Surface plasmon resonance study of the molecular recognition between polymerase and DNA containing various mismatches and conformational changes of DNA-protein complexes

Although surface plasmon resonance (SPR) biosensor technique has been used to study protein-protein interactions and to detect conformational changes of proteins, it has not been shown whether the SPR biosensor can be used to study a complex kinetic system such as the protein-DNA binding, which sometimes involves several binding steps as well as dynamic conformational changes of the complexes. In this study, we have used SPR biosensor and T7 polymerase as the model system to study the interactions of the polymerase with a series of DNA template-primer duplexes containing different number of mismatches and GC contents at various positions near the primer 3'-end. In general, the binding constants measured by the SPR are several magnitudes smaller than those determined in solution, indicating the limitation of the surface-based technique for measuring solution-based interactions. However, the distinct polymerase binding profiles obtained for DNA duplexes differed by as low as a single mismatch suggest that the SPR data can be used for relative comparison purpose among a set of experiments carried out under identical conditions. The successful fitting of the binding profiles using the established translocation model also demonstrated that SPR can be used to monitor conformational changes, as well as to derive relative kinetic values, within a complicated DNA-protein interaction system. The results also demonstrated that SPR biosensor may be used as a sensitive technique for studying molecular recognition events, such as single-base discrimination involved in protein-DNA interactions.     

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed ''ligand'') is immobilized onto a sensor chip surface, while the other (the ''analyte'') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method''s high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter''s cognate substrate binding protein).     

Biosensor-surface plasmon resonance: quantitative analysis of small molecule-nucleic acid interactions

Surface plasmon resonance (SPR)-biosensor techniques directly provide essential information for the study and characterization of small molecule-nucleic acid interactions, and the use of these methods is steadily increasing. The method is label-free and monitors the interactions in real time. Both dynamic and steady-state information can be obtained for a wide range of reaction rates and binding affinities. This article presents the basics of the SPR technique, provides suggestions for experimental design, and illustrates data processing and analysis of results. A specific example of the interaction of a well-known minor groove binding agent, netropsin, with DNA is evaluated by both kinetic and steady-state SPR methods. Three different experiments are used to illustrate different approaches and analysis methods. The three sets of results show the reproducibility of the binding constants and agreement from both steady-state and kinetic analyses. These experiments also show that reliable kinetic information can be obtained, even with difficult systems, if the experimental conditions are optimized to minimize mass transport effects. Limitations of the biosensor-SPR technique are also discussed to provide an awareness of the care needed to conduct a successful experiment.     

Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review of new surface plasmon resonance technologies

Surface plasmon resonance (SPR) biosensors have enabled a wide range of applications in which researchers can monitor biomolecular interactions in real time. Owing to the fact that SPR can provide affinity and kinetic data, unique features in applications ranging from protein-peptide interaction analysis to cellular ligation experiments have been demonstrated. Although SPR has historically been limited by its throughput, new methods are emerging that allow for the simultaneous analysis of many thousands of interactions. When coupled with new protein array technologies, high-throughput SPR methods give users new and improved methods to analyze pathways, screen drug candidates and monitor protein-protein interactions.     

Surface plasmon resonance: a useful technique for cell biologists to characterize biomolecular interactions

Surface plasmon resonance (SPR) is a powerful technique for monitoring the affinity and selectivity of biomolecular interactions. SPR allows for analysis of association and dissociation rate constants and modeling of biomolecular interaction kinetics, as well as for equilibrium binding analysis and ligand specificity studies. SPR has received much use and improved precision in classifying protein–protein interactions, as well as in studying small-molecule ligand binding to receptors; however, lipid–protein interactions have been underserved in this regard. With the field of lipids perhaps the next frontier in cellular research, SPR is a highly advantageous technique for cell biologists, as newly identified proteins that associate with cellular membranes can be screened rapidly and robustly for lipid specificity and membrane affinity. This technical perspective discusses the conditions needed to achieve success with lipid–protein interactions and highlights the unique lipid–protein interaction mechanisms that have been elucidated using SPR. It is intended to provide the reader a framework for quantitative and confident conclusions from SPR analysis of lipid–protein interactions.     

Computational and experimental investigation of DNA repair protein photolyase interactions with low molecular weight drugs

This paper reports the previously unknown interactions between eight low molecular weight commercially available drugs (130–800 Da) and DNA repair protein photolyase using computational docking simulations and surface plasmon resonance (SPR) experiments. Theoretical dissociation constants, K_d, obtained from molecular docking simulations were compared with the values found from SPR experiments. Among the eight drugs analyzed, computational and experimental values showed similar binding affinities between selected drug and protein pairs. We found no significant differences in binding interactions between pure and commercial forms of the drug lornoxicam and DNA photolyase. Among the eight drugs studied, prednisone, desloratadine, and azelastine exhibited the highest binding affinity (K_d = 1.65, 2.05, and 8.47 μM, respectively) toward DNA photolyase. Results obtained in this study are promising for use in the prediction of unknown interactions of common drugs with specific proteins such as human clock protein cryptochrome. Copyright © 2013 John Wiley & Sons, Ltd.     

Semiquantitative and quantitative analysis of protein–DNA interactions using steady-state measurements in surface plasmon resonance competition experiments

One method commonly used to characterize protein–DNA interactions is surface plasmon resonance (SPR). In a typical SPR experiment, chip-bound DNA is exposed to increasing concentrations of protein; the resulting binding data are used to calculate a dissociation constant for the interaction. However, in cases in which knowledge of the specificity of the interaction is required, a large set of DNA variants has to be tested; this is time consuming and costly, in part because of the requirement for multiple SPR chips. We have developed a new protocol that uses steady-state binding levels in SPR competition experiments to determine protein-binding dissociation constants for a set of DNA variants. This approach is rapid and straightforward and requires the use of only a single SPR chip. Additionally, in contrast to other methods, our approach does not require prior knowledge of parameters such as on or off rates, using an estimate of the wild-type interaction as the sole input. Utilizing relative steady-state responses, our protocol also allows for the rapid, reliable, and simultaneous determination of protein-binding dissociation constants of a large series of DNA mutants in a single experiment in a semiquantitative fashion. We compare our approach to existing methods, highlighting specific advantages as well as limitations.     

Molecular interactions in the insulin-like growth factor (IGF) axis: a surface plasmon resonance (SPR) based biosensor study

This review describes a comprehensive analysis of a surface plasmon resonance (SPR)-based biosensor study of molecular interactions in the insulin-like growth factor (IGF) molecular axis. In this study, we focus on the interaction between the polypeptide growth factors IGF-I and IGF-II with six soluble IGF binding proteins (IGFBP 1-6), which occur naturally in various biological fluids. We have describe the conditions required for the accurate determination of kinetic rate constants for these interactions and highlight the experimental and theoretical pitfalls, which may be encountered in the early stages of such a study. We focus on IGFBP-5 and describe a site-directed mutagenesis study, which examines the contribution of various residues in the protein to high affinity interaction with IGF-I and -II. We analyse the interaction of IGFBP-5 (and IGFBP-3) with heparin and other biomolecules and describe experiments, which were designed to monitor multi-protein complex formation in this molecular axis.     

Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet

ForteBio’s Octet optical biosensor harnesses biolayer interferometry to detect and quantify molecular interactions using disposable fiber-optic biosensors that address samples from an open shaking microplate without any microfluidics. We recruited a monoclonal antibody against a panel of peptides to compare the Octet directly with Biacore’s well-established 3000 platform and Bio-Rad’s recently launched ProteOn XPR36 array system, which use surface plasmon resonance (SPR) to detect the binding of one analyte over four surfaces and six analytes over six surfaces, respectively. A sink method was used to prevent analyte from rebinding the ligand-coated Octet tips and enabled us to extract accurate kinetic rate constants, as judged by their close agreement with those determined by SPR. Although the Octet is not sensitive enough to detect the binding of small molecules directly, it can access their affinities indirectly via solution competition experiments. We conducted similar experiments on the SPR instruments to validate these measurements. The Octet is emerging as a versatile complement to other more sophisticated biosensors, and the ProteOn provides high-quality data near the sensitivity of Biacore but in a more multiplexed format. Our results provide a benchmark for assessing the performance of the above-mentioned sensors.     

Kinetic analysis of interaction of BRCA1 tandem breast cancer c-terminal domains with phosphorylated peptides reveals two binding conformations

Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants ( K d) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the K d values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.     

Disruption of shape complementarity in the ribosomal protein L1–RNA contact region does not hinder specific recognition of the RNA target site

The formation of a specific and stable complex between two (macro)molecules implies complementary contact surface regions. We used ribosomal protein L1, which specifically binds a target site on 23S rRNA, to study the influence of surface modifications on the protein?RNA affinity. The threonine residue in the universally conserved triad Thr?Met?Gly significant for RNA recognition and binding was substituted by phenylalanine, valine and alanine, respectively. The crystal structure of the mutant Thr217Val of the isolated domain I of L1 from Thermus thermophilus (TthL1) was determined. This structure and that of two other mutants, which had been determined earlier, were analysed and compared with the structure of the wild type L1 proteins. The influence of structural changes in the mutant L1 proteins on their affinity for the specific 23S rRNA fragment was tested by kinetic experiments using surface plasmon resonance (SPR) biosensor analysis. Association rate constants undergo minor changes, whereas dissociation rate constants displayed significantly higher values in comparison with that for the wild type protein. The analysed L1 mutants recognize the specific RNA target site, but the mutant L1?23S rRNA complexes are less stable compared to the wild type complexes. Copyright © 2010 John Wiley & Sons, Ltd.     

Probing the impact of GFP tagging on Robo1-heparin interaction

Green fluorescent proteins (GFPs) and their derivatives are widely used as markers to visualize cells, protein localizations in in vitro and in vivo studies. The use of GFP fusion protein for visualization is generally thought to have negligible effects on cellular function. However, a number of reports suggest that the use of GFP may impact the biological activity of these proteins. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins mediating diverse patho-physiological processes. In the heparin-based interactome studies, heparin-binding proteins are often prepared as GFP fusion proteins. In this report, we use surface plasmon resonance (SPR) spectroscopy to study the impact of the GFP tagging on the binding interaction between heparin and a heparin-binding protein, the Roundabout homolog 1 (Robo1). SPR reveals that heparin binds with higher affinity to Robo1 than GFP-tagged Robo1 and through a different kinetic mechanism. A conformational change is observed in the heparin-Robo1 interaction, but not in the heparin-Robo1-GFP interaction. Furthermore the GFP-tagged Robo1 requires a shorter (hexasaccharide) than the tag-free Robo1 (octadecasaccharide). These data demonstrate that GFP tagging can reduce the binding affinity of Robo1 to heparin and hinder heparin binding-induced Robo1 conformation change.     

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.     

Bayesian Estimation of the Active Concentration and Affinity Constants Using Surface Plasmon Resonance Technology

Surface plasmon resonance (SPR) has previously been employed to measure the active concentration of analyte in addition to the kinetic rate constants in molecular binding reactions. Those approaches, however, have a few restrictions. In this work, a Bayesian approach is developed to determine both active concentration and affinity constants using SPR technology. With the appropriate prior probabilities on the parameters and a derived likelihood function, a Markov Chain Monte Carlo (MCMC) algorithm is applied to compute the posterior probability densities of both the active concentration and kinetic rate constants based on the collected SPR data. Compared with previous approaches, ours exploits information from the duration of the process in its entirety, including both association and dissociation phases, under partial mass transport conditions; do not depend on calibration data; multiple injections of analyte at varying flow rates are not necessary. Finally the method is validated by analyzing both simulated and experimental datasets. A software package implementing our approach is developed with a user-friendly interface and made freely available.     

Quantitative analysis of CUG-BP1 binding to RNA repeats

CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-3-like factors (CELF) family of RNA-binding proteins, and is involved in myotonic dystrophy type 1 (DM1). Several mRNA targets of CUG-BP1 have been identified, including the insulin receptor, muscle chloride channel, and cardiac troponin T. On the other hand, CUG-BP1 has only a weak affinity for CUG repeats. We conducted quantitative-binding assays to assess CUG-BP1 affinities for several repeat RNAs by surface plasmon resonance (SPR). Although we detected interactions between CUG-BP1 and CUG repeats, other UG-rich sequences actually showed stronger interactions. Binding constants of CUG-BP1 for RNAs indicated that the affinity for UG repeats was far stronger than for CUG repeats. We also found that N-terminal deletion mutant of CUG-BP1 has UG repeat-binding activity in a yeast three-hybrid system, although C-terminal deletion mutant does not. Our data indicates that CUG-BP1 specifically recognized UG repeats, probably through cooperative binding of RNA recognition motifs at both ends of the protein. This is the first report of a binding constant for CUG-BP1 calculated in vitro.     

Application of surface plasmon resonance for analysis of protein-protein interactions in the G protein-mediated signal transduction pathway

Hundreds of extracellular stimuli are received by cells via the pathways consisting of three basic components: cell-surface receptors, heterotrimeric G proteins, and intracellular effector enzymes or ion channels. A number of additional molecules, including G protein-coupled receptor kinases (GRKs), phosducin and Ca(2+)-binding proteins modulate signal transduction through these cascades. Understanding how these universal pathways work requires a detailed analysis of the interactions between these proteins. The recently emerged technology of surface plasmon resonance (SPR) can study protein-protein interactions by measuring not only the equilibrium binding constants, but also the association and dissociation rates. This article reviews experimental design used by researchers to analyze different components of the G protein pathway by SPR and focuses on the insights this technique provides regarding the kinetics, structure-function aspects and regulation of specific molecular events in the cascade.     

Kinetic determinations of molecular interactions using Biacore--minimum data requirements for efficient experimental design

Reliable kinetic estimates can be obtained from significantly less data than is commonly used today, particularly in the characterization of 1:1 interactions involving low molecular weight compounds and proteins. We have designed a rational and cost-effective strategy to determine kinetic constants using Biacore's surface plasmon resonance-based biosensors and show that the number of measurements necessary for accurate kinetic determinations can be greatly reduced, increasing sample throughput and saving sample material. Simulated and measured data for a range of possible 1:1 interactants were studied to find the minimum requirements of a data set for kinetic analysis. The results showed that kinetic constants in the region 10(4) < k(a) < 10(7) M(-1) s(-1) (association) and 10(-4) < k(d) < 10(-1) s(-1) (dissociation) could easily be determined in a 1:1 interaction model. Owing to the information-dense nature of Biacore data, only two sample concentrations were necessary to reliably determine the kinetics. A standard sample concentration series consisting of 10-fold dilutions between approximately 10 microM and approximately 1 nM consistently provided at least two concentrations with sufficient information about the interaction in this region. Determinations of the constants became increasingly unreliable outside this region. If the rate constants prove to be outside the specified region or the data fits poorly to the 1:1-MTL model, more experiments are required. General recommendations for the design of a cost-effective assay to deliver reliable kinetic measurements are provided.     

Transglutaminase cross-linking properties of the small proline-rich 1 family of cornified cell envelope proteins. Integration with loricrin

Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no alpha or beta structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.     

Altered amelogenin self-assembly based on mutations observed in human X-linked amelogenesis imperfecta (AIH1)

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin, a 180-amino acid hydrophobic protein that self-assembles to form nanospheres. We have used independent technical methods, consisting of the yeast two-hybrid (Y2H) assay and surface plasmon resonance (SPR), to demonstrate the importance of amelogenin self-assembly domains. In addition, we have analyzed mutations in amelogenin observed in patients with amelogenesis imperfecta who demonstrate defects in enamel formation. Assessments of self-assembly of these mutant amelogenins by either SPR or Y2H assay yield concordant data. These data support the conclusion that the amelogenin amino-terminal self-assembly domain is essential to the creation of an enamel extracellular organic matrix capable of directing mineral formation. It also suggests that a pathway through which point mutations in the amelogenin protein can adversely impact on the formation of the enamel organ is by disturbing self-assembly of the organic matrix. These data support the utilization of the Y2H assay to search for protein interactions among extracellular matrix proteins that contribute to biomineralization and provide functional information on protein-protein and protein-mineral interactions.