: A group of six principal investigators at LRI, who have a wide range of NIH funded projects that specifically demand for Biosensor methods to quantitate molecular recognition events in real time to advance the aims of their research projects. The primary user group aim to exploit the BIAcore technology of analysis for a variety of projects summarized as follows: (i) Characterization of the kinetics and binding affinities of the regulatory subunit (RII) of c-AMP-dependent PKA II for various A-kinase anchoring proteins (AKAPs) expressed in heart. Binding affinities of mutated RII domain (hydrophobic amino acids) as ligands and AKAPs as analyte will be determined to dissect the role of beta-adrenergic responses in cardiac hypertrophy and failure, (ii) Several signaling proteins, including eNOS, are known to interact with caveolin-1, oligomeric integral membrane protein, that appears to serve as the structural """"""""scaffold"""""""" within caveolae. BIAcore 3000 analysis will be used to quantitate binding interactions and kinetics between Cav-1 an separate eNOS domains as well as the full-length eNOS enzyme; Biotinylated Cav-1 scafolding domain peptide will be immobilized on the sensor chip and titrated in eNOS reductase, eNOS oxgenase, as well as eNOS full length protein as the interacting protein; (iii) to determine the kd and stoichiometry of the interaction of intracellular signaling molecules (tallin, calcium integrin binding protein) with the biotinylated peptides of beta3 integrins alphaII 6, beta3', (alphaVbeta 3) cytoplasmic tails liganded to sensor chips for elucidating the integrin signal transduction pathway; (iv) Biosensor technology will be used to identify potentially interacting proteins for the various I kappa B Kinase (IKK) complexes utilizing the BIAcore 3000 and mas spectrometers. Cellular proteins from wild type MEFs, IKKalpha' MEFs, IKKbeta' MEFs, IKKalpha/KKbeta' MEFs and IKKgamma' MEFS prepared from the various cells after stimulation with proinflammatory cytokine (TNFalpha) will be incubated with each of the three types of IKK complexes attached to biosensor chips. It will allow us to dissect the mechanisms of activation of the IKK in response to proinflammatory cytokines and pathogen infection; (v) to characterize the energetic process that drive the binding properties of double stranded RNA (ds-RNA) to dsRNA binding proteins using the kinetic constants of these interactions, (vi) To study the kinetic interactions of the cytoplasmic domains of transmembrane proteases of the matix metalloprotease (MMP) and A Disintegrin and Metalloprotease domain (ADAM) family members. Since the MMP-16 cytoplasmic extension is only 20 a a long, we will use synthetic peptides with single amino acid substitutions to refine the critical residues required for the interaction using BIAcore. Our approach will be to synthesize the biotinylated peptides and immobilizing them directionally to sensor chips. In this way, the authentic presentation of the cytoplasmic domain as visualized from the interior of the cell will be maintained.
