probe techniques. Here we present the high-yield, reproducible preparation of large-area monolayer junctions, assembled on a Si platform, of proteins of three different families: azurin (Az), a blue-copper ET protein, bacteriorhodopsin (bR), a membrane protein-chromophore complex with a proton pumping function, and bovine serum albumin (BSA). We achieve highly reproducible electrical current measurements with these three types of monolayers using appropriate top electrodes. Notably, the current-voltage (I-V) measurements on such junctions show relatively minor differences between Az and bR, even though the latter lacks any known ET function. Electron Transport (ETp) across both Az and bR is much more efficient than across BSA, but even for the latter the measured currents are higher than those through a monolayer of organic, C18 alkyl chains that is about half as wide, therefore suggesting transport mechanism(s) different from the often considered coherent mechanism. Our results show that the employed proteins maintain their conformation under these conditions. The relatively efficient ETp through these proteins opens up possibilities for using such biomolecules as current-carrying elements in solid-state electronic devices.

Spin-dependent photoelectron transmission and spin-dependent electrochemical studies were conducted on purple membrane containing bacteriorhodopsin (bR) deposited on gold, aluminum/ aluminum-oxide, and nickel substrates. The result indicates spin selectivity in electron transmission through the membrane. Although the chiral bR occupies only about 10% of the volume of the membrane, the spin polarization found is on the order of 15%. The electrochemical studies indicate a strong dependence of the conduction on the protein's structure. Denaturation of the protein causes a sharp drop in the conduction through the membrane.electron transfer | electrochemistry | magnetic effect | chirality T he role of the electron spin in chemistry and biology has been receiving much attention because of a plausible relation to electromagnetic field effects on living organisms (1), and due to the seemingly importance of the earth's magnetic field on birds and fish navigation (2). Part of the difficulty in studying the subject arises from the lack of a physical model that can rationalize these phenomena. Recently, the chiral-induced spin selectivity (CISS) effect was observed in electron transmission and conduction through organic molecules (3). The spin selectivity was observed for photoelectron transmission through monolayers of double-stranded DNA adsorbed on gold (4). Another study discovered a spin dependence in the conduction through single molecules of double-stranded DNA. In this configuration, one end of the molecule was adsorbed on a Ni substrate, whereas the other was attached to a gold nanoparticle (5).The CISS effect may provide a novel approach for better understanding the role of electron spin in biological systems. The studies mentioned above led to several questions, including the actual role played by the gold substrate in the overall spinfiltering process. Gold exhibits a very large spin orbit coupling; hence, one may wonder whether gold itself affects the CISS phenomenon. In addition, the interface between gold and the thiol group, through which the molecules are attached to the gold, may play a role. Because many of the past studies were performed with DNA, an important question arises whether CISS is a general effect or possibly a special property of DNA. CISS was only observed for double-stranded DNA, whereas for single-stranded molecules, no spin selectivity was found. On the one hand, this was attributed to the lack of ordered monolayers (4, 6); on the other hand, a theoretical model, proposed to rationalize the CISS effect, predicted that a double-helix structure (7) was needed for CISS to occur, whereas other approaches do not emphasize this need (8). Finally, because many of the past studies were performed in vacuum or in ambient air, it is of importance to probe to what extent the effect persists in solutions, which are more relevant to biology. The present study aims at answering the above questions in an attempt to establish CISS as a general phenomenon.For the present study, we chose bacteriorhodopsin (...

Factors affecting the C = N stretching frequency of protonated retinal Schiff base (RSBH+) were studied with a series of synthetic chromophores and measured under different conditions. Interaction of RSBH+ with nonconjugated positive charges in the vicinity of the ring moiety or a planar polyene conformation (in contrast to the twisted retinal conformation in solution) shifted the absorption maxima but did not affect the C = N stretching frequency. The latter, however, was affected by environmental perturbations in the vicinity of the Schiff base linkage. Diminished ion pairing (i.e., of the positively charged nitrogen to its anion) achieved either by substituting a more bulky counteranion or by designing models with a homoconjugation effect lowered the C = N stretch energy. Decreasing solvation of the positively charged nitrogen leads to a similar trend. These effects in the vicinity of the Schiff base linkage also perturb the deuterium isotope effect observed upon deuteriation of the Schiff base. The results are interpreted by considering the mixing of the C = N stretching and C = N-H bending vibration. The C = N mode is shifted due to electrostatic interaction with nonconjugated positive charges in the vicinity of the Schiff base linkage, an interaction that does not influence the isotope effect. Weak hydrogen bonding between the Schiff base linkage in bacteriorhodopsin (bR) and its counteranion or, alternatively, poor solvation of the positively charged Schiff base nitrogen can account for the C = N stretching frequency of 1640 cm-1 and the deuterium isotope effect of 17 cm-1 observed in this pigment.(ABSTRACT TRUNCATED AT 250 WORDS)

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...

Sub-10-fs laser pulses are used to impulsively photoexcite bacteriorhodopsin (BR) suspensions and probe the evolution of the resulting vibrational wave packets. Fourier analysis of the spectral modulations induced by transform-limited as well as linearly chirped excitation pulses allows the delineation of excited- and ground-state contributions to the data. On the basis of amplitude and phase variations of the modulations as a function of the dispersed probe wavelength, periodic modulations in absorption above 540 nm are assigned to ground-state vibrational coherences induced by resonance impulsive Raman spectral activity (RISRS). Probing at wavelengths below 540 nm-the red edge of the intense excited-state absorption band-uncovers new vibrational features which are accordingly assigned to wave packet motions along bound coordinates on the short-lived reactive electronic surface. They consist of high- and low-frequency shoulders adjacent to the strong C=C stretching and methyl rock modes, respectively, which have ground-state frequencies of 1008 and 1530 cm-1. Brief activity centered at approximately 900 cm-1, which is characteristic of ground-state HOOP modes, and strong modulations in the torsional frequency range appear as well. Possible assignments of the bands and their implication to photoinduced reaction dynamics in BR are discussed. Reasons for the absence of similar signatures in the pump-probe spectral modulations at longer probing wavelengths are considered as well.

New information concerning the photochemical dynamics of bacteriorhodopsin (BR) is obtained by impulsively stimulating emission from the reactive fluorescent state. Depletion of the excited-state fluorescence leads to an equal reduction in production of later photoproducts. Accordingly, chromophores which are forced back to the ground state via emission do not continue on in the photocycle, conclusively demonstrating that the fluorescent state is a photocycle intermediate. The insensitivity of depletion dynamics to the "dump" pulse timing, throughout the fluorescent states lifetime, and the biological inactivity of the dumped population suggest that the fluorescent-state structure is constant, well-defined, and significantly different than that where crossing to the ground state takes place naturally. In conjunction with conclusions from comparing the photophysics of BR with those of synthetic analogues containing "locked" retinals, present results show that large-amplitude torsion around C13=C14 is required to go between the above structures.

The primary light-induced events in the photosynthetic retinal protein bacteriorhodopsin (bR) are investigated by ultrafast optical spectroscopy over the 440-1000 nm spectral range. The study compares the early dynamics of the native all-trans pigment bR 570 with those of two synthetic analogues, bR5.12 and bR5.13, in which isomerization around the critical C 13 dC 14 bond is blocked by a five-membered ring into all-trans and 13-cis configurations, respectively. Nearly identical spectral evolution is observed in both native and artificial systems over the first 100-200 fs of probe delay. During this period stimulated near-IR (∼900 nm) emission, and intense ∼460 nm absorption bands, due to analogous fluorescent I states (denoted as I 460 , I5.12 and I5.13, respectively), appear concurrently within 30 fs. In all systems continuous spectral shifting over tens of femtoseconds is observed in the 500-700 nm range. Native bR goes on to produce the J 625 absorption band within ∼1 ps, which is accompanied by disappearance of the I 460 emission and absorption features. In bR5.12 and bR5.13, aside from minor spectral modifications, the analogous dramatic changes associated with I5.12 and I5.13 are arrested beyond the first ∼100 fs, reverting uniformly to the initial ground state with exponential time constants of 19 ps and 11 ps, respectively. Analysis of the data calls for a major revision of models previously put forward for the primary events in bacteriorhodopsin. The close likeness of initial transient spectral evolution in both native and artificial pigments, despite the locking of the active isomerization coordinate in the synthetic chromophores, demonstrates that in bR 570 the ultrafast changes in transmission leading to I 460 , previously believed to involve C 13 dC 14 torsion, must be associated with other modes. The detailed comparison conducted here also identifies which of the later spectral changes in the native system requires torsional flexibility in C 13 dC 14. Similarity of 660 nm probing data in both synthetic and native chromophores demonstrates that the sub-picosecond dynamic features uncovered at this probing wavelength commonly attributed to the evolution of J 625 , are not, as previously thought, reliable measures of all-trans S 13-cis isomerization dynamics.

Interfacing functional proteins with solid supports for device applications is a promising route to possible applications in bio-electronics, -sensors, and -optics. Various possible applications of bacteriorhodopsin (bR) have been explored and reviewed since the discovery of bR. This tutorial review discusses bR as a medium for biomolecular optoelectronics, emphasizing ways in which it can be interfaced, especially as a thin film, solid-state current-carrying electronic element.