Purchase of BIAcore 3000 Instrument

描述(由申请人提供)：LRI的六名主要研究人员，他们有一系列由NIH资助的项目，这些项目特别需要生物传感器方法来实时量化分子识别事件，以推进他们研究项目的目标。主要用户群体的目标是利用Biaccore技术分析总结如下的各种项目：(I)表征c-AMP依赖的PKA II的调节亚单位(RII)与在心脏中表达的各种A-激酶锚定蛋白(AKAP)的动力学和结合亲和力。突变的RII结构域(疏水氨基酸)作为配体和AKAP作为分析物的结合亲和力将被确定为剖析β-肾上腺素能反应在心肌肥大和衰竭中的作用。(Ii)已知包括eNOS在内的几个信号蛋白与小窝内的结构“支架”-1-寡聚整膜蛋白相互作用。Biacore 3000分析将用于定量Cav-1一个单独的eNOS结构域以及全长eNOS酶之间的结合作用和动力学；生物素化的Cav-1 scafolding结构域肽将固定在传感器芯片上，并在eNOS还原酶、eNOS加氧酶以及eNOS全长蛋白中滴定为相互作用蛋白；(Iii)确定细胞内信号分子(Tallin、钙整合素结合蛋白)与β3整合素α6、β3‘、βV3的生物素化多肽相互作用的kd和化学计量学；(4)将利用Biacore 3000和MAS光谱仪，利用生物传感器技术确定各种I kappa B激酶(IKK)复合体可能相互作用的蛋白质。将不同细胞经促炎细胞因子(TNFpha)刺激后制备的野生型MEF、IKKalpha‘MEF、IKKbeta’MEF、IKKalpha/KKbeta‘MEF和IKKGamma’MEF的细胞蛋白分别与附着在生物传感器芯片上的三种IKK复合体孵育。这将使我们能够剖析IKK在致炎细胞因子和病原体感染反应中的激活机制；(V)利用双链RNA(ds-RNA)与dsRNA结合蛋白的相互作用的动力学常数来表征驱动双链RNA(ds-RNA)与dsRNA结合蛋白的能量过程；(Vi)研究基质金属蛋白酶(Matix Metals Protease，MMPs)和去整合素和金属蛋白酶结构域(ADAM)家族成员跨膜蛋白的细胞质结构域的动力学相互作用。由于基质金属蛋白酶-16的细胞质延伸长度只有20a，我们将使用带有单一氨基酸取代的合成肽来提炼使用Biacore进行相互作用所需的关键残基。我们的方法将是合成生物素化的多肽，并将它们定向固定到传感器芯片上。这样，从细胞内部可见的细胞质结构域的真实呈现将保持不变。

DESCRIPTION (provided by the applicant): 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.