Carbohydrate-lectin interactions assessed by surface plasmon resonance

The specificity, the strength, the kinetics and some thermodynamic parameters of sugar-protein interactions are easily assessed by surface plasmon resonance (SPR). This paper intends to present both theoretical and practical considerations. This includes: the principle of SPR, the analysis according to Langmuir and Scatchard, the problems linked either to mass transport limitation, to the heterogeneity of the immobilized ligand density or to the non-linearity due to cluster effects. The non-linearity may be taken into account by either one of two ways: the fractal or the Sips approaches that have been developed with the aim of linearizing the data. In addition, selected data obtained by using either immobilized carbohydrates or immobilized lectins are summarized. The SPR has also been found useful to collect information concerning oligosaccharide structure as well as lectin-sugar specificity and to develop new tools with medical applications. Finally, a series of practical considerations are gathered in the hope of avoiding some of the common pitfalls arising in sugar-lectin interaction studies based on the use of SPR.     

Evaluation of alpha-D-mannopyranoside glycolipid micelles-lectin interactions by surface plasmon resonance method

It is established that achieving higher binding affinities in carbohydrate-protein interactions requires multivalent presentations of the sugar ligands at the receptor binding site. Several inhibition, calorimetric, mass balance, and other studies have reiterated the beneficial effects of molecular level clustering of the sugar ligands for tight binding to the receptors. We have undertaken an effort to study the multivalent effects involving larger assemblies, represented by micelles, and their lectin interactions. The micelles were constituted with monomer bearing one- or two-sugar moieties at the monomolecular level and with varying the distances between the sugar moieties. Micellar aggregation studies and dynamic light scattering (DLS) studies afforded details of the aggregation numbers and the hydrodynamic diameters of various glycolipid (GL) micelles. The GL micelles were used as analytes of surface plasmon resonance (SPR) experiments on a lectin concanavalin A (Con A)-immobilized surface. SPR studies of the micelle-lectin interactions demonstrate that the ligand-receptor binding can be fit into the bivalent analyte model of interaction. Furthermore, micelles formed from two-sugar containing GLs are able to elicit favorable kinetic association rate constants in comparison to the micelles constituted with one-sugar containing GLs. The kinetic rate constants across the micelles and the effect of the sugar valencies in the GLs are discussed.     

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.     

Study of the subunit interactions in myosin phosphatase by surface plasmon resonance.

The interactions of the catalytic subunit of type 1 protein phosphatase (PP1c) and the N-terminal half (residues 1-511) of myosin phosphatase target subunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobilized on streptavidin-biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of PP1c was: MYPT11-296 > MYPT11-38 > MYPT123-38. No binding was detected with MYPT11-34, suggesting a critical role for residues 35-38, i.e. the PP1c binding motif. Binding of residues 1-22 was inferred from: a higher affinity binding to PP1c for MYPT11-38 compared to MYPT123-38, as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT11-38, but not by MYPT123-38. Residues 40-296 (ankyrin repeats) in MYPT11-296 inhibited the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nM), whereas MYPT11-38, MYPT123-38 or MYPT11-34 were without effect. MYPT140-511, which alone did not bind to PP1c, showed facilitated binding to the complexes of PP1c-MYPT11-38 and PP1c-MYPT123-38. The inhibitory effect of MYPT140-511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT11-38. The binding of MYPT1304-511 to complexes of PP1c and MYPT11-38, or MYPT11-296, was detected by SPR. These results suggest that within the N-terminal half of MYPT1 there are at least four binding sites for PP1c. The essential interaction is with the PP1c-binding motif and the other interactions are facilitated in an ordered and cooperative manner.     

Vesicle fusion studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Substrate-supported planar lipid bilayers are generated most commonly by the adsorption and transformation of phospholipid vesicles (vesicle fusion). We have recently demonstrated that simultaneous measurements of surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) are highly informative for monitoring lipid membranes on solid substrates. SPR and SPFS provide information on the amount and topography of adsorbed lipid membranes, respectively. In this study, the vesicle fusion process was studied in detail by measuring SPR-SPFS at a higher rate and plotting the obtained fluorescence intensity versus film thickness. We could track the initial adsorption of vesicles, the onset of vesicle rupture occurring at certain vesicle coverage of the surface, and the autocatalytic transformation into planar bilayers. We also monitored vesicle fusion of the same vesicle suspensions by quartz crystal microbalance with dissipation monitoring (QCM-D). We compared the results obtained from SPR-SPFS and QCM-D to highlight the unique information provided by SPR-SPFS.     

Antibody-antigen interactions measured by surface plasmon resonance: global fitting of numerical integration algorithms

The interactions between adenylate kinase (AK) and a monoclonal antibody against AK (McAb3D3) were examined by means of optical biosensor technology, and the sensograms were fitted to four models using numerical integration algorithms. The interaction of a solution of McAb3D3 with immobilized AK follows a double exponential function and the data fitted well to an inhomogeneous ligand model. The interaction of a solution AK with immobilized McAb3D3 follows a single exponential function and the data fitted well to a pseudo-first order reaction model. The true association constants of AK binding to McAb3D3 in solution were obtained from competition BIAcore measurements. The difference in results obtained with solid-phase BIAcore and competition BIAcore may be due to rebinding of the dissociated analyte to the immobilized surface. The results obtained with BIAcore are compared to those obtained by ELISA methods. We suggest that the best method for analysis of BIAcore data is direct, global fitting of sensorgrams to numerical integration algorithms corresponding to the different possible models for binding.     

Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping.

Surface plasmon resonance (SPR) detection requires no labeling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated SPR instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosampler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two-site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti-mouse IgG1 (RAMG1) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, the ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMG1 surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi-site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV-1 core protein p24 were used in all experiments.     

Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing

Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.     

Substrate-supported phospholipid membranes studied by surface plasmon resonance and surface plasmon fluorescence spectroscopy

Substrate-supported planar lipid bilayer membranes are attractive model cellular membranes for biotechnological applications such as biochips and sensors. However, reliable fabrication of the lipid membranes on solid surfaces still poses significant technological challenges. In this study, simultaneous surface plasmon resonance (SPR) and surface plasmon fluorescence spectroscopy (SPFS) measurements were applied to the monitoring of adsorption and subsequent reorganization of phospholipid vesicles on solid substrates. The fluorescence intensity of SPFS depends very sensitively on the distance between the gold substrate and the fluorophore because of the excitation energy transfer to gold. By utilizing this distance dependency, we could obtain information about the topography of the adsorbed membranes: Adsorbed vesicles could be clearly distinguished from planar bilayers due to the high fluorescence intensity. SPSF can also incorporate various analytical techniques to evaluate the physicochemical properties of the adsorbed membranes. As an example, we demonstrated that the lateral mobility of lipid molecules could be estimated by observing the recovery of fluorescence after photobleaching. Combined with the film thickness information obtained by SPR, SPR-SPFS proved to be a highly informative technique to monitor the lipid membrane assembly processes on solid substrates.     

Application of surface plasmon resonance toward studies of low-molecular-weight antigen-antibody binding interactions

Methods for studying low-molecular-weight antigen-antibody binding interactions using surface plasmon resonance detection are presented. The experimental parameters most relevant to studies of low-molecular-weight antigen-antibody binding interactions are discussed. Direct kinetic analysis of the binding interactions is most informative, providing both apparent association and dissociation rate constants from which equilibrium constants can be calculated. Equilibrium analysis, including steady-state and solution affinity studies, offers an alternative approach to direct kinetic analysis when knowledge of the individual kinetic rate constants is not required or difficult to determine. The various methods are illustrated by studies of an anti-T(4) Fab fragment binding interaction with several thyroxine analogs. The methods utilized were dependent on the affinity of the interaction. The high-affinity anti-T(4) Fab fragment/l-T(4) binding interaction was evaluated using direct kinetic analysis. An intermediate affinity anti-T(4) Fab fragment/l-T(3) binding interaction was evaluated using a combination of direct kinetic analysis, steady-state analysis, and solution affinity analysis. The relatively weak anti-T(4) Fab fragment/l-T(2) binding interaction was evaluated using steady-state and solution affinity analysis protocols. Several thyroxine tracers that could not be immobilized to a biosensor surface were also evaluated via the solution affinity format. In cases where a given binding interaction was examined using multiple methods the results were comparable.     

Analysis of protein interactions on protein arrays by a wavelength interrogation-based surface plasmon resonance biosensor

We have investigated whether surface plasmon resonance (SPR) sensors based on the wavelength interrogation are able to analyze protein interactions on protein arrays. The spectral SPR sensor was self-constructed and its detection limit, expressed as the minimal refractive index variation, was calculated to be 6.6x10(-5) with the signal fluctuation of 1.0x10(-5). The protein array surface was modified by a mixed thiol monolayer to immobilize proteins. Protein arrays were analyzed by the line-scanning mode of the SPR sensor, which scanned every 100 microm along the central line of array spots and the scanned results were presented by color spectra from blue to red. Glutathione S-transferase (GST)-rac1 caused a concentration-dependent increase of SPR wavelength shift on protein arrays. The surface structure of the protein arrays was analyzed by atomic force microscopy. Specific interactions of antigens with antibodies were analyzed on the protein arrays by using three antibodies and eight proteins. These results suggest that the wavelength interrogation-based SPR sensor can be used as the biosensor for the high-throughput analysis of protein interactions on protein arrays.     

Selective toxin-lipid membrane interactions of natural, haemolytic Scyphozoan toxins analyzed by surface plasmon resonance

A comparison of the molecular interaction of natural Scyphozoan lysins with their bioactivity in a haemolytic assay was performed by establishing an efficient, automatable and reproducible procedure for the measurement of protein-membrane interactions. The toxin-membrane interactions were analyzed utilising a chip-based technology with immobilized liposomes as artificial cell membranes. The technique was established with streptolysin O as a cholesterol-selective model toxin and its cholesterol-selectivity has been proven. The haemolytic potency of protein fractions derived from the venom of the jellyfish Aurelia aurita and Cyanea capillata was tested and EC50 values of 35.3 μg/mL and 43.1 μg/mL against sheep and 13.5 μg/mL and 8.8 μg/mL against rabbit erythrocytes were measured. Cell membrane binding as a first step in the haemolytic process was analyzed using the Biacore^® technology. Major cell membrane lipids (cholesterol, sphingomyelin and phosphatidylcholine) were immobilized as pure liposomes and in binary mixtures. A preference for cholesterol and sphingomyelin of both jellyfish species was demonstrated. The specificity of the method was proven with a non-haemolytic A. aurita protein fraction that did not express a lipid binding. Additionally, an inactivated C. capillata lysine with negligible haemolytic activity showed a remaining but reduced adsorption onto lipid layers. The binding level of the lytic venom fraction of these dominant boreal jellyfish species increased as a function of protein concentration. The binding strength was expressed in RU50 values ranging from 12.4 μg/mL to 35.4 μg/mL, which were in the same order of magnitude as the EC50 values in the haemolytic assay.     

Analysis of protein interactions on protein arrays by a novel spectral surface plasmon resonance imaging

We presented a novel surface plasmon resonance (SPR) imaging method for analysis of protein arrays based on a wavelength interrogation-based SPR biosensor. The spectral imaging was performed by the combination of position control and resonance wavelengths calculated from SPR reflectivity spectra. The imaging method was evaluated by analyzing interactions of glutathione S-transferase-fusion proteins with their antibodies. Antigen-antibody interactions were successfully analyzed on glutathione S-transferase-fusion protein arrays by using the spectral imaging method, and the results were confirmed by a parallel analysis using a previously used spectral SPR biosensor based on wavelength interrogation. Specific binding of anti-Rac1 and anti-RhoA to Rac1 and RhoA on the protein arrays was qualitatively and quantitatively analyzed by the spectral SPR imaging. Thus, it was suggested that the novel spectral SPR imaging was a useful tool for the high-throughput analysis of protein-protein interactions on protein arrays.     

Investigation of the interaction between DnaK and DnaJ by surface plasmon resonance spectroscopy.

Hsp70 chaperones assist protein folding through ATP-regulated transient association with substrates. Substrate binding by Hsp70 is controlled by DnaJ co-chaperones which stimulate Hsp70 to hydrolyze ATP and, consequently, to close its substrate binding cavity allowing trapping of substrates. We analyzed the interaction of the Escherichia coli Hsp70 homologue, DnaK, with DnaJ using surface plasmon resonance (SPR) spectroscopy. Resonance signals of complex kinetic characteristics were detected when DnaK was passed over a sensor chip with coupled DnaJ. This interaction was specific as it was not detected with a functionally defective DnaJ mutant protein, DnaJ259, that carries a mutation in the HPD signature motif of the conserved J-domain. Detectable DnaK-DnaJ interaction required ATP hydrolysis by DnaK and was competitively inhibited by chaperone substrates of DnaK. For DnaK mutant proteins with amino acid substitutions in the substrate binding cavity that affect substrate binding, the strength of detected interaction with DnaJ decreased proportionally with increased strength of the substrate binding defects. These findings indicate that the detected response signals resulted from DnaJ and ATP hydrolysis-dependent association of DnaJ as substrate for DnaK. Although not considered as physiologically relevant, this association allowed us to experimentally unravel the mechanism of DnaJ action. Accordingly, DnaJ stimulates ATP hydrolysis only after association of a substrate with the substrate binding cavity of DnaK. Further analysis revealed that this coupling mechanism required the J-domain of DnaJ and was also functional for natural DnaK substrates, and thus is central to the mechanism of action of the DnaK chaperone system.     

Surface plasmon resonance in protein-membrane interactions

Surface plasmon resonance (SPR) has become one of the most important techniques for studying macromolecular interactions. The most obvious advantages of SPR over other techniques are: direct and rapid determination of association and dissociation rates of binding process, no need for labelling of protein or lipids, and small amounts of sample used in the assay (often nM concentrations of proteins). In biochemistry, SPR is used mainly to study protein-protein interactions. On the other hand, protein-membrane interactions, although crucial for many cell processes, are less well studied. Recent advances in the preparation of stable membrane-like surfaces and the commercialisation of sensor chips has enabled widespread use of SPR in protein-membrane interactions. One of the most popular is Biacore's L1 sensor chip that allows capture of intact liposomes or even subcellular preparations. Lipid specificity of protein-membrane interactions can, therefore, be easily studied by manipulating the lipid composition of the immobilised membrane. The number of published papers has increased steadily in the last few years and the examples include domains or proteins that participate in cell signalling, pore-forming proteins, membrane-interacting peptides, coagulation factors, enzymes, amyloidogenic proteins, prions, etc. This paper gives a brief overview of different membrane-mimetic surfaces that can be prepared on the surface of SPR chips, properties of liposomes on the surface of L1 chips and some selected examples of protein-membrane interactions studied with such system.     

Integrated-optic biosensor by electro-optically modulated surface plasmon resonance

We present a new integrated-optic surface plasmon resonance (SPR) biosensor based on electro-optical modulation. The SPR characteristics for the analyte concentration detection can be electro-optically modulated by applying the voltage on the electrodes of the biosensor fabricated on lithium niobate, which is an excellent electro-optic material. Two measurement methods, electro-optically modulated SPR spectral measurement and electro-optically modulated SPR intensity measurement, are demonstrated and their measurands are the SPR wavelength and the output optical intensity, respectively. Human serum albumin is coated on the gold film surface of the proposed biosensor to detect the concentration of beta-blocker, which is a remedy for heart disease. As the applied voltage increases such that the effective index of guided mode rises, the SPR wavelength shifts toward the long wavelength side and the output optical intensity at the wavelength of 632.8 nm diminishes. The linear regression slope of the relation between the measurand and the applied voltage is dependent on the analyte concentration and can be used to determine the concentration variation. Experimental results measured by the electro-optically modulated SPR methods are compared with those measured by the conventional spectral and intensity methods, and the effects of waveguide width on the biosensor performance are discussed.     

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.