The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A critical question has been whether EL2 forms a reversible gate that opens upon activation or acts as a rigid barrier. Distance measurements using solid-state 13C NMR spectroscopy between the retinal chromophore and the β4 strand of EL2 show the loop is displaced from the retinal binding site upon activation, and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular ends of transmembrane helices H4, H5 and H6. NMR measurements further reveal that structural changes in EL2 are coupled to the motion of helix H5 and breaking of the ionic lock that regulates activation. These results provide a comprehensive view of how retinal isomerization triggers helix motion and activation in this prototypical G protein-coupled receptor.

A central vision in molecular electronics is the creation of devices with functional molecular components that may provide unique properties. Proteins are attractive candidates for this purpose, as they have specific physical (optical, electrical) and chemical (selective binding, self-assembly) functions and offer a myriad of possibilities for (bio-)chemical modification. This Progress Report focuses on proteins as potential building components for future bioelectronic devices as they are quite efficient electronic conductors, compared with saturated organic molecules. The report addresses several questions: how general is this behavior; how does protein conduction compare with that of saturated and conjugated molecules; and what mechanisms enable efficient conduction across these large molecules? To answer these questions results of nanometer-scale and macroscopic electronic transport measurements across a range of organic molecules and proteins are compiled and analyzed, from single/few molecules to large molecular ensembles, and the influence of measurement methods on the results is considered. Generalizing, it is found that proteins conduct better than saturated molecules, and somewhat poorer than conjugated molecules. Significantly, the presence of cofactors (redox-active or conjugated) in the protein enhances their conduction, but without an obvious advantage for natural electron transfer proteins. Most likely, the conduction mechanisms are hopping (at higher temperatures) and tunneling (below ca. 150-200 K).

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.

We review the status of protein-based molecular electronics. First, we define and discuss fundamental concepts of electron transfer and transport in and across proteins and proposed mechanisms for these processes. We then describe the immobilization of proteins to solid-state surfaces in both nanoscale and macroscopic approaches, and highlight how different methodologies can alter protein electronic properties. Because immobilizing proteins while retaining biological activity is crucial to the successful development of bioelectronic devices, we discuss this process at length. We briefly discuss computational predictions and their connection to experimental results. We then summarize how the biological activity of immobilized proteins is beneficial for bioelectronic devices, and how conductance measurements can shed light on protein properties. Finally, we consider how the research to date could influence the development of future bioelectronic 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 (...

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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)

Activation of the visual pigment rhodopsin is caused by 11-cis to -trans isomerization of its retinal chromophore. High-resolution solid-state NMR measurements on both rhodopsin and the metarhodopsin II intermediate show how retinal isomerization disrupts helix interactions that lock the receptor off in the dark. We made 2D dipolar-assisted rotational resonance NMR measurements between 13 C-labels on the retinal chromophore and specific 13 C-labels on tyrosine, glycine, serine, and threonine in the retinal binding site of rhodopsin. The essential aspects of the isomerization trajectory are a large rotation of the C20 methyl group toward extracellular loop 2 and a 4-to 5-Å translation of the retinal chromophore toward transmembrane helix 5. The retinal-protein contacts observed in the active metarhodopsin II intermediate suggest a general activation mechanism for class A G proteincoupled receptors involving coupled motion of transmembrane helices 5, 6, and 7.G protein-coupled receptors (GPCRs) are a large superfamily of membrane receptors that have seven transmembrane (TM) helices and respond to a wide array of signaling ligands. Similarities in the sequences of these receptors have led to the idea that they share a common activation mechanism, whereas sequence diversity is correlated with the specificity of different receptors for different ligands and G proteins. With the exception of the visual pigment rhodopsin (1, 2), the structures of GPCRs are unknown. The rhodopsin crystal structure shows that the TM helices are locked in an inactive conformation by interhelical interactions involving conserved amino acids. The general model of GPCR activation that has emerged over the past few years is that ligand-binding, or retinal isomerization in the case of the visual pigments, disrupts these interactions and drives a rigid body movement of one or more of the TM helices (3, 4).Helix-helix interactions in rhodopsin are mediated by two sets of conserved amino acids. The highly conserved signature amino acids have sequence identities of Ͼ80% across the family of class A GPCRs. These amino acids are generally highly polar, aromatics, or proline. The group-conserved amino acids are small and͞or weakly polar (Gly, Ala, Ser, Thr, and Cys) (5). They have low individual sequence identities but are highly conserved (Ͼ80%) when considered as a group. These amino acids are involved in mediating close helix contacts and facilitating interhelical hydrogen bonding (6).The location of the group-conserved amino acids largely in the interfaces of TM helices H1-H4 of rhodopsin has suggested that these helices are locked in a stable structure that does not change significantly upon receptor activation (5). There are fewer group-conserved amino acids in H5-H7. H5 and H7 are unusual in containing conserved prolines that facilitate interactions with the H1-H4 core by exposing the i-4 backbone carbonyl for interhelical hydrogen bonding. H6 is unusual in that the TM region is composed of conserved aromatic and large hydrophobic amino acids ...