Biomimetic Membranes for Multi-Redox Center Proteins.

His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs). An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA) terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM). MRPs, such as the cytochrome c oxidase (CcO) from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs) from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced resonance Raman spectroscopy (SERRS), were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs). The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM) and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

Silica nanoparticles for the oriented encapsulation of membrane proteins into artificial bilayer lipid membranes.

An artificial bilayer lipid membrane system is presented, featuring the oriented encapsulation of membrane proteins in a functionally active form. Nickel nitrilo-triacetic acid-functionalized silica nanoparticles, of a diameter of around 25 nm, are used to attach the proteins via a genetically engineered histidine tag in a uniform orientation. Subsequently, the proteins are reconstituted within a phospholipid bilayer, formed around the particles by in situ dialysis to form so-called proteo-lipobeads (PLBs). With a final size of about 50 nm, the PLBs can be employed for UV/vis spectroscopy studies, particularly of multiredox center proteins, because the effects of light scattering are negligible. As a proof of concept, we use cytochrome c oxidase (CcO) from P. denitrificans with the his tag genetically engineered to subunit I. In this orientation, the P side of CcO is directed to the outside and hence electron transfer can be initiated by reduced cytochrome c (cc). UV/vis measurements are used in order to determine the occupancy by CcO molecules encapsulated in the lipid bilayer as well as the kinetics of electron transfer between CcO and cc. The kinetic data are analyzed in terms of the Michaelis-Menten kinetics showing that the turnover rate of CcO is significantly decreased compared to that of solubilized protein, whereas the binding characteristics are improved. The data demonstrate the suitability of PLBs for functional cell-free bioassays of membrane proteins.

Proteo-lipobeads for the oriented encapsulation of membrane proteins.

As a surrogate of live cells, proteo-lipobeads are presented, encapsulating functional membrane proteins in a strict orientation into a lipid bilayer. Assays can be performed just as on live cells, for example using fluorescence measurements. As a proof of concept, we have demonstrated proton transport through cytochrome c oxidase.

Time-resolved surface-enhanced IR-absorption spectroscopy of direct electron transfer to cytochrome c oxidase from R. sphaeroides.

Time-resolved surface-enhanced IR-absorption spectroscopy triggered by electrochemical modulation has been performed on cytochrome c oxidase from Rhodobacter sphaeroides. Single bands isolated from a broad band in the amide I region using phase-sensitive detection were attributed to different redox centers. Their absorbances changing on the millisecond timescale could be fitted to a model based on protonation-dependent chemical reaction kinetics established previously. Substantial conformational changes of secondary structures coupled to redox transitions were revealed.

Electron transfer kinetics of cytochrome c probed by time-resolved surface-enhanced resonance Raman spectroscopy.

An improved setup including a measuring cell was designed for time-resolved surface-enhanced resonance Raman (SERR) spectroscopy. The cell is based on a rotating disk electrode (RDE) made from electrochemically roughened Ag. Cytochrome c (cc) adsorbed on a monolayer of mercaptoethanol is investigated with respect to heterogeneous electron transfer. Cyclic voltammograms and potential-dependent static SERR spectra indicate cc to be electroactive on the Ag electrode. The standard redox potential was found to be 234 mV. Time-resolved SERR spectra were then measured triggered by periodic potential pulses changing the protein between the oxidized and reduced state at a frequency of 10 Hz. Monoexponential functions obtained from the intensity of the band at 1361 cm-1 plotted versus time yielded the rate constants of heterogeneous electron transfer to be k(ox) = 46 +/- 7 s(-1) and k(red) = 84 +/- 20 s(-1). These relatively low rates are in line with the orientation of cc on the mercaptoethanol-modified Ag electrode. In this case the heme cleft pointed away from the surface thus hampering electron transfer.

In situ monitoring of the catalytic activity of cytochrome C oxidase in a biomimetic architecture.

Cytochrome c oxidase (CcO) from Paracoccus denitrificans was immobilized in a strict orientation via a his-tag attached to subunit I on a gold film and reconstituted in situ into a protein-tethered bilayer lipid membrane. In this orientation, the cytochrome c (cyt c) binding site is directed away from the electrode pointing to the outer side of the protein-tethered bilayer lipid membrane architecture. The CcO can thus be activated by cyt c under aerobic conditions. Catalytic activity was monitored by impedance spectroscopy, as well as cyclic voltammetry. Cathodic and anodic currents of the CcO with cyt c added to the bulk solution were shown to increase under aerobic compared to anaerobic conditions. Catalytic activity was considered in terms of repeated electrochemical oxidation/reduction of the CcO/cyt c complex in the presence of oxygen. The communication of cyt c bound to the CcO with the electrode is discussed in terms of a hopping mechanism through the redox sites of the enzyme. Simulations supporting this hypothesis are included.

Electronic wiring of a multi-redox site membrane protein in a biomimetic surface architecture.

Bioelectronic coupling of multi-redox-site membrane proteins was accomplished with cytochrome c oxidase (CcO) as an example. A biomimetic membrane system was used for the oriented immobilization of the CcO oxidase on a metal electrode. When the protein is immobilized with the CcO binding side directed toward the electrode and reconstituted in situ into a lipid bilayer, it is addressable by direct electron transfer to the redox centers. Electron transfer to the enzyme via the spacer, referred to as electronic wiring, shows an exceptionally high rate constant. This allows a kinetic analysis of all four consecutive electron transfer steps within the enzyme to be carried out. Electron transfer followed by rapid scan cyclic voltammetry in combination with surface-enhanced resonance Raman spectroscopy provides mechanistic and structural information about the heme centers. Probing the enzyme under turnover conditions showed mechanistic insights into proton translocation coupled to electron transfer. This bioelectronic approach opens a new field of activity to investigate complex processes in a wide variety of membrane proteins.

Activity of membrane proteins immobilized on surfaces as a function of packing density.

A systematic study of the influence of the packing density of proteins on their activity is performed with cytochrome c oxidase (CcO) from R. sphaeroides as an example. The protein was incorporated into a protein-tethered bilayer lipid membrane and CcO was genetically engineered with a histidine-tag, attached to Subunit II, and then tethered by an interaction with functionalized thiol compounds bound to a gold electrode. The packing density was varied by diluting the functionalized thiol with a nonfunctionalized thiol that does not bind to the enzyme. After attaching the CcO to the gold surface, a lipid bilayer was formed to incorporate the tethered proteins. The reconstituted protein-lipid bilayer was characterized by surface enhanced infrared reflection absorption spectroscopy (SEIRAS), electrochemical impedance spectroscopy, surface plasmon resonance, and atomic force microscopy. The activity of the proteins within the reconstituted bilayer was probed by direct electrochemical electron injection and was shown to be very sensitive to the packing density of protein molecules. At low surface density of CcO, the bilayer did not effectively form, and protein aggregates were observed, whereas at very high surface density, very little lipid is able to intrude between the closely packed proteins. In both of these cases, redox activity, measured by the efficiency to accept electrons, is low. Redox activity of the enzyme is preserved in the biomimetic structure but only at a moderate surface coverage in which a continuous lipid bilayer is present and the proteins are not forced to aggregate. Electrostatic and other interaction forces between protein molecules are held responsible for these effects.

Modeling ion transport in tethered bilayer lipid membranes. 1. Passive ion permeation.

Ion transport across tethered bilayer lipid membranes (tBLMs) is modeled using a hybrid network description which combines potential-dependent rate equations with passive electrical elements. Passive permeation of ions is described by the integrated Nernst-Planck equation. Simulations based on this model are performed with the network simulation program SPICE (simulation program with integrated circuit emphasis). Electrochemical impedance spectra of tBLMs are simulated with this algorithm and challenged by spectra measured with tBLMs submersed in 0.1 M KCl solution and subjected to various potential differences. It is found that the simulated spectra can only satisfactorily represent the experimental data if the permeability coefficients of the ions are dependent on the membrane potential. It is concluded that the mechanism of passive ion transport across the tBLM seems to follow the transient pore model rather than the solubility-diffusion model. This algorithm can be easily extended to include ion transport processes due to channels, carriers, or pumps incorporated into the tBLM.

Proteo-lipobeads to encapsulate cytochrome c oxidase from Paracoccus denitrificans.

Proteo-lipobeads (PLBs) are investigated as cell-free model systems to encapsulate membrane proteins such as ion channels and transporters. PLBs are based on nickel nitrile tri-acetic acid (Ni-NTA)-functionalized agarose beads, onto which membrane proteins (MP) are bound via histidine(his)-tag. Composite beads thus obtained (subsequently called proteobeads) are dialyzed in the presence of lipid micelles to form PLBs. As an example we employed cytochrome c oxidase from P. denitrificans with a his-tag fused to the C-terminus of subunitI. In this orientation the P side of CcO faces the outside of the PLB and hence protons are released to the outer aqueous phase, when electron transfer is initiated by light excitation of Ru complexes. Proton release kinetics was probed by fluorescence microscopy using the pH-sensitive sensor molecule fluorescein DHPE inserted into the lipid layer. In order to monitor the generation of membrane potentials we performed a FLIPR assay on the CcO embedded in PLBs using the FRET pair CC2-DMPE/DiSBAC2(3). The combined results show that PLBs can be used as a model system designed to quantify the kinetic parameters of membrane proteins. In addition, the FLIPR assay demonstrates the feasibility of PLBs for high throughput screening applications.

pH and Potential Transients of the bc1 Complex Co-Reconstituted in Proteo-Lipobeads with the Reaction Center from Rb. sphaeroides.

His-tag technology is employed to bind membrane proteins, such as the bc1 complex and the reaction center (RC) from Rhodobacter sphaeroides, to spherical as well as planar surfaces in a strict orientation. Subsequently, the spherical and planar surfaces are subjected to in situ dialysis to form proteo-lipobeads (PLBs) and protein-tethered bilayer membranes, respectively. PLBs based on Ni-nitrileotriacetic acid-functionalized agarose beads that have diameters ranging from 50 to 150 µm are used to assess proton release and membrane potential parameters by confocal laser-scanning microscopy. The pH and potential transients are thus obtained from bc1 activated by the RC. To assess the turnover of bc1 excited by the RC in a similar setting, we used the planar surface of an attenuated total reflection crystal modified with a thin gold layer to carry out time-resolved surface-enhanced IR absorption spectroscopy triggered by flash lamp excitation. The experiments suggest that both proteins interact in a cyclic manner in both environments. The activity of the proteins seems to be preserved in the same manner as that in chromatophores or reconstituted in liposomes.

Kinetics of cytochrome c oxidase from R. sphaeroides initiated by direct electron transfer followed by tr-SEIRAS.

Time-resolved surface-enhanced IR-absorption spectroscopy (tr-SEIRAS) has been performed on cytochrome c oxidase from Rhodobacter sphaeroides. The enzyme was converted electrochemically into the fully reduced state. Thereafter, in the presence of oxygen, the potential was switched to open circuit potential (OCP). Under these conditions, the enzyme is free to undergo enzymatic oxidation in the absence of an external electric field. Tr-SEIRAS was performed using the step-scan technique, triggered by periodic potential pulses switching between - 800mV and OCP. Single bands were resolved in a broad band in the amide I region using phase sensitive detection. Amplitudes of these bands were analyzed as a function of time. Time constants in the ms time scale were considered in terms of conformational changes of the protein secondary structures associated with the enzymatic turnover of the protein.

Active site structure and redox processes of cytochrome c oxidase immobilised in a novel biomimetic lipid membrane on an electrode.

Membrane-bound cytochrome c oxidase was attached to an electrode via a His-tag linker and studied by surface enhanced resonance Raman spectroscopy, demonstrating intact redox site structures and electron transfer between the electrode and the immobilized enzyme.

Phase-sensitive detection in modulation excitation spectroscopy applied to potential induced electron transfer in cytochrome c oxidase.

Cytochrome c oxidase (CcO) from Rhodobacter sphaeroides was investigated by modulated excitation surface-enhanced infrared-absorption spectroscopy (SEIRAS). Sequential electron transfer (ET) within CcO was initiated by electrochemical excitation. During modulated excitation by periodic potential pulses with frequencies between 20 and 500 Hz, time-resolved infrared spectra were measured by the step-scan technique, with time resolution in the millisecond range. Conformational changes of the protein structure as a result of ET lead to rather complex SEIRA spectra with many overlapping bands embedded in a broad background signal. Phase-sensitive detection (PSD) was used to separate single components within the broad band of overlapping structural bands in the amide I region. PSD is able to extract the periodic response of single components with the same frequency as the excitation from noise or from static background and therefore enhances the signal-to-noise ratio. Moreover, PSD enables validation of the fit model used for the deconvolution of overlapping bands by analyzing phase lags of single components acquired at different stimulation frequencies. Phase lags between the evaluated vibrational components and the modulated excitation increase with increasing excitation frequencies, an inherent prerequisite of this evaluation method.

2D-SEIRA spectroscopy to highlight conformational changes of the cytochrome c oxidase induced by direct electron transfer.

Potentiometric titrations of the cytochrome c oxidase (CcO) immobilized in a biomimetic membrane system were followed by two-dimensional surface-enhanced IR absorption spectroscopy (2D SEIRAS) in the ATR-mode. Direct electron transfer was employed to vary the redox state of the enzyme. The CcO was shown to undergo a conformational transition from a non-activated to an activated state after it was allowed to turnover in the presence of oxygen. Differences between the non-activated and activated state were revealed by 2D SEIRA spectra recorded as a function of potential. The activated state was characterized by a higher number of correlated transitions as well as a higher number of amino acids associated with electron transfer.

Self-assembled tethered bimolecular lipid membranes.

This chapter describes some of the strategies developed in our group for designing, constructing and structurally and functionally characterizing tethered bimolecular lipid membranes (tBLM). We introduce this platform as a novel model membrane system that complements the existing ones, for example, Langmuir monolayers, vesicular liposomal dispersions and bimolecular ("black") lipid membranes. Moreover, it offers the additional advantage of allowing for studies of the influence of membrane structure and order on the function of integral proteins, for example, on how the composition and organization of lipids in a mixed membrane influence the ion translocation activity of integral channel proteins. The first strategy that we introduce concerns the preparation of tethered monolayers by the self-assembly of telechelics. Their molecular architecture with a headgroup, a spacer unit (the "tether") and the amphiphile that mimics the lipid molecule allows them to bind specifically to the solid support thus forming the proximal layer of the final architecture. After fusion of vesicles that could contain reconstituted proteins from a liposomal dispersion in contact to this monolayer the tethered bimolecular lipid membrane is obtained. This can then be characterized by a broad range of surface analytical techniques, including surface plasmon spectroscopies, the quartz crystal microbalance, fluorescence and IR spectroscopies, and electrochemical techniques, to mention a few. It is shown that this concept allows for the construction of tethered lipid bilayers with outstanding electrical properties including resistivities in excess of 10 MOmega cm2. A modified strategy uses the assembly of peptides as spacers that couple covalently via their engineered sulfhydryl or lipoic acid groups at the N-terminus to the employed gold substrate, while their C-terminus is being activated afterward for the coupling of, for example, dimyristoylphosphatidylethanol amine (DMPE) lipid molecules via the NH2 moiety of their headgroups. It is demonstrated that these membranes are well suited for the in situ synthesis of membrane protein by a cell-free expression approach. The vectorial integration of an in vitro synthesized odorant receptor, OR5 from the rat, is demonstrated by means of antibodies that specifically bind to a tag at the N-terminus of the receptor and is read out by surface plasmon fluorescence spectroscopy. A completely different strategy employs his-tagged membrane proteins in their solubilized form binding to a surface-attached Ni(+)-NTA monolayer generating a well-oriented protein layer the density of which can be easily controlled by online monitoring the binding (assembly) step by surface plasmon spectroscopy. Moreover, the attachment of the his-tag to either the C- or the N-terminus allows for the complete control of the protein orientation. After the exchange of the detergent micelle by a lipid bilayer via a surface dialysis procedure an electrically very well isolating protein-tethered membrane is formed. We show that this "wiring" of the functional units allows for the (external) manipulation of the oxidation state of the redox-protein cytochrome c Oxidase by the control of the potential applied to the gold substrate which is used as the working electrode in an electrochemical attachment.

In situ PM-IRRAS studies of an archaea analogue thiolipid assembled on a au(111) electrode surface.

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) has been applied to determine the conformation, orientation, and hydration of a monolayer of 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-dl-alpha-lipoic acid ester (DPTL) self-assembled at a gold electrode surface. This Archaea analogue thiolipid has been recently employed to build tethered lipid bilayers. By synthesizing DPT(d16)L, a DPTL molecule with a deuterium substituted tetraethylene glycol spacer, it was possible to differentiate the C-H stretch vibrations of the phytanyl chains from the tetraethylene glycol spacer and acquire the characteristic IR spectra for the chains, spacer, and lipoic acid headgroup separately. Our results show that the structure of the monolayer displays remarkable stability in a broad range of electrode potentials and that the phytanyl chains remain in a liquid crystalline state. The tetraethylene glycol chains are coiled, and the IR spectrum for this region shows that it is in the disordered state. The most significant result of this study is the information that in contrast to expectations the spacer region is poorly hydrated. Our results have implications for the design of a tethered lipid membrane based on this thiolipid.