Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics

The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a “fit-for-purpose” approach in instrument selection for biosensor studies.     

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.     

GEC1, a protein related to GABARAP, interacts with tubulin and GABA(A) receptor

We have previously identified in uterine cells a novel estrogen-regulated gene called gec1. GEC1 presents 87% identity with GABARAP which, so far, was the only protein found to associate with tubulin and GABA(A) receptor. We demonstrated then that GEC1 interacts in vitro with tubulin and GABA(A) receptor, and promotes tubulin assembly and microtubule bundling. Since all polyclonal antibodies failed in discrimination of both proteins GEC1 and GABARAP, a GEC1-GFP fusion protein was used to specifically localize GEC1. GEC1-GFP was distributed over the cytoplasm in perinuclear vesicles with a scattered pattern. Overall, our data show that GEC1 could be a new member of the GABARAP family involved in the transport of GABA(A) receptor.     

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.     

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.     

Studies of small molecule interactions with protein phosphatases using biosensor technology

Reversible protein phosphorylation of serine, threonine, and tyrosine residues by protein kinases and phosphatases is important for the regulation of cellular signal transduction and controls many cellular functions. Disturbances in this regulation have been implicated in a growing number of diseases, making kinases and phosphatases useful targets for therapeutic intervention. The suitability of surface plasmon resonance (SPR) technology has been widely demonstrated in many drug discovery applications. A novel and straightforward methodology is presented for analyzing small molecule binding to two serine/threonine phosphatases, PP1 and PP2B (calcineurin), and to the prototypic tyrosine phosphatase, PTP1B. Emphasis was placed on investigating the immobilization conditions of the phosphatases by using reducing conditions, inhibitors and metal ions. A comparison of inhibitor binding, either to phosphatase (PP2B) alone or in complex with the regulatory protein subunit calmodulin, revealed different kinetics. The methodology was also used to test inhibitor specificity toward different phosphatases. Inhibition of regulatory protein PP-inhibitor-2 binding to PP1 by a small molecule inhibitor was demonstrated. This type of information, together with data on compound binding that is independent of enzyme activity and in which affinities are resolved into kinetic rate constants, may be of great significance for the development of highly specific and high-affinity phosphatase inhibitors.     

Kinetic studies of small molecule interactions with protein kinases using biosensor technology

Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are few examples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for better developed assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study small molecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composed assay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds to immobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and competition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor binding and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. This might be of key importance for the development of effective kinase inhibitors.     

New dimensions in the study of protein complexes using quantitative mass spectrometry

Mass spectrometry combined with affinity purification techniques has evolved as a prime tool for the in-depth study of distinct protein complexes and protein-protein interactions. It fueled proteome-wide studies leading to the establishment of intricate cellular protein interaction networks. Recent innovative advancements in quantitative protein mass spectrometry act as driving force for the design of ingenious strategies in interaction proteomics facilitating the acquisition of interaction data with improved accuracy and, most intriguingly, the elucidation of functional aspects by monitoring transient interactions as well as dynamic changes in composition, stoichiometry, localization and post-translational modification of protein complexes under various conditions.     

Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays

Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of β-d-glucose (negative control), α-d-mannose (conA-responsive), β-d-galactose (RCA₁₂₀-responsive) and N-acetyl-β-d-glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA₁₂₀ was passed over the array surface. Limited or no binding was observed for the non-cognate ligands. SPR imaging of an array of 40 sialylated and unsialylated glycans established the binding preference of hSiglec7 for α2-8-linked disialic acid structures over α2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than α2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10–20 μg of lectin per experiment. The SPR imaging technique was also able to establish selective binding to the preferred glycan ligand when analyzing crude culture supernatant containing 10–20 μg of recombinant hSiglec7-Fc. Our results show that SPR imaging provides results that are in agreement with those obtained from fluorescence based carbohydrate arrays but with the added advantage of label-free analysis.     

Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies

We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24 h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1 h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000 Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.     

Synthesis,DNA and protein interactions and human topoisomerase inhibition of novel Spiroacridine derivatives

Nine new spiroacridine derivatives were synthetized by introducing cyano-N-acylhydrazone group between the acridine and phenyl-substituted rings followed by spontaneous cyclization. The new compounds were assayed for their DNA binding properties, human topoisomerase IIα inhibition and bovine serum albumin (BSA) interaction. Besides, docking analysis were performed in order to better understanding the biomolecule-compounds interactions. All compounds interacted with BSA which was demonstrated by the fluorescence suppression constant of 10⁴?M^?1. Compounds with chloro and NO₂ substituents at that para-position on phenyl ring demonstrated the best results for BSA interaction. DNA binding constant determined by UV–vis data demonstrated high values for AMTAC-11 and AMTAC-14, 1.1?×?10⁸?M^?1 and 4.8?×?10⁶?M^?1, respectively, and all others presented constant values of 10⁵?M^?1. AMTAC-06 with chloro at para-position on phenyl ring presented a topoisomerase II inhibition of 84.34% in comparison to the positive controls used. Docking studies indicated that AMTAC-06 is able to intercalate the DNA base pairs at topoisomerase IIα active site, preventing DNA connection after break, in a process known as poisoning. Topoisomerase enzyme inhibition result was correlated to BSA interaction profile, since AMTAC-06 showed the best results in both analysis. The findings obtained here proved that methoxy or chloro substitution on phenyl ring at para-position is fundamental for in vitro activity of new spiroacridine derivatives, and indicates that AMTAC-06 is a promising entity and should serve as a lead compound in the development of new DNA and protein binders, as well as human topoisomerase II inhibitors.     

Surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of HPV, E6, E6AP, and p53

We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein-protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein-protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein-protein interactions where multiple proteins are involved.     

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

Identifying and characterizing protein interactions are fundamental steps toward understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the colocalization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin α (Impα), and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified colocalization assay is sensitive enough to detect protein interactions with K_d values that span over four orders of magnitude (1 nM to 15 μM). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris.     

Construction of a 19F-lectin biosensor for glycoprotein imaging by using affinity-guided DMAP chemistry

In this study, assisted by affinity-guided DMAP strategy, we developed a novel ¹⁹F-modified lectin as a biosensor for specific detection and imaging of glycoproteins. Exploited the large chemical shift anisotropy property of ¹⁹F nuclei, glycoproteins detected by our ¹⁹F-biosensor are signatured by broadened peaks in ¹⁹F NMR, hence enabled the distinction between glycoproteins and small molecule saccharides. Such signal on/off switching was also applied to glycoprotein imaging by ¹⁹F MRI.     

Real-time quantitation of viral replication and inhibitor potency using a label-free optical biosensor

Real-time detection of viral replication inside cells remains a challenge to researchers. The Epic^® System is a high-throughput, label-free optical detection platform capable of measuring molecular interaction in a biochemical assay, as well as integrated cellular response from measurement of cellular dynamic mass redistribution (DMR) in a cell-based assay. DMR has previously been used to measure cell signaling upon receptor stimulation. In this report, we present the first example of Epic^® measurement of viral replication-induced cellular response and demonstrate that this system is extremely powerful not only for the sensitive and quantitative detection of viral replication inside cells but also for screening of viral inhibitors. By comparing with conventional assays used for the measurement of viral replication, we show that the Epic^® response has many advantages including sensitivity, high throughput, real-time quantification and label-free detection. We propose that the Epic^® system for measurement of integrated cellular response will be an excellent method for elucidating steps in viral replication as well as for the high-throughput screening of inhibitors of rhinovirus and other viruses.     

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.     

Faster and more accurate global protein function assignment from protein interaction networks using the MFGO algorithm

MOTIVATION: Predicting protein function accurately is an important issue in the post-genomic era. To achieve this goal, several approaches have been proposed deduce the function of unclassified proteins through sequence similarity, co-expression profiles, and other information. Among these methods, the global optimization method (GOM) is an interesting and powerful tool that assigns functions to unclassified proteins based on their positions in a physical interactions network [Vazquez, A., Flammini, A., Maritan, A. and Vespignani, A. (2003) Global protein function prediction from protein-protein interaction networks, Nat. Biotechnol., 21, 697-700]. To boost both the accuracy and speed of GOM, a new prediction method, MFGO (modified and faster global optimization) is presented in this paper, which employs local optimal repetition method to reduce calculation time, and takes account of topological structure information to achieve a more accurate prediction. CONCLUSION: On four proteins interaction datasets, including Vazquez dataset, YP dataset, DIP-core dataset, and SPK dataset, MFGO was tested and compared with the popular MR (majority rule) and GOM methods. Experimental results confirm MFGO's improvement on both speed and accuracy. Especially, MFGO method has a distinctive advantage in accurately predicting functions for proteins with few neighbors. Moreover, the robustness of the approach was validated both in a dataset containing a high percentage of unknown proteins and a disturbed dataset through random insertion and deletion. The analysis shows that a moderate amount of misplaced interactions do not preclude a reliable function assignment.     

Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins: detection probes and affinity standards

Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four Cu(I) ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their Cu(I) affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with Cu(I) to yield distinct 1:2 chromatophoric complexes [Cu(I)L(2)](3-) with formation constants β(2) = 10(17.2) and 10(19.8) m(-2), respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu(+) concentrations from 10(-12) to 10(-19) m. Dtt binds Cu(I) with K(D) ～10(-15) m at pH 7, but it is air-sensitive, and its Cu(I) affinity varies with pH. The Cu(I) binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity Cu(I) binding and the individual quantitative affinities (K(D) values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind Cu(I) with sub-femtomolar affinities, consistent with tight control of labile Cu(+) concentrations in living cells.     

Mapping protein interactions by combining antibody affinity maturation and mass spectrometry

Mapping protein interactions by immunoprecipitation is limited by the availability of antibodies recognizing available native epitopes within protein complexes with sufficient affinity. Here we demonstrate a scalable approach for generation of such antibodies using phage display and affinity maturation. We combined antibody variable heavy (V_H) genes from target-specific clones (recognizing Src homology 2 (SH2) domains of LYN, VAV1, NCK1, ZAP70, PTPN11, CRK, LCK, and SHC1) with a repertoire of 10⁸ to 10⁹ new variable light (V_L) genes. Improved binders were isolated by stringent selections from these new “chain-shuffled” libraries. We also developed a predictive 96-well immunocapture screen and found that only 12% of antibodies had sufficient affinity/epitope availability to capture endogenous target from lysates. Using antibodies of different affinities to the same epitope, we show that affinity improvement was a key determinant for success and identified a clear affinity threshold value (60 nM for SHC1) that must be breached for success in immunoprecipitation. By combining affinity capture using matured antibodies to SHC1 with mass spectrometry, we identified seven known binding partners and two known SHC1 phosphorylation sites in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells. These results demonstrate that antibodies capable of immunoprecipitation can be generated by chain shuffling, providing a scalable approach to mapping protein–protein interaction networks.     

Proinsulin C-peptide interaction with protein tyrosine phosphatase 1B demonstrated with a labeling reaction

Based on nickel-catalyzed cross-labeling where binding partners become biotinylated, we have studied molecular interactions with an N-terminally fused GGH-tag proinsulin C-peptide. Since C-peptide has been reported to influence phosphatase activity in intact cells, we employed this method to study possible binding of the peptide to protein tyrosine phosphatase 1B (PTP-1B). C-peptide was found to interact with PTP-1B (and for control, also with antibodies to C-peptide), as did also the N- and C-terminal fragments of C-peptide which have sequence similarities with PTP-1B binding proteins. The labeling data combined with enzyme activity analysis indicate a functional interaction between acidic regions of C-peptide and specific sites of PTP-1B. Results highlight the importance of possible phosphatase/C-peptide roles in diabetes, and the usefulness of the cross-labeling reaction also for acidic peptides like C-peptide.