Synthesis of phosphono-CheY and phosphono-VHR

描述（由申请人提供）：本项目的目标是确定两种信号转导蛋白Che-Y和VHR的结构，这两种蛋白可能与人类疾病相关。这项工作的独特策略是使用蛋白质的化学修饰来产生每种蛋白质的瞬时磷酸化形式的稳定类似物，否则很难研究。具体地，半胱氨酸残基用膦酰基甲基CH 2 PO 3修饰。将通过液相色谱法和质谱法确认膦酰基甲基半胱氨酸残基的存在。蛋白质的结构将通过X射线晶体学确定。第一种蛋白质是CheY，这是细菌游向更理想环境所必需的蛋白质，这一过程被称为趋化性。Phosphono-CheY由CheY的突变形式和三氟甲磺酸膦甲酯合成。膦酰基-CheY的结构将与其伴侣FliM复合确定。还可以确定与磷酸酶复合的膦酰基-CheY的结构，所述磷酸酶被设计为使CheY返回其去磷酸化状态，以获得对信号终止过程的了解。趋化性的破坏可以使生物体的致病性降低。第二种蛋白质是VHR，一种双特异性磷酸酶。蛋白酪氨酸磷酸酶的失调在许多人类疾病中起作用，包括癌症和糖尿病。蛋白酪氨酸磷酸酶通过产生半胱氨酰磷酸中间体（随后水解）来分解磷蛋白。当半胱氨酸残基被膦酰基甲基修饰成膦酰基甲基半胱氨酸时，VHR失活。可逆抑制剂可防止失活。确定这种膦酰基甲基半胱氨酸中间体的稳定类似物的结构可以提供有关该反应机制的信息，并可以为基于结构的药物设计提供目标。 公共卫生相关性：该项目可能会导致更好地了解如何通过干扰细菌运动来减少细菌的发病机制。此外，该项目将提供有关细胞生长信号如何打开和关闭的见解。这些信号传导过程中的错误是许多人类疾病的基础，包括糖尿病。

DESCRIPTION (provided by applicant): The goal of this project is to determine the structures of two signal transduction proteins, Che-Y and VHR, that are potentially relevant to human diseases. The unique strategy of this work is the use of chemical modification of the proteins to create stable analogs of transient, phosphorylated forms of each protein that would be otherwise difficult to study. Specifically, a cysteine residue is modified with a phosphonomethyl group, CH2PO3. The presence of the phosphonomethylcysteine residue will be confirmed by liquid chromatography and mass spectrometry. The structures of the proteins will be determined by x-ray crystallography. The first protein is CheY, a protein necessary for bacteria to swim toward a more desirable environment, a process known as chemotaxis. Phosphono-CheY was synthesized from a mutant form of CheY and phosphonomethytrifluoromethanesulfonate. The structure of phosphono-CheY will be determined in complex with its partner, FliM. The structure of phosphono-CheY complexed with phosphatases designed to return CheY to its dephosphorylated state may also be determined to obtain insight into the signal termination process. Disruption of chemotaxis may render an organism less pathogenic. The second protein is VHR, a dual-specificity phosphatase. Dysregulation of protein tyrosine phosphatases plays a role in many human diseases, including cancer and diabetes. Protein tyrosine phosphatase enzymes break down phosphoproteins via the creation of a cysteinyl phosphate intermediate that is subsequently hydrolyzed. VHR is inactivated when the cysteine residue is modified with a phosphonomethyl group to become phosphonomethylcysteine. Reversible inhibitors protect against inactivation. Determining the structure of a stable analog of this phosphonomethylcysteine intermediate may provide information about the mechanism of this reaction and may provide a target for structure-based drug design. PUBLIC HEALTH RELEVANCE: This project may lead to a better understanding of how to decrease bacterial pathogenesis by interfering with bacterial motility. Further, this project will provide insights into how cellular growth signals are turned on and off. Errors in these signaling processes underlie many human diseases, including diabetes.