NMR STUDIES OF SPECIFICITY SWITCHING IN SRC SH2

可逆蛋白质磷酸化参与多种生物过程，包括新陈代谢、细胞增殖和分化、膜运输、基因表达、运动、记忆和学习。然而，人们对磷酸化调节蛋白质功能的生物物理机制知之甚少。本研究的目的是以鸡c-Src的Src同源2(SH2)结构域为模型系统，阐明这些机制。主要目标是：1)确定在自然界中观察到的SH2结构域中特定酪氨酸残基(Y213)的磷酸化对改变配基特异性的结构、动力学和功能的影响，以及ii)确定这种特异性开关是否可以通过加入丝氨酸或苏氨酸的触发机制以细胞周期依赖或组织特异性的方式改变。多维核磁共振波谱将用于该模型体系的结构确定和动态表征。滴定微量热法将作为一种功能分析来评估配体的特异性和亲和力的变化。这些实验的结合将有助于全面了解可逆蛋白质磷酸化对Src SH2特异性开关的生物物理影响。这些信息可以用来设计蛋白质，当与基因治疗相结合时，将允许在细胞周期的特定阶段或不同组织类型中引入分子开/关开关。这种方法在帮助开发新的抗癌治疗药物方面具有巨大的潜力。

DESCRIPTION Reversible protein phosphorylation is involved in a diverse array of biological processes, including metabolism, cell proliferation and differentiation, membrane transport, gene expression, locomotion, memory, and learning. Yet very little is known about the biophysical mechanisms at work in regulation of protein function by phosphorylation. The goal of the proposed research is to elucidate these mechanisms, using the Src Homology 2 (SH2) domain from chicken c-Src as a model system. The main objectives are i) to determine the structural, dynamic, and functional effects of phosphorylation of a specific Tyrosine residue (Y213) within the SH2 domain that has been observed in nature to alter ligand specificity, and ii) to ascertain if this specificity switch can be modified in a cell cycle dependent or tissue specific manner by incorporating a Ser or Thr trigger mechanism. Multi-dimensional NMR spectroscopy will be employed for structure determination and dynamic characterization of this model system. Titration microcalorimetry will be used as a functional assay to evaluate changes in ligand specificity and affinity. The combination of these experiments will help provide a comprehensive understanding of the biophysical effects of reversible protein phosphorylation on Src SH2 specificity switching. This information can be used to design proteins that, when coupled with gene therapy, will allow the introduction of molecular on/off switches in specific stages of the cell cycle or in different tissue types. This approach has great potential in aiding development of novel anti-cancer therapeutics.