Direct measurement of VDAC-actin interaction by surface plasmon resonance

VDAC - a mitochondrial channel involved in the control of aerobic metabolism and apoptosis - interacts in vitro and in vivo with a wide repertoire of proteins including cytoskeletal elements. A functional interaction between actin and Neurospora crassa VDAC was reported, excluding other VDAC isoforms. From a recent genome-wide screen of the VDAC interactome, we found that human actin is a putative ligand of yeast VDAC. Since such interaction may have broader implications for various mitochondrial processes, we probed it with Surface Plasmon Resonance (SPR) technology using purified yeast VDAC (YVDAC) and rabbit muscle G-actin (RGA). We show that RGA binds to immobilized YVDAC in a reversible and dose-dependent manner with saturating kinetics and an apparent K_D of 50 μg/ml (1.2 μM actin). BSA does not bind VDAC regardless of the concentrations. Alternatively, VDAC binds similarly to immobilized RGA but without saturating kinetics. VDAC being known to interact with itself, this latter interaction was directly measured to interpret the RGA signals. VDAC could bind to VDAC without saturating kinetics as expected if higher order binding occurred, and could account for maximally 66% of the non-saturating behavior of VDAC binding onto RGA. Hence, actin-VDAC interactions are not a species-specific oddity and may be a more general phenomenon, the role of which ought to be further investigated.     

Direct measurement of VDAC-actin interaction by surface plasmon resonance

VDAC--a mitochondrial channel involved in the control of aerobic metabolism and apoptosis--interacts in vitro and in vivo with a wide repertoire of proteins including cytoskeletal elements. A functional interaction between actin and Neurospora crassa VDAC was reported, excluding other VDAC isoforms. From a recent genome-wide screen of the VDAC interactome, we found that human actin is a putative ligand of yeast VDAC. Since such interaction may have broader implications for various mitochondrial processes, we probed it with Surface Plasmon Resonance (SPR) technology using purified yeast VDAC (YVDAC) and rabbit muscle G-actin (RGA). We show that RGA binds to immobilized YVDAC in a reversible and dose-dependent manner with saturating kinetics and an apparent K(D) of 50 microg/ml (1.2 microM actin). BSA does not bind VDAC regardless of the concentrations. Alternatively, VDAC binds similarly to immobilized RGA but without saturating kinetics. VDAC being known to interact with itself, this latter interaction was directly measured to interpret the RGA signals. VDAC could bind to VDAC without saturating kinetics as expected if higher order binding occurred, and could account for maximally 66% of the non-saturating behavior of VDAC binding onto RGA. Hence, actin-VDAC interactions are not a species-specific oddity and may be a more general phenomenon, the role of which ought to be further investigated.     

Studying the assembly of multicomponent protein and ribonucleoprotein complexes using surface plasmon resonance

The assembly of large macromolecular complexes is an important aspect of cellular organization and metabolism. Interactions involving such complexes in principle follow the same rules as the interactions between single proteins or other macromolecules and can therefore be investigated using similar approaches. We have developed protocols employing standard surface plasmon resonance technology that allow the investigation of interactions involving complex macromolecular structures. The principal experimental challenges arise from the possibility of parallel reactions where partially assembled or dissociated subcomplexes form a significant proportion of the molecule population and from an increased likelihood of unspecific binding events owing to the larger surface and statistically higher number of charged areas on multisubunit assemblies. Ways to experimentally avoid or, where this is not possible, to control for these complications are discussed.     

Screening protein refolding using surface plasmon resonance

Surface plasmon resonance (SPR) measurements were used to screen refolding conditions to identify a physicochemical environment which gives an acceptable refolding yield for samples of glutathione-S-transferase (GST) denatured in 6 M guanidine hydrochloride and 32 mM dithiothreitol. The SPR measurements were performed on carboxymethylcellulose coated chips that could accommodate two separate flow paths. One side of the chip was derivatized with immobilized glutathione and the other with goat anti-GST antibody. This created a dual-derivatized chip capable of showing both the presence of GST and providing a measure of enzyme activity. The dual-derivatized chip could be regenerated using a two-step washing procedure and reused to analyze multiple samples from a screening study of protein refolding conditions. SPR measurements have been shown to be suitable for screening protein refolding conditions due to the high sensitivity, ease of chip regeneration and the ability to incorporate a control in the experimental design. The combination of such advantages with the high-throughput automated SPR systems currently available may be a valuable approach to determine conditions suitable for protein refolding following insoluble expression in a bacterial host.     

Direct analysis of a GPCR-agonist interaction by surface plasmon resonance

Despite their clinical importance, detailed analysis of ligand binding at G-protein coupled receptors (GPCRs) has proved difficult. Here we successfully measure the binding of a GPCR, neurotensin receptor-1 (NTS-1), to its ligand, neurotensin (NT), using surface plasmon resonance (SPR). Specific responses were observed between NT and purified, detergent-solublised, recombinant NTS-1, using a novel configuration where the biotinylated NT ligand was immobilised on the biosensor surface. This SPR approach shows promise as a generic approach for the study of ligand interactions with other suitable GPCRs.     

Characterization of sialyltransferase mutants using surface plasmon resonance

Sialyltransferases are enzymes responsible for the important sialylation of glycoconjugates. Since crystal structures are not available, other tools are needed to study enzymatic mechanisms. As a model, we used human alpha2,6-sialyltransferase. A putative acceptor-binding domain containing the small and the very small sialyl motifs was randomly mutated. This resulted in enzymes with altered enzymatic activity. Affinity chromatography demonstrated that their binding to donor substrate was maintained. To illustrate the role of the mutated domain in acceptor binding, a method based on surface plasmon resonance was set up. Only at low salt and high acceptor concentration was association of wild-type ST6GalI with asialofetuin demonstrated. As expected, this interaction was affected by cytidine 5'-monophospho-N-acetylneuraminic acid, the donor substrate, which proves the specificity of the interaction. Different types of mutants were found. For some, the drop in activity could be explained by loss in affinity for the acceptor. For others, the catalytic center, but not the acceptor-binding site, was affected. Neither acceptor binding nor catalytic activity were limited to the sialyl motifs. To our knowledge, this is the first example in which surface plasmon resonance is successfully used to demonstrate the binding of a glycosyltransferase to its natural acceptor.     

Kinetic studies of RNA-protein interactions using surface plasmon resonance

Although structural, biochemical, and genetic studies have provided much insight into the determinants of specificity and affinity of proteins for RNA, little is currently known about the kinetics that underlie RNA-protein interactions. Protein-RNA complexes are dynamic, and the kinetics of binding and release could influence many processes, such as the ability of RNA-binding proteins to compete for binding sites, the sequential assembly of ribonucleoprotein complexes, and the ability of bound RNA to move between cellular compartments. Therefore, to attain a complete and biologically relevant understanding of RNA-protein interactions, complex formation must be studied not only in equilibrated reactions, but also as a dynamic process. BIACORE, a surface plasmon resonance-based biosensor technology, allows intermolecular interactions to be measured in real time, and can provide both equilibrium and kinetic information about complex formation. This technology is a powerful tool with which to study the dynamics of RNA-protein interactions. We have used BIACORE extensively to obtain detailed insight into the interaction between RNA and proteins carrying RNA recognition motif domains. Here we discuss the physical principles on which BIACORE is based, and the required instrumentation. We describe how to design well-controlled RNA-protein interaction experiments aimed at yielding high-quality data, and outline the steps required for data analysis. In addition, we present examples to illustrate how kinetic studies have provided us with unique insights into the interaction of the spliceosomal U1A protein and the neuronal HuD protein with their respective RNA targets.     

Functional demonstration of connexin-protein binding using surface plasmon resonance

Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.     

Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance

It is well established that IgG from rheumatoid arthritis (RA) patients are less galactosylated than IgG from normal individuals. Determination of agalacto-IgG may therefore aid in diagnosis and treatment of RA. The decrease in galactosylation of IgG leads to an increase in terminal N-acetylglucosamine residues, which can be detected using a specific lectin from Psathyrella velutina. In the present study IgG from RA and control serum was purified using affinity chromatography. The samples were then, after reduction, analyzed on a BIOCORE® 2000 system with immobilized Psathyrella velutina lectin. Using this technique it was possible to discriminate between IgG from RA patients and IgG from control individuals with respect to its content of IgG with terminal N-acetylglucosamine. The affinity biosensor technique makes it possible to detect binding without labeling or using secondary antibodies.     

Real time analysis of intact organelles using surface plasmon resonance

Membrane proteins remain refractory to standard protein chip analysis. They are typically expressed at low densities in distinct subcellular compartments, their biological activity can depend on assembly into macromolecular complexes in a specific lipid environment. We report here a real-time, label-free method to analyze membrane proteins inserted in isolated native synaptic vesicles. Using surface plasmon resonance-based biomolecular interaction analysis (Biacore), organelle capture from minute quantities of 10,000 g brain supernatant (1-10 microg) was monitored. Immunological and morphological characterization indicated that pure intact synaptic vesicles were immobilized on sensor chips. Vesicle chips were stable for days, allowing repetitive use with multiple analytes. This method provides an efficient way in which to characterize organelle membrane components in their native context. Organelle chips allow a broad range of measurements, including interactions of exogenous ligands with the organelle surface (kinetics, Kd), and protein profiling.     

Real-time monitoring of odorant-induced cellular reactions using surface plasmon resonance

Surface plasmon resonance (SPR) is a powerful technique for measuring molecular interaction in real-time. SPR can be used to detect molecule to cell interactions as well as molecule to molecule interactions. In this study, the SPR-based biosensing technique was applied to real-time monitoring of odorant-induced cellular reactions. An olfactory receptor, OR I7, was fused with a rho-tag import sequence at the N-terminus of OR I7, and expressed on the surface of human embryonic kidney (HEK)-293 cells. These cells were then immobilized on a SPR sensor chip. The intensity of the SPR response was linearly dependent on the amount of injected odorant. Among all the aldehyde containing odorants tested, the SPR response was specifically high for octanal, which is the known cognate odorant for the OR I7. This SPR response is believed to have resulted from intracellular signaling triggered by the binding of odorant molecules to the olfactory receptors expressed on the cell surface. This SPR system combined with olfactory receptor-expressed cells provides a new olfactory biosensor system for selective and quantitative detection of volatile compounds.     

High-throughput affinity ranking of antibodies using surface plasmon resonance microarrays

A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.     

Immunosensor for detection of Legionella pneumophila using surface plasmon resonance

Immunosensor using surface plasmon resonance (SPR) onto self-assembled protein G layer was developed for the detection of Legionella pneumophila. A self-assembled protein G layer on gold (Au) surface was fabricated by adsorbing a mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol (molar ratio of 1:2) and the activation process for chemical binding between free amine (-NH(2)) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) in series. The formation of self-assembled protein G layer on Au substrate and the binding of antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of self-assembled protein G layer on Au substrate and monoclonal antibody against L. pneumophila immobilized on protein G were performed by atomic force microscope (AFM). The immunosensor for detection of L. pneumophila using SPR was developed and its detection limit could find up to 10(5) cells/ml.     

Investigations into self-association of vancomycin covalent dimers using surface plasmon resonance technology.

Covalent dimers of vancomycin linked through the vancosamine sugar moieties of the glycopeptide antibiotic have been synthesized in one step in 67-69% yield. The propensity for self-association of these and related vancomycin covalent dimers is evaluated using surface plasmon resonance technology.     

Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface

Complexes involving three DNA strands were used to demonstrate that the single-cycle kinetics (SCK) method, which consists in injecting sequentially samples at increasing concentrations and until now used exclusively to investigate bimolecular complexes by surface plasmon resonance, can be extended to the kinetic analysis of ternary complexes. DNA targets, B, were designed with sequences of variable lengths on their 3' sides that recognise a surface-immobilized biotinylated DNA anchor, A. These targets displayed on their 5' sides sequences that recognise DNA oligonucleotides of variable lengths, C, namely the analytes. Combinations of B and C DNA oligonucleotides on A generated ternary complexes each composed of two Watson-Crick helices displaying different kinetic properties. The target-analyte B-C duplexes were formed by sequentially injecting three increasing concentrations of the analytes C during the dissociation phase of the target B from the anchor A. The sensorgrams for the target-analyte complexes dissociating from the functionalized surface were successfully fitted by the SCK method while the target dissociated from the anchor, i.e. on a decaying surface. Within the range of applicability of the method which is driven by the rate of dissociation of the target from the anchor, the rate and equilibrium constants characteristic of these target-analyte duplexes of the ternary complexes did not depend on how fast the targets dissociated from the immobilized DNA anchor. In addition the results agreed very well with those obtained when such duplexes were analysed directly as bimolecular complexes, i.e. when the target, modified with a biotin, was directly immobilized onto a streptavidin sensor chip surface rather than captured by an anchor. Therefore the method we named SCKODS (Single-Cycle Kinetics On a Decaying Surface) can also be used to investigate complexes formed during a dissociation phase, in a ternary complex context. The SCKODS method can be combined with the SCK one to fully characterize the two bimolecular complexes of a ternary complex.     

Multi-analyte surface plasmon resonance biosensing

Surface plasmon resonance (SPR) biosensors are affinity sensing devices exploiting a special mode of electromagnetic field-surface plasmon-polariton-to detect the binding of analyte molecules from a liquid sample to biomolecular recognition elements immobilized on the surface of the sensor. In this paper, we review advances of SPR biosensor technology towards detection systems for the simultaneous detection of multiple analytes (multi-analyte detection). In addition, we report application of a recently developed multichannel SPR sensor based on spectroscopy of surface plasmons and wavelength division multiplexing of sensing channels to multi-analyte detection.     

Detection of immuno-complex formation via surface plasmon resonance on gold-coated diffraction gratings

The sensitivity of surface plasmon resonance techniques to changes in local interfacial refractive index has been exploited to detect immuno-complex formation in two model biochemical systems. A gold-coated diffraction grating has been used to excite surface plasmons at the gold/solution interface to which either human immunoglobulin G or the immunoglobulin fraction of sheep antiserum to human serum albumin was physically adsorbed. The complementary proteins, either affinity purified goat antihuman-IgG IgG or human serum albumin was subsequently specifically bound by immuno-complex formation. The binding reactions could be followed with respect to time.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

Direct detection of acetylcholinesterase inhibitor binding with an enzyme-based surface plasmon resonance sensor

Acetylcholinesterase (AChE) inhibitors are potentially lethal but also have applications as therapeutic drugs for neurodegenerative diseases such as Alzheimer’s. Enzyme inhibitor binding are difficult to be detected directly by surface plasmon resonance (SPR) due to their small molecular weight. In this article, we describe the detection of AChE inhibitor binding by SPR without the use of competitive binding or antibodies. AChE was immobilized on the gold surface of an SPR sensor through covalent attachment to a self-assembled monolayer (SAM) of a COOH-terminated alkanethiol. The activity of the immobilized protein and the surface density were determined by using a standard photometric assay. Binding of two reversible inhibitors, which are used as therapeutic drugs, was detectable by SPR without the need to further modify the surface or the use of other reagents. The binding affinities (K_A) obtained from the fits were 3.8 × 10³ M⁻¹ for neostigmine and 1.7 × 10³ M⁻¹ for eserine, showing a higher affinity of the sensor for neostigmine. We believe that the SPR sensor’s ability to detect these inhibitors is due to conformational changes of the enzyme structure on inhibitor binding.