Purchase of BIAcore 3000 Instrument

购买BIAcore 3000仪器

基本信息

批准号：
6577502
负责人：
Satya P. Yadav
金额：
$ 27.5万
依托单位：
CLEVELAND CLINIC FOUNDATION
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-04-01 至 2004-03-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：LRI 的六名主要研究人员组成的小组，他们拥有广泛的 NIH 资助项目，这些项目特别需要生物传感器方法来实时定量分子识别事件，以推进其研究项目的目标。主要用户组旨在利用 BIAcore 分析技术进行各种项目，概述如下： (i) 表征 c-AMP 依赖性 PKA II 的调节亚基 (RII) 对各种 A- 的动力学和结合亲和力激酶锚定蛋白（AKAP）在心脏中表达。将确定突变 RII 结构域（疏水性氨基酸）作为配体和 AKAP 作为分析物的结合亲和力，以剖析 β-肾上腺素能反应在心脏肥大和衰竭中的作用。(ii) 已知包括 eNOS 在内的几种信号蛋白与Caveolin-1，寡聚整合膜蛋白，似乎充当小凹内的结构“支架”。 BIAcore 3000 分析将用于定量 Cav-1、单独的 eNOS 结构域以及全长 eNOS 酶之间的结合相互作用和动力学；生物素化的 Cav-1 支架结构域肽将固定在传感器芯片上，并在 eNOS 还原酶、eNOS 加氧酶以及作为相互作用蛋白的 eNOS 全长蛋白中滴定； (iii) 确定细胞内信号分子（tallin、钙整合素结合蛋白）与与传感器芯片配体的 β3 整合素 αII 6、β3'、（αVβ 3）细胞质尾部的生物素化肽相互作用的 kd 和化学计量，以阐明整合素信号转导通路； (iv) 生物传感器技术将用于利用 BIAcore 3000 和质谱仪识别各种 I kappa B 激酶 (IKK) 复合物的潜在相互作用蛋白质。来自野生型 MEF、IKKalpha' MEF、IKKbeta' MEF、IKKalpha/KKbeta' MEF 和 IKKgamma' MEFS 的细胞蛋白将与附着的三种类型的 IKK 复合物中的每一种一起孵育，这些蛋白是在用促炎细胞因子 (TNFalpha) 刺激后从各种细胞中制备的。到生物传感器芯片。它将使我们能够剖析 IKK 响应促炎细胞因子和病原体感染的激活机制； (v) 使用这些相互作用的动力学常数来表征驱动双链 RNA (ds-RNA) 与 dsRNA 结合蛋白的结合特性的能量过程， (vi) 研究跨膜蛋白酶的细胞质结构域的动力学相互作用基质金属蛋白酶 (MMP) 和解整合素和金属蛋白酶结构域 (ADAM) 家族成员。由于 MMP-16 细胞质延伸长度仅为 20 个氨基酸，因此我们将使用具有单个氨基酸取代的合成肽来精炼使用 BIAcore 相互作用所需的关键残基。我们的方法是合成生物素化肽并将它们定向固定到传感器芯片上。通过这种方式，将保持从细胞内部观察到的细胞质结构域的真实呈现。