Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.     

Kinetic analysis and fragment screening with Fujifilm AP-3000

We evaluated the performance of Fujifilm’s new AP-3000 surface plasmon resonance biosensor for kinetic analysis and fragment screening. Using carbonic anhydrase II as a model system, we characterized a set of 10 sulfonamide-based inhibitors that range in molecular mass from 98 to 341 Da and approximately 10,000-fold in affinity (0.4 mM to 20 nM). Although the data collected from the AP-3000 were generally similar to those collected using a Biacore T100, the AP-3000’s stop-flow analyte delivery system complicated the shapes of the association- and dissociation-phase binding responses. We illustrate how reasonable estimates of the kinetic rate constants can be extracted from AP-3000 data by limiting data analysis to only the regions of the responses collected during flow conditions. We also provide an example of the results obtained for a fragment-screening study with the AP-3000, which is the ideal application of this technology.     

Biacore analysis with stabilized G-protein-coupled receptors

Using stabilized forms of β₁ adrenergic and A_2A adenosine G-protein-coupled receptors, we applied Biacore to monitor receptor activity and characterize binding constants of small-molecule antagonists spanning more than 20,000-fold in affinity. We also illustrate an improved method for tethering His-tagged receptors on NTA (carboxymethylated dextran preimmobilized with nitrilotriacetic acid) chips to yield stable, high-capacity, high-activity surfaces as well as a novel approach to regenerate receptor binding sites. Based on our success with this approach, we expect that the combination of stabilized receptors with biosensor technology will become a common method for characterizing members of this receptor family.     

High-resolution characterization of antibody fragment/antigen interactions using Biacore T100

A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.     

Label-enhanced surface plasmon resonance applied to label-free interaction analysis of small molecules and fragments

Surface plasmon resonance (SPR) is a well-established method for studying interactions between small molecules and biomolecules. In particular, SPR is being increasingly applied within fragment-based drug discovery; however, within this application area, the limited sensitivity of SPR may constitute a problem. This problem can be circumvented by the use of label-enhanced SPR that shows a 100-fold higher sensitivity as compared with conventional SPR. Truly label-free interaction data for small molecules can be obtained by applying label-enhanced SPR in a surface competition assay format. The enhanced sensitivity is accompanied by an increased specificity and inertness toward disturbances (e.g., bulk refractive index disturbances). Label-enhanced SPR can be used for fragment screening in a competitive assay format; the competitive format has the added advantage of confirming the specificity of the molecular interaction. In addition, label-enhanced SPR extends the accessible kinetic regime of SPR to the analysis of very fast fragment binding kinetics. In this article, we demonstrate the working principles and benchmark the performance of label-enhanced SPR in a model system—the interaction between carbonic anhydrase II and a number of small-molecule sulfonamide-based inhibitors.     

Strategies for the identification and purification of ligands for orphan biomolecules

Summary The isolation of related genes with evolutionary conserved motifs by the application of polymerase chain reaction-based molecular biology techniques, or from database searching strategies, has facilitated the identification of new members of protein families. Many of these protein molecules will be involved in protein-protein interactions (e.g. growth factors, receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellular process. However, the precise biological function and specific binding partners of these novel proteins are frequently unknown, hence they are known as ‘orphan’ molecules. Complementary technologies are required for the identification of the specific ligands or receptors for these and other orphan proteins (e.g., antibodies raised against crude biological extracts or whole cells). We describe herein several alternative strategies for the identification, purification and characterisation of orphan peptide and protein molecules, specifically the synergistic use of micropreparative HPLC and biosensor techniques. These authors made equivalent contributions.     

Exploring "one-shot" kinetics and small molecule analysis using the ProteOn XPR36 array biosensor

A ProteOn XPR36 parallel array biosensor was used to characterize the binding kinetics of a set of small molecule/enzyme interactions. Using one injection with the ProteOn's crisscrossing flow path system, we collected response data for six different concentrations of each analyte over six different target protein surfaces. This "one-shot" approach to kinetic analysis significantly improves throughput while generating high-quality data even for low-molecular-mass analytes. We found that the affinities determined for nine sulfonamide-based inhibitors of the enzyme carbonic anhydrase II were highly correlated with the values determined using isothermal titration calorimetry. We also measured the temperature dependence (from 15 to 35 degrees C) of the kinetics for four of the inhibitor/enzyme interactions. Our results illustrate the potential of this new parallel-processing biosensor to increase the speed of kinetic analysis in drug discovery and expand the applications of real-time protein interaction arrays.     

Surface plasmon resonance characterization of calspermin-calmodulin binding kinetics

We cloned, expressed, and purified a chimeric fusion between a soluble green fluorescent protein (smGFP) and the calmodulin binding protein calspermin. We have shown that the fusion protein, labeled smGN, has a K(i) in the calmodulin-dependent cyclic nucleotide phosphodiesterase activity assay of 1.97 nM, i.e., 3800 times smaller than that of the commonly used calmodulin inhibitor W7. Association and dissociation rate constants (k(a) and k(d)) and the dissociation equilibrium constant (K(D)) of smGN for calmodulin were determined using surface plasmon resonance (SPR). The k(a)=1.24 x 10(6)M(-1)s(-1), the k(d)=5.49 x 10(-3)s(-1), and the K(D)=4.42 x 10(-9)M. We also found that the GFP moiety was important for successfully binding calspermin to the surface of the CM5 flow cell at a sufficiently high concentration for SPR, and that this procedure may be used for SPR analysis of other acidic polypeptides, whose pI< or =4. To determine whether smGN might also bind to other calmodulin-like proteins in a heterologous system, we purified proteins from a plant total cell extract or a plant total protein extract by affinity chromatography against smGN. The purified proteins were identified as calmodulins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry, indicating a high level of specificity. We conclude that the high affinity and specific binding between smGN and calmodulin make it an easily localized recombinant alternative to chemical calmodulin inhibitors.     

Screening antibody-antigen interactions in parallel using Biacore A100

Label-free optical biosensor technology has become a standard tool for characterizing monoclonal antibodies for therapeutic and diagnostic applications. The availability of high-quality binding data at an early stage greatly improves the ability to select antibodies for further development. This article shows how Biacore A100, a protein interaction array system, is capable of providing high-quality data with increased throughput. In a 12-h automated run, we analyzed 386 crude hybridoma samples to identify those with the desired kinetic profiles. Selected antibodies were further characterized by higher resolution analysis, and binding interactions were studied under a range of buffer conditions. We demonstrate how this new parallel processing system significantly expands the throughput of protein interaction analysis while maintaining data quality.     

Solubilization, stabilization, and purification of chemokine receptors using biosensor technology

Establishing solubilization conditions for membrane-associated receptors is often a tedious empirical process. Here we describe a novel application of SPR biosensor technology to screen solubilization conditions automatically and to assess receptor activity directly. We focus on two chemokine receptors, CXCR4 and CCR5, which are important in HIV cell invasion. The autosampler in Biacore 3000 permitted whole cells expressing C-terminally tagged receptors to be automatically lysed under a given solubilization condition and the lysates to be injected over an antibody surface. The total amount of solubilized receptor could be quantitated from the antibody capture level, whereas the amount of active receptor could be quantitated using a subsequent injection of conformationally sensitive antibody or protein. Using this approach, we identified detergent/lipid/buffer combinations that enhanced and maintained receptor activity. We also used the biosensor to demonstrate CD4-dependent binding of gp120 to solubilized CCR5 and to develop affinity chromatography-based purification methods that increased receptor activity more than 300%. Together, these results illustrate the benefits of using the biosensor as a tool for isolating functional membrane receptors and for analyzing ligand/receptor interactions.     

Estimation of analyte concentration by surface plasmon resonance-based biosensing using parameter identification techniques

Surface plasmon resonance-based biosensors have been applied to the determination of macromolecule concentration. Up to now, the proposed experimental approaches have relied either on the generation of a calibration curve that exploits only a few data points from each sensorgram or on multiple injections of the unknown sample at various flow rates. In this article, we show that prior knowledge of the kinetic parameters related to the interaction of the species with a given partner could advantageously reduce the number of injections required by both aforementioned methods, thereby reducing experimental time while maintaining a good level of confidence on the determined concentrations.     

Determination of half-maximal inhibitory concentration using biosensor-based protein interaction analysis

Half-maximal inhibitory concentration (IC₅₀) is the most widely used and informative measure of a drug's efficacy. It indicates how much drug is needed to inhibit a biological process by half, thus providing a measure of potency of an antagonist drug in pharmacological research. Most approaches to determine IC₅₀ of a pharmacological compound are based on assays that utilize whole cell systems. While they generally provide outstanding potency information, results can depend on the experimental cell line used and may not differentiate a compound's ability to inhibit specific interactions. Here we show using the secreted Transforming Growth Factor-β (TGF-β) family ligand BMP-4 and its receptors as example that surface plasmon resonance can be used to accurately determine IC₅₀ values of individual ligand-receptor pairings. The molecular resolution achievable wih this approach can help distinguish inhibitors that specifically target individual complexes, or that can inhibit multiple functional interactions at the same time.     

A global benchmark study using affinity-based biosensors

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.     

Development of biosensor-based assays to identify anti-infective oligosaccharides

It is now well accepted that milk oligosaccharides can have a direct inhibitory effect on pathogenic microorganisms by interfering with their adhesion to human cells. Many free oligosaccharides from milk are considered to be soluble receptor analogs of epithelial cell surface carbohydrates and, thus, function as receptor decoys to which pathogens can bind instead of the host. In reality, there are few rapid methods to screen for such oligosaccharides, and much of the research in this area has centered on using human cell line models of infection that are time-consuming. Therefore, a quick and sensitive method is required for detecting the binding of microorganisms to milk oligosaccharides. Our study describes a number of biosensor-based methods to achieve these aims. Our approach involved the exposure of whole bacterial cells to the well-characterized human milk oligosaccharide, 2′-fucosyllactose, immobilized to a pretreated gold chip surface. The technique was validated by screening a range of pathogenic bacteria, including Campylobacter jejuni, to which 2′-fucosyllactose is known to bind. Where binding was detected, its specificity was confirmed by preincubation studies using unlabeled 2′-fucosyllactose. The techniques described represent a quick, cost-effective, and highly reproducible detection method for identifying anti-infective oligosaccharides.     

Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors

Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of a fusion partner that was inserted into the third intracellular loop. Using chimeras of the human β(2)-adrenergic and human A(2A) adenosine receptors, we present the methodology and data for the initial selection of an expanded set of fusion partners for crystallizing GPCRs. In particular, use of the thermostabilized apocytochrome b(562)RIL as a fusion partner displays certain advantages over previously utilized fusion proteins, resulting in?a significant improvement in stability and structure of GPCR-fusion constructs.     

SPR生物传感器及其应用进展

基于表面等离子体共振 (SPR)技术的光学生物传感器是进行生物分子相互作用分析的一种先进手段。与传统的超速离心、荧光法等相比 ,它具有实时检测、无需标记、耗样最少等特点 ,在药物筛选、临床诊断、食物及环境监控和膜生物学等领域中的新兴应用日益扩大 ,并且已成为生命科学和制药研究的一种标准的生物物理学工具。综述了近几年国际上生物传感器的应用进展情况 ,并简要展望了该技术的发展和应用前景     

Computational design of a new hydrogen bond network and at least a 300-fold specificity switch at a protein-protein interface

The redesign of protein-protein interactions is a stringent test of our understanding of molecular recognition and specificity. Previously we engineered a modest specificity switch into the colicin E7 DNase-Im7 immunity protein complex by identifying mutations that are disruptive in the native complex, but can be compensated by mutations on the interacting partner. Here we extend the approach by systematically sampling alternate rigid body orientations to optimize the interactions in a binding mode specific manner. Using this protocol we designed a de novo hydrogen bond network at the DNase-immunity protein interface and confirmed the design with X-ray crystallographic analysis. Subsequent design of the second shell of interactions guided by insights from the crystal structure on tightly bound water molecules, conformational strain, and packing defects yielded new binding partners that exhibited specificities of at least 300-fold between the cognate and the non-cognate complexes. This multi-step approach should be applicable to the design of polar protein-protein interactions and contribute to the re-engineering of regulatory networks mediated by protein-protein interactions.     

G蛋白偶联受体与G蛋白相互作用的最新研究进展

传统观念认为,在激动剂作用下,G蛋白偶联受体(GPCRs)能够激活G蛋白的α亚基,从而使Gα亚基与Gβγ亚基分离,被激活的Gα亚基通过信号转导进一步参与细胞的生理过程。但是,最新研究发现GPCRs和G蛋白存在多种偶联关系,GPCRs不仅能够激活Gα亚基,还可以与Gβγ亚基相互靠近,甚至会使G蛋白亚基构象发生重排而不分离,这对于疾病发病机制的研究及新的药物靶点的发现具有重要意义。就GPCRs与G蛋白之间的相互作用以及最新研究技术作一简要综述。     

Kinetic analysis of a high-affinity antibody/antigen interaction performed by multiple Biacore users

To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.