PROTOONCOPROTEIN REGULATION--NMR STUDIES OF C SRC

原癌蛋白Src在细胞内信号转导中起关键作用 转导，并被认为与几种癌症有关，包括 乳腺癌和结肠癌。这项拟议研究的主旨是 将结构、动力学和热力学测量相结合来研究 SH3和SH2配体结合的基本原理 该蛋白的结构域，并测试该蛋白的功能片段 SRC监管设备在原封不动中准确模拟功能 蛋白。所采用的模型系统表示处于“开”状态的Src， 其中SH3和SH2结构域均可用于形成激活的信令 与其他蛋白质的复合体，有些与SH3和SH2结合 域名。核磁共振波谱提供了一种独特的检测配体的能力 在包括蛋白质、配体和溶剂的体系中结合 作为自由态和束缚态之间不受阻碍的交换。核磁共振还能使 结合能的焓和熵分量将与 蛋白质主链和侧链的结构和动态变化 在配体结合时，并允许确定速率常数 当KOF处于确定的快速到中级时间范围时交换 通过束缚态和自由态之间的化学位移差异。 将这一信息与来自 等温滴定量热法(ITC)允许详细描述 从独特的角度看焓和熵的结合决定因素 这包括蛋白质在游离态和动态态的结构 束缚态。虽然目前的大小限制阻碍了核磁共振研究 完整的Src，蛋白质大小不受ITC测量的限制。因此， 广泛使用的切除功能域方法的有效性 将对该系统进行结构、动力学和约束性研究 通过将核磁共振和ITC应用于分离的和共价连接的SH3和 Sh2结构域，并将ITC的结果与 完好无损的源这一系统提供了一个极好的机会来关联 蛋白质的高分辨结构和动力学 易于表征的函数，并测试使用的有效性 各个功能域，以研究它们之间的关系 结构、动力和功能。拟议的研究将提供 对新型抗癌药物开发有价值的见解和指导 以Src监管机构为目标的代理。

The proto-oncoprotein Src plays a key role in intracellular signal transduction and has been implicated in several carcinomas including breast and colon cancer. The main thrust of the proposed research is to combine structure, dynamics, and thermodynamics measurements to study the fundamental principles governing ligand binding to the SH3 and SH2 domains of this protein, and to test whether functional fragments of the Src regulatory apparatus accurately mimic function in the intact protein. The model systems employed represent Src in the "on" state, where both SH3 and SH2 domains are available to form activated signaling complexes with other proteins, some which bind to both SH3 and SH2 domains. NMR spectroscopy offers the unique ability to examine ligand binding in a system that includes protein, ligand and solvent as well as unhindered exchange between free and bound states. NMR also enables enthalpic and entropic components of binding energy to be related to structural and dynamic changes of the protein backbone and side chains upon ligand binding, and enables determination of rate constants for exchange when koff is in the fast to intermediate time scale determined by chemical shift differences between the bound and free states. Combining this information with thermodynamics measurements from isothermal titration calorimetry (ITC) allows a detailed description of enthalpic and entropic binding determinants from a unique perspective that includes the structure and dynamics of the protein in both free and bound states. While current size limitations prevent NMR studies an intact Src, protein size is not limited in ITC measurements. Hence, the validity of the widely used approach of excising functional domains for structural, dynamics, and binding studies will be tested for this system by applying NMR and ITC to the separate and covalently linked SH3 and SH2 domains, and by comparing the ITC results with those obtained for intact Src. This system offers an excellent opportunity to correlate the high resolution structure and dynamics of a protein to well defined and easily characterized functions, and to test the validity of using individual functional domains to investigate relationships between structure, dynamics and function. The proposed studies will provide valuable insights and guidance for development of novel anti-cancer agents that target the Src regulatory apparatus.