Scanning probe microscopic studies of the oriented attachment and membrane reconstitution of cytochrome C oxidase to a gold electrode

Scanning probe microscopy was used to monitor the resulting surface of the oriented incorporation of cytochrome c oxidase into electrode supported lipid bilayer at four crucial stages with molecular resolution. We were able to reveal the formation of a densely packed monolayer of the active ester dithio(succiniimidylepropionate) (DTSP) and the covalent linkage of the nitrilotriacetic acid (NTA) to the thiol anchored DTSP by scanning tunneling microscopy. Atomic force microscopy investigations showed that the detergent solubilized oxidase is immobilized as monomers and small aggregates via histidine residues. Finally, the reconstitution of the proteins within the supported membrane was verified. The amount of oxidase immobilized within the solid supported membrane was estimated.     

Biochemical applications of surface-enhanced infrared absorption spectroscopy

An overview is presented on the application of surface-enhanced infrared absorption (SEIRA) spectroscopy to biochemical problems. Use of SEIRA results in high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude and has the potential to enable new studies, from fundamental aspects to applied sciences. This report surveys studies of DNA and nucleic acid adsorption to gold surfaces, development of immunoassays, electron transfer between metal electrodes and proteins, and protein–protein interactions. Because signal enhancement in SEIRA uses surface properties of the nano-structured metal, the biomaterial must be tethered to the metal without hampering its functionality. Because many biochemical reactions proceed vectorially, their functionality depends on proper orientation of the biomaterial. Thus, surface-modification techniques are addressed that enable control of the proper orientation of proteins on the metal surface. Figure Surface enhanced infrared absorption spectroscopy (SEIRAS) on the studies of tethered protein monolayer (cytochrome c oxidase and cytochrome c) on gold substrate (left), and its potential induced surface enhanced infrared difference absorption (SEIDA) spectrum     

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.     

Membrane protein selectively oriented on solid support and reconstituted into a lipid membrane

Mimetic functional membranes on solid support are now emerging for the development of membrane biosensor or for the study of membrane-mediated processes and should have an important impact on biodiagnostics. We established a method to reconstitute a membrane protein into a lipid membrane in a selective orientation on a solid support. Membrane protein OprM, a component of OprM-MexA-MexB multidrug efflux pump, solubilized in detergent was immobilized via its extracellular domain on aminosilane-modified silica surface. The oriented protein was reconstituted into a lipid membrane by detergent removal. The membrane protein reconstitution process carried out on silica nanoparticles and on planar silica surfaces was followed by cryo-electron microscopy (cryo-EM) and quartz crystal microbalance with dissipation monitoring (QCM-D) respectively. The selective protein orientation on aminosilane-modified silica surface was assessed by cryo-EM and was compared to the nonspecific protein deposition on silica surface. Finally, the binding of MexA, a periplasmic component of the tripartite efflux complex, was monitored with QCM-D on the oriented OprM protein monolayer. The large adsorbed mass gave a direct evidence of the high affinity of MexA with the periplasmic helical part of OprM.     

In situ monitoring of the orientated assembly of strep-tagged membrane proteins on the gold surface by surface enhanced infrared absorption spectroscopy

Surface enhanced infrared absorption spectroscopy (SEIRAS) has been employed to monitor the orientated assembly of a strep-tagged membrane protein on the gold surface via a streptavidin/biotin interlayer. The high surface sensitivity of SEIRAS allows for tracking the individual assembling steps on the molecular level. The sequence of surface modification steps comprises: (i) cross-linking of biotin to the self-assembled monolayer of cysteamine along the gold surface; (ii) adsorption of streptavidin to and desorption from the biotin layer; and (iii) adsorption of the strep-tagged membrane protein ecgltP (glutamate transporter of E. coli) on the streptavidin/biotin layer. The analysis of the SEIRA spectra reveals that the biotin layer undergoes a phase transition from an isotropic orientation to a densely packed layer during coupling to the cysteamine monolayer. Formation of the densely packed layer weakens the interaction between streptavidin and the biotin layer but yields a binding specificity of 80%. The specificity of strep-tagged ecgltP to the streptavidin layer is with 60% only modest. Nevertheless, the streptavidin/biotin interlayer reveals a higher regeneration propensity than the His-tag/Ni-NTA interlayer.     

Amperometric detection of superoxide dismutase at cytochrome c-immobilized electrodes: xanthine oxidase and ascorbate oxidase incorporated biopolymer membrane for in-vivo analysis.

Amperometric measurement of superoxide dismutase (SOD) was carried out at cytochrome c-immobilized monolayers and ascorbate oxidase (AOD)/xanthine oxidase (XOD)/cytochrome c- and (AOD, XOD)/cytochrome c-multilayers. Cytochrome c was covalently immobilized on mercaptopropionic acid-containing self-assembled monolayers on gold. A biopolymer membrane of poly-L-lysine confining XOD and AOD was cast on the monolayer of cytochrome c. While both the cytochrome c-immobilized monolayer and multilayer electrodes show anodic current responses to the generation of superoxide radical, the sensitivity of the multilayer system for the detection of superoxide radical was high relative to that of the monolayer system. In the case of the cytochrome c-multilayer electrodes, the generation of superoxide radical near the sensing element, cytochrome c, resulted in high sensitivity for the detection of superoxide. The use of a XOD and AOD-incorporated poly-L-lysine membrane enabled the detection of the generation of superoxide radical in the presence of L-ascorbic acid. Though L-ascorbic acid could scavenge superoxide radical, the biopolymer membrane confined with AOD will oxidize any L-ascorbic acid that permeated into the membrane. By using the multilayer electrodes, one could measure the activity of SOD in the presence of L-ascorbic acid.     

Formation of a protein monomolecular layer by a combined technique of LB and SA methods

This paper describes a technique developed for the formation of the self-assembled protein monomolecular layer. The main idea is a direct transfer of protein molecules consisting of a Langmuir-Blodgett (LB) film onto the surface of another chemically modified metal substrate and induction of the spontaneous formation of a self-assembled monolayer (SAM) by chemisorption without protein aggregates. In the present experiments, a cytochrome c (cyt c) SAM on a gold substrate was prepared by incubating a EDC/MUA-modified gold substrate overlaid with a cyt c LB film in a phosphate buffer solution. Scanning tunneling microscopy (STM) image of a cyt c SAM shows that the size of cyt c clusters in the most part of the substrate is approximately 5 nm, indicating the cyt c monomolecular layer. The rectifying property of a cyt c monolayer was confirmed to remain by an asymmetric I-V curve in the applied bias from -1 V to 1 V.     

Electrochemically induced surface-enhanced infrared difference absorption (SEIDA) spectroscopy of a protein monolayer

Functional infrared difference spectra of a protein monolayer are recorded by exploiting the surface enhancement exerted by a modified gold surface. The noble metal is used as the working electrode to induce redox-linked structural changes in the single layer of cytochrome c. The observed differences in the relative band intensities as compared to the difference spectrum of cytochrome c in solution are attributed to orientational effects of the adsorbed protein.     

QCM and EC-SPR Studies of Cytochrome cSelf-assembled on Au Electrode and Enhancement of SPR Signal by Au Nanoparticles

Quartz crystal microbalance(QCM) and cyclic voltammetry(CV) were used to characterize the monolayer of cytochrome c(Cyt c), which was adsorbed on gold film modified with alkanethiol mixed monolayer. A direct comparison of protein surface coverages calculated from QCM and cyclic voltammetric measurements illustrates that the ratio of the electroactive Cyt c to the total surface-confined Cyt cis 34%, which suggests that the orientation is a main factor affecting the electroactivity of Cyt c. Moreover, surface plasmon resonance(SPR) measurement combined with CV “in situ” was used to investigate the conformational change of Cyt c in the redox process. Besides, Au nanoparticles(Au NPs) were adsorbed on the surface of Cyt c. The result indicates that Au NPs promote electron transfer between Cyt c and the gold electrode, and SPR result suggests Au NPs enhance SPR signal.     

Determination of the orientation of adsorbed cytochrome C on carboxyalkanethiol self-assembled monolayers by in situ differential modification

The contact domain utilized by horse cytochrome c when adsorptively bound to a C(10)COOH self-assembled monolayer (SAM) was delineated using a chemical method based on differential modification of surface amino acids. Horse cytochrome c was adsorbed at low ionic strength (pH 7.0, 4.4 mM potassium phosphate) onto 10 microm diameter gold particles coated with HS(CH(2))(10)COOH SAMs. After in situ modification of lysyl groups by reductive Schiff-base methylation, the protein was desorbed, digested using trypsin, and the peptide mapped using LC/MS. Relative lysyl reactivities were ascertained by comparing the resulting peptide frequencies to control samples of solution cytochrome c modified to the same average extent. The least reactive lysines in adsorbed cytochrome c were found to be 13, 72, 73, 79, and 86-88, consistent with a contact region located up and to the left (Met-80 side) of the solvent-exposed heme edge (conventional front face view). The most reactive lysines were 39, 53, 55, and 60, located on the lower backside. The proposed orientation features a heme tilt angle of approximately 35-40 degrees with respect to the substrate surface normal. Factors that can complicate or distort data interpretation are discussed, and the generality of differential modification relative to existing in situ methods for protein orientation determination is also addressed.     

Protein adsorption on supported phospholipid bilayers

Quartz crystal microbalance with dissipation (QCM-D) measurements were used to investigate the adsorption of human fibrinogen, human serum albumin, bovine hemoglobin, horse heart cytochrome c, human immunoglobulin (hIgG), and 10% fetal bovine serum on supported bilayers of egg-phosphatidylcholine (eggPC) lipids. For comparison the adsorption of fibrinogen and hIgG to eggPC bilayers was also studied with surface plasmon resonance (SPR). The supported bilayers were formed in situ by vesicle adhesion and spontaneous fusion onto a SiO(2) surface. The supported lipid bilayer is highly protein resistant: The irreversible adsorption measured with the QCM-D technique was below the detection level, while reversible protein adsorption was detected for all the proteins in the range 0.3-4% of the saturation coverage on a hydrophobic thiol monolayer on gold. The adsorbed amounts were slightly higher for the SPR measurements. Possible mechanisms for the protein resistance of eggPC bilayers are briefly discussed.     

Orientational control of the physiological reaction of cytochrome c oxidase tethered to a gold electrode

The physiological reaction of a membrane protein is reconstituted on a solid-supported electrode by orientational control via the position of an affinity tag. Recombinant cytochrome c oxidase (CcO) from Rhodobacter sphaeroides is immobilized on a chemically modified gold surface via the affinity of a histidine tag (His-tag) to a nickel chelating nitrilotriacetic acid surface. Control of the orientation is achieved by the adsorption of CcO through the His-tag engineered into the two opposite sites of the membrane protein surface. After reconstitution into a lipid layer, the functionality of this enzyme film electrode is probed by surface-enhanced infrared absorption spectroscopy and cyclic voltammetry. We demonstrate that cytochrome c (Cc) binds and initiates the catalytic reaction of CcO only when the latter is orientated with subunit II facing the bulk aqueous phase while Cc does not interact with the oppositely orientated CcO. We infer from the observed catalytic dioxygen reduction at potentials below 240 mV (vs a normal hydrogen electrode) that reduced Cc mediates electron input into CcO in a way similar to the physiological pathway. The quantitative analysis of the IR spectra indicates the presence of an inactive population of Cc bound to CcO at equal amounts as the redox-active population. This methodological approach demonstrates that the orientation of the membrane protein can be controlled depending on the position of the affinity tag. The approach is considered to be of general applicability as the introduction of affinity tags is routine in current biochemistry.     

Visualization of red-ox proteins on the gold surface using enzymatic polypyrrole formation

We describe a new method for the visualization of the activity of red-ox proteins on a gold interface. Glucose oxidase was selected as a model system. Surfaces were modified by adhesion of glucose oxidase on (a) electrochemically cleaned gold; (b) gold films modified with gold nanoparticles, (c) a gold surface modified with self-assembled monolayer, and (d) covalent immobilization of protein on the gold surface modified with a self-assembled monolayer. The simple optical method for the visualization of enzyme on the surfaces is based on the enzymatic formation of polypyrrole. The activity of the enzyme was quantified via enzymatic formation of polypyrrole, which was detected and investigated by quartz microbalance and amperometric techniques. The experimental data suggest that the enzymatic formation of the polymer may serve as a method to indicate the adhesion of active redox enzyme on such surfaces.

Conductive wiring of immobilized photosynthetic reaction center to electrode by cytochrome C

The photosynthetic reaction center (RC) found in photosynthetic bacteria is one of the most advanced photoelectronic devices developed by nature. However, after immobilization on the electrode surface, the efficiency of electron transfer (ET) between the RC and the electrode is relatively low. This inefficiency has limited the possibility of using the RC for technological applications. Here we show that photoinduced electron transfer between the immobilized RC and a gold electrode can be increased by several tens-fold by incorporation of cytochrome c into the RC-self-assembled monolayer (SAM)-electrode complex. The effect does not depend on the initial redox state of the cytochrome and seems to be the result of the formation of a complex between the RC and the cytochrome c serving as an ET wire. This observation opens the possibility for electrochemical analysis of the special pair in the RC protein that is deeply buried inside the protein globe and is barely electrically addressable from the electrode surface.     

细胞色素c在微带金电极上的直接电化学

报道了一种新的促进剂4,6-二甲基-2-巯基嘧啶(DMMP)对细胞色素c(Cyt.c) 电化反应的促进作用,用红外光谱和光电子能对DMMP 在金电极表面形成的单分子膜进行了表征.循环伏安实验表明Cyt.c在DMMP修饰微电极上能发生准可逆的电化学反应,异相电子传递速率常数K~5为6.6×10^-^3cm/s,对DMMP修饰膜的稳定性进行了考察;讨论了Cyt.c发生电化学反应的异相电子传递速率常数K~5受电极, 表面促进剂的修饰量以及空气中氧影响的机理.     

Immobilization of acetylcholinesterase in lipid membranes deposited on self-assembled monolayers

Human red blood cell acetylcholinesterase was incorporated into planar lipid membranes deposited on alkanethiol self-assembled monolayers (SAMs) on gold substrates. Activity of the protein in the membrane was detected with a standard photometric assay and was determined to be similar to the protein in detergent solution or incorporated in lipid vesicles. Monolayer and bilayer lipid membranes were generated by fusing liposomes to hydrophobic and hydrophilic SAMs, respectively. Liposomes were formed by the injection method using the lipid dimyristoylphosphatidylcholine (DMPC). The formation of alkanethiol SAMs and lipid monolayers on SAMs was confirmed by sessile drop goniometry, ellipsometry, and electrochemical impedance spectroscopy. In this work, we report acetylcholinesterase immobilization in lipid membranes deposited on SAMs formed on the gold surface and compare its activity to enzyme in solution.     

Direct wiring of cytochrome c's heme unit to an electrode: electrochemical studies

A novel strategy for the immobilization of cytochrome c on the surface of chemically modified electrodes is demonstrated and used to investigate the protein's electron-transfer kinetics. Mixed monolayer films of alkanethiols and omega-terminated alkanethiols (terminated with pyridine, imidazole, or nitrile groups that are able to ligate with the heme) are used to adsorb cytochrome c to the surface of gold electrodes. The use of mixed films, as opposed to pure films, allows the concentration of adsorbed cytochrome to remain dilute and ensures a higher degree of homogeneity in their environment. The adsorbed protein is studied using electrochemical methods and scanning tunneling microscopy.     

Functional vibrational spectroscopy of a cytochrome c monolayer: SEIDAS probes the interaction with different surface-modified electrodes

Electrochemically induced infrared difference spectra of cytochrome c on various chemically modified electrodes (CMEs) are recorded by exploiting the surface-enhancement exerted by a granular gold film. We have recently developed surface-enhanced infrared difference absorption spectroscopy (SEIDAS), which provides acute sensitivity to observe the minute enzymatic change of a protein on the level of a monolayer. By these means, we demonstrate that the relative band intensities in the potential-induced difference spectra of adsorbed cytochrome c are significantly dependent on the type of CME used (mercaptopropionic acid, mercaptoethanol, 4,4'-dithiodipyridine, or L-cysteine). These differences are attributed to the altered interaction of cytochrome c with the headgroup of the various CMEs leading to variations in surface orientation and relative distance from the surface. Nevertheless, the peak positions of the observed bands are identical among the CMEs employed. This implies that the internal conformational changes induced by the redox reaction of the adsorbed cytochrome c are not disturbed by the interaction with the CME and that full functionality of the protein is retained. Finally, we critically discuss our results within the framework of the different models for cytochrome c adsorption on CMEs.     

Direct Electrochemistry of Cytochrome c Covalently Immobilized on ω-Carboxyalkanethiol Monolayer Electrode and its Biosensor Applications

The study of direct electron transfer (ET) between solid electrodes and proteins or enzymes has been attracting considerable research interest for several decades since it represents a basic feature for the application of biocatalysts in chemical sensors and other electrical devices. We have been focusing our research interest on the use of SAMs for the study of diffusionless, direct electrochemistry of cytochromes. In the present paper, we report electrochemistry of cytochrome c covalently immobilized on ω-carboxyalkanethiol monolayer electrodes. A carboxylic acid terminated monolayer was utilized to provide an uniform surface for attaching cytochrome c, and characterization of the redox reaction of the protein was made with using cyclic voltammetric and electrochemical impedance measurements.     

Using scanning electrochemical microscopy (SECM) to measure the electron-transfer kinetics of cytochrome c immobilized on a COOH-terminated alkanethiol monolayer on a gold electrode

Cytochrome c was electrostatically immobilized onto a COOH-terminated alkanethiol self-assembled monolayer (SAM) on a gold electrode at ionic strengths of less than 40 mM. Scanning electrochemical microscopy (SECM) was used to simultaneously measure the electron transfer (ET) kinetics of the bimolecular ET between a solution-based redox mediator and the immobilized protein and the tunneling ET between the protein and the underlying gold electrode. Approach curves were recorded with ferrocyanide as a mediator at different coverages of cytochrome c and at different substrate potentials, allowing the measurement of k(BI) = 2 x 10(8) mol(-1) cm3 s(-1) for the bimolecular ET and k degrees = 15 s(-1) for the tunneling ET. The kinetics of ET was also found to depend on the immobilization conditions of cytochrome c: covalent attachment gave slightly slower tunneling ET values, and a mixed CH3/COOH-terminated ML gave faster tunneling ET rates. This is consistent with previous studies and is believed to be related to the degree of mobility of cyt c in its binding configuration and its orientation with respect to the underlying electrode surface.