Protein complexes involved in signal transduction

参与信号转导的蛋白质复合物

• 批准号: 7118201
• 负责人: PHILIP MATSUMURA
• 金额: $ 35.76万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-04-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118201
• 关键词: DNA binding protein; Escherichia coli; X ray crystallography; bacterial genetics; bacterial proteins; binding sites; biological signal transduction; chemotaxis; cilium /flagellum motility; fibronectins; gene mutation; genetic transcription; nuclear magnetic resonance spectroscopy; phosphorylation; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): Our understanding of the Escherichia coli chemotaxis system has progressed during the last 30 or so years. We understand the biochemistry of the binding of the ligands to the transmembrane receptors, the path of phosphate through the two-component signaling system, and the behavior of the flagellar motor in response to the signal. To gain further insights into this complex signal transduction system, the questions of how the receptors generate the initial signal, how this signal is modulated, and how the signal is received by the flagellar motor will be addressed in the future. This proposal focuses on the modulation of the intracellular signal and the interface of this signal with the flagellar motor. We have taken the approach of studying the higher order structure of various protein complexes in order to gain insight into mechanisms of transmission of the chemotaxis signal and of transcriptional regulation. We will focus on the following Specific Aims 1) We will determine how activated CheY binds to the flagellar switch component, FliM. We have found that by forming a complex of a mutant CheY and FliM, we have been able to obtain soluble FliM for the first time after many years of effort. This and other mutant CheY/FliM complexes will be used for NMR and crystallography. 2) We will determine the role the CheAs/CheZ interaction in chemotaxis. A 42 amino acid fragment of the amino terminal end of CheAs ('P1) has been shown to bind and activate CheZ in vitro. The fibronectin display peptides that bind 'P1 will be used in vivo to inactivate CheAs functions without affecting CheAL functions. 3) We will use our technique of isolating stable CheAL/CheA*s/ CheW complexes to test the possibility that CheAs can bind CheW in a phosphorylating complex and then switch to bind CheZ after phosphorylation. 4) We will determine a cognate partner of FlhD for regulation of FlhD dependent-FIhC independent promoters. Once this is established, we will solve the crystal structures of the FlhDC/DNA complex and FlhD/X/DNA complex to determine how complex formation alters DNA binding specificity.

描述（由申请人提供）：我们对大肠杆菌趋化系统的理解在过去30年左右的时间里取得了进展。我们了解配体与跨膜受体结合的生物化学，通过两组分量信号系统磷酸盐的路径以及响应信号的鞭毛运动的行为。为了进一步了解这种复杂的信号转导系统，有关受体如何生成初始信号，如何调制该信号以及如何通过鞭毛电动机接收的信号的问题将在将来解决。该提案重点介绍了细胞内信号的调制以及该信号与鞭毛电动机的界面。我们采取了研究各种蛋白质复合物的高阶结构的方法，以深入了解趋化信号传播和转录调控的机制。我们将重点关注以下特定目标1）我们将确定激活的chey如何与鞭毛开关组件Flim结合。我们发现，通过形成一个突变体Chey和Flim的络合物，经过多年的努力，我们能够首次获得可溶性FLIM。这种和其他突变体的Chey/Flim复合物将用于NMR和晶体学。 2）我们将确定Cheas/Chez相互作用在趋化性中的作用。已显示出一个42个氨基末端的氨基酸片段（'p1）在体外结合并激活Chez。纤连蛋白显示结合'P1的肽将在体内使用，以使Cheal功能失活的功能，而不会影响Cheal功能。 3）我们将使用我们的技术分离稳定的Cheal/ Chea*S/ Chew配合物来测试Cheas可以在磷酸化复合物中结合咀嚼的可能性，然后在磷酸化后切换到CHEZ。 4）我们将确定FLHD的同源伴侣，以调节FLHD依赖性-FIHC独立启动子。一旦建立了这一点，我们将解决FLHDC/DNA复合物和FLHD/X/DNA复合物的晶体结构，以确定复合形成如何改变DNA结合特异性。