Thomas A. Morton

Extending the range of rate constants available from BIACORE: interpreting mass transport-influenced binding data.

Surface-based binding assays are often influenced by the transport of analyte to the sensor surface. Using simulated data sets, we test a simple two-compartment model to see if its description of transport and binding is sufficient to accurately analyze BIACORE data. First we present a computer model that can generate realistic BIACORE data. This model calculates the laminar flow of analyte within the flow cell, its diffusion both perpendicular and parallel to the sensor surface, and the reversible chemical reaction between analyte and immobilized reactant. We use this computer model to generate binding data under a variety of conditions. An analysis of these data sets with the two-compartment model demonstrates that good estimates of the intrinsic reaction rate constants are recovered even when mass transport influences the binding reaction. We also discuss the conditions under which the two-compartment model can be used to determine the diffusion coefficient of the analyte. Our results illustrate that this model can significantly extend the range of association rate constants that can be accurately determined from BIACORE.

KINETIC ANALYSIS OF MACROMOLECULAR INTERACTIONS USING SURFACE PLASMON RESONANCE BIOSENSORS

Surface plasmon resonance based biosensors are being used to define the kinetics of a wide variety of macromolecular interactions. As the popularity of this approach grows, experimental design and data analysis methods continue to evolve. These advances are making it possible to accurately define the assembly mechanisms and rate constants associated with macromolecular interactions.

Binding Interactions of Human Interleukin 5 with Its Receptor α Subunit LARGE SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS

Human interleukin 5 (hIL5) and soluble forms of its receptor α subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor α subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-Å resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor α subunit (shIL5Rα). Additionally, the relative abundance of ligand and receptor in the hIL5·shIL5Rα complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 n M, respectively, for the binding of hIL5 to shIL5Rα and to a chimeric form of the receptor containing shIL5Rα fused to the immunoglobulin Fc domain (shIL5Rα-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kdvalues from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor α subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5Rα in Drosophila membranes was found to be 6 n M, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the α receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5·receptor αβ complex and signal transduction.

New Opportunities for Using Immobilized Ligands to Characterize Macromolecular Recognition and Design Recognition Molecules

Abstract Characterization of protein interactions and interaction sites can provide a means both to learn about molecular recognition and assembly processes in biology and to identify and evaluate new recognition molecules of practical use in biotechnology. We have had a long-standing interest in using immobilized ligands as analytical tools for characterizing recognition mechanisms of proteins and other biological macromolecules. This interest started with analytical affinity chromatography (AAC). Recently, a newcomer among analytical technologies using immobilized ligands has appeared, namely the surface plasmon resonance (SPR) biosensor. The SPR biosensor, as AAC, enables detection and measurement of noncovalent interaction of a soluble macromolecule with a solid phase containing covalently attached ligand. Importantly, the biosensor offers several unique features including access to kinetics (hence deeper mechanistic understanding), ability to analyze molecules in mixtures (hence access to more biologically relevant conditions), and real-time observation of the interaction process (hence ability to observe interacting molecules as they form or are added). Recent results with HIV proteins including p24 self assembly and CD4-gpl20 interactions, as well as with interleukin 5 and its receptor reflect some of the growing uses of both AAC and the SPR biosensor as macromolecular recognition tools. Overall, the advent of the SPR biosensor and the likely follow-up development of other automated devices promise to stimulate evolution of the analytical use of immobilized ligands that started with AAC into a broad-based analytical solid phase science for the field of biomolecular recognition.