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.     

Analysis of small-molecule interactions using Biacore S51 technology

Biacore S51 is a new surface plasmon resonance-based biosensor developed by Biacore AB (Uppsala, Sweden). The instrument was engineered specifically to support small-molecule drug discovery and development. The platform includes increased sensitivity, larger sample handling capabilities, and automated data processing to improve throughput. Compared to previously released Biacore instruments, the most significant design change relates to the introduction of the hydrodynamic-addressing flow cell. This design allows two reaction surfaces and a reference surface to be placed within the same flow cell, thereby improving data quality and extending the kinetic range of the instrument. Using a set of small-molecule inhibitors of the enzyme carbonic anhydrase II, we tested the reproducibility, sensitivity, and dynamic range of the biosensor. Given the S51's performance capabilities, it should play an active role in secondary screening by providing high-resolution information for small-molecule/target interactions.     

Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30,000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators’ instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1 ± 1.6 × 10⁶ M⁻¹ s⁻¹ and 6.7 ± 2.5 × 10⁻² s⁻¹, respectively, which corresponded to an average equilibrium dissociation constant (K_D) of 2.6 ± 1.4 × 10⁻⁸ M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.     

Kinetic analysis of beta-amyloid fibril elongation

We used surface plasmon resonance biosensors to evaluate the kinetics associated with the initial events of beta-amyloid (Abeta) fibril elongation. Fibrils were immobilized on the sensor chip surface and extended by exposure to soluble Abeta(1-40) peptide. The fibril surfaces bound Congo red, a marker for beta sheet structures, and exhibited a slow linear background decay that is consistent with fibril depolymerization. Sonicated fibrils supported elongation better than unsonicated fibrils, which is consistent with fibril extension reactions. The kinetic data revealed that peptide association and dissociation occurred in multiple steps. Kinetic rate constants for fibril extension were determined by globally fitting the response data with a three-step polymerization model. In the first step, the soluble peptide binds to the growing fibril tip in a readily reversible reaction. The subsequent steps likely allow bound peptide to be stabilized into the growing fiber through postbinding transitional events. Using a mutant peptide, F19P Abeta(1-40), we illustrate how the biosensor assay can be used to probe structure/function relationships of fibril elongation.     

Survey of the year 2004 commercial optical biosensor literature

The year 2004 represents a milestone for the biosensor research community: in this year, over 1000 articles were published describing experiments performed using commercially available systems. The 1038 papers we found represent an approximately 10% increase over the past year and demonstrate that the implementation of biosensors continues to expand at a healthy pace. We evaluated the data presented in each paper and compiled a 'top 10' list. These 10 articles, which we recommend every biosensor user reads, describe well-performed kinetic, equilibrium and qualitative/screening studies, provide comparisons between binding parameters obtained from different biosensor users, as well as from biosensor- and solution-based interaction analyses, and summarize the cutting-edge applications of the technology. We also re-iterate some of the experimental pitfalls that lead to sub-optimal data and over-interpreted results. We are hopeful that the biosensor community, by applying the hints we outline, will obtain data on a par with that presented in the 10 spotlighted articles. This will ensure that the scientific community at large can be confident in the data we report from optical biosensors.     

Identification by photoaffinity labeling of fatty acid-binding protein as a potential warfarin receptor in rat liver

Two different photoaffinity analogs of 4-hydroxy coumarin, 3-(p-azidobenzyl)-4-hydroxycoumarin (AzBHC) and 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin (AzISAHC), are being used in the identification of warfarin-binding proteins present in mammalian tissue (Myszka, D. G., and Swenson, R. P. (1990) Biochem. Biophys. Res. Commun. 172, 415-422; Myszka, D. G., and Swenson, R. P. (1991) J. Biol. Chem. 266, 4789-4797). In this study, [14C]AzBHC, but not [125I]AzISAHC, was observed to specifically label a 15,000-dalton protein present in both the microsomal and cytosolic fractions of rat liver. Pretreatment of the crude protein samples with warfarin or dicoumarol completely protected the 15-kDa protein from modification by [14C]AzBHC, indicating that this photoaffinity reagent is specifically labeling a coumarin-binding protein. 4-Hydroxycoumarin itself and AzISAHC were unable to block the incorporation of this photoaffinity probe. The 15-kDa protein was isolated by two-dimensional electrophoresis and subjected to amino-terminal sequence analysis. The first 20 amino acid residues analyzed were found to be identical with the amino-terminal sequence of rat liver fatty acid-binding protein (L-FABP) (Gordon J. I., Alpers, D. H., Ockner, R. K., and Strauss, A. W. (1983) J. Biol. Chem. 258, 3356-3363). Photoaffinity labeling and protection experiments carried out on purified preparations of L-FABP paralleled the labeling results obtained in the microsomes and cytosol, confirming that L-FABP is capable of specifically binding AzBHC, warfarin, and dicoumarol. Oleic acid, an established ligand for L-FABP, can compete with the binding of the photoaffinity probe; however, it was less effective in protecting the protein than warfarin. The specificity of labeling of crude liver fractions by warfarin photoaffinity analogs reported here as well as the high concentration of FABP in liver tissue together suggest that this protein may represent a major hepatic receptor responsible for the uptake and/or transport of various oral 4-hydroxycoumarin-based anticoagulant drugs.     

Two functionally distinct steps mediate high affinity binding of U1A protein to U1 hairpin II RNA

Binding of the U1A protein to its RNA target U1 hairpin II has been extensively studied as a model for a high affinity RNA/protein interaction. However, the mechanism and kinetics by which this complex is formed remain largely unknown. Here we use real-time biomolecular interaction analysis to dissect the roles various protein and RNA structural elements play in the formation of the U1A.U1 hairpin II complex. We show that neutralization of positive charges on the protein or increasing the salt concentration slows the association rate, suggesting that electrostatic interactions play an important role in bringing RNA and protein together. In contrast, removal of hydrogen bonding or stacking interactions within the RNA/protein interface, or reducing the size of the RNA loop, dramatically destabilizes the complex, as seen by a strong increase in the dissociation rate. Our data support a binding mechanism consisting of a rapid initial association based on electrostatic interactions and a subsequent locking step based on close-range interactions that occur during the induced fit of RNA and protein. Remarkably, these two steps can be clearly distinguished using U1A mutants containing single amino acid substitutions. Our observations explain the extraordinary affinity of U1A for its target and may suggest a general mechanism for high affinity RNA/protein interactions.     

BIACORE J: a new platform for routine biomolecular interaction analysis

SPR biosensor technology continues to evolve. The recently released platform from Biacore AB (Uppsala, Sweden), BIACORE J, is designed for the routine analysis of biomolecular interactions. Using an antibody-protein A and a ligand-receptor system, we demonstrate the utility of BIACORE J in determining active concentration and binding affinities. The results from these studies illustrate the high sensitivity of the instrument and its ability to generate reproducible binding responses. The BIACORE J is easy to operate and useful in diverse applications, making SPR technology widely accessible as a research tool.     

Unraveling regulatory networks in plant defense using microarrays

DNA microarrays are being used to comprehensively examine gene expression networks during the plant defense response that is triggered when a plant encounters a pathogen or an elicitor molecule. In addition to identifying new genes induced during defense, these studies are providing new insights into the complex pathways governing defense gene regulation.