Synthesis of phosphono-CheY and phosphono-VHR

描述(申请人提供)：这个项目的目标是确定两个信号转导蛋白，Che-Y和VHR的结构，这两个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创建每种蛋白质的瞬时磷酸化形式的稳定类似物，否则将难以研究。具体地说，半胱氨酸残基用磷酸亚甲基CH2PO3修饰。磷酸甲基半胱氨酸残基的存在将通过液相色谱和质谱仪进行确认。蛋白质的结构将通过X射线结晶学来确定。第一种蛋白质是Chey，这是细菌游向更理想的环境所必需的蛋白质，这一过程被称为趋化性。以乙酰胆碱酯酶(Chey)和三氟甲磺酸膦甲酯为原料，合成了一种突变型的膦-胆碱酯酶。亚膦-Chey的结构将在与其伙伴Flim的络合物中确定。还可以确定磷酸-Chey与旨在使Chey返回其去磷酸化状态的磷酸酶复合体的结构，以深入了解信号终止过程。化学趋化性的破坏可能会使生物体的致病性降低。第二种蛋白是VHR，一种双特异性磷酸酶。蛋白酪氨酸磷酸酶的失调在包括癌症和糖尿病在内的许多人类疾病中扮演着重要角色。蛋白质酪氨酸磷酸酶通过产生半胱氨酰磷酸中间体来分解磷蛋白，半胱氨酰磷酸中间体随后被水解。当半胱氨酸残基被磷酸亚甲基修饰为磷酸甲基半胱氨酸时，VHR被灭活。可逆抑制剂可防止失活。确定该中间体的稳定类似物的结构可提供有关该反应机理的信息，并可为基于结构的药物设计提供靶点。 公共卫生相关性：该项目可能有助于更好地理解如何通过干扰细菌的运动来减少细菌的致病作用。此外，该项目将提供有关细胞生长信号如何开启和关闭的见解。这些信号传递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.