A group of seven qualifying Primary Users have detailed a broad range of experimental plans that specifically employ BIAcore analysis as summarized herein: (i) Characterization of association and dissociation rate constants for the interaction of immunoglobulin E (IgE) and the high affinity IgE receptor (FcepsilonRI) is a major goal of the PI. The effect of point mutations on alterations of k/on and/or k/off values will be determined by measurement of phage-displayed peptide binding to sensor chips C1 and/or F1 covalently derivatized with either recombinant IgE receptor ectodomain or IgE; (ii) Determination of the kinetics of the interaction between the T cell receptor (TCR) and its ligands, specifically MHC class I-peptide complexes. To extend our recently published study of peptide/Class I-TCR binding kinetics, which showed a correlation between the affinity of TCR for particular ligands and the biological outcome, we plan to determine TCR-ligand recognition in lipid monolayers using the HPA sensor chip to model cell-cell TCR-MHC/peptide mediated interactions; (iii) The superoxide generating system of neutrophils is a membrane-associated enzyme complex, NADPH oxidase, which consists of an integral membrane protein, flavocytochrome b245 and cytosolic proteins p47phox, p67phox and Rac. BIAcore analysis will be used to study the molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how their affinity is affected by anionic lipids, ionic strength and GTP; (iv) The precise role of Rac in the foregoing system is still unknown, but one current hypothesis is that the association of GTP-bound Rac with p67phox increases the affinity of this protein for the flavocytochrome. This will be tested by determining the kinetics and affinity of binding of p67phox to immobilized, purified, relipidated flavocytochrome in the presence and absence of Rac-GTP in the analyze solution; (v) The energetic processes that drive the binding reactions for DNA and RNA will be determined using the zinc finger protein TFIIA, and truncated or mutated derivatives, in solution with either 5S DNA or 5S RNA immobilized on a BIAcore chip; (vi) Surface plasmon resonance technology will be utilized to study fibroblast growth factor (FGF) receptor- mediated signal transduction. Several proteins, identified using the two-hybrid system, show affinity for the cytoplasmic domain of FGFR-1. The binding components from the 2 hybrid analysis will now be analyzed using the BIAcore system to determine the kinetic binding parameters. (vii) Autoantibody binding to wildtype and mutant fibrillarin, in the presence and absence of mercury, using the BIAcore machine. Quantitation of Hg-dependent changes of antibody k/on and dissociation (k/off) from fibrillarin immobilized on the biosensor chip will then be assessed.
