Molecular basis of PII function

PII 功能的分子基础

• 批准号: 7417652
• 负责人: GARY Paul ROBERTS
• 金额: $ 20.3万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2008-06-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7417652
• 关键词: Affect; Affinity; Antibiotics; Behavior; Binding; Biochemical; Biochemical Genetics; Carbon; Cell physiology; Class; Condition; Cytoplasm; Data; Dissociation; Employee Strikes; Environment; Equilibrium; Excision; Glutamine; Growth; Hand; Homologous Gene; In Vitro; Lead; Light; Measures; Membrane; Membrane Proteins; Modification; Molecular; Molecular Conformation; Nature; Nitrogen; Numbers; Organism; Personal Satisfaction; Physiological; Process; Production; Prokaryotic Cells; Property; Reaction; Regulation; Role; Signal Transduction; Structure; Testing; Toxin; Uridine Monophosphate; Variant; alpha ketoglutarate; base; conformer; improved; in vitro Assay; in vivo; interest; microbial; mutant; nitrogen metabolism; pathogen; receptor; research study; response; small molecule; success; tool

GlnB and its homologs are integrators of signals of nitrogen and carbon status in almost all prokaryotic cells, including all major pathogens. GlnB can exist in two extreme forms, one that signals nitrogen sufficiency and one that signals nitrogen deficiency. The equlibrium between those forms is affected by binding (carbon-rich) 2-ketoglutarate and ATP, and by covalent modification of GlnB in response to low levels of (nitrogen-rich) glutamine. These various forms of GlnB then interact differentially with a critical set of receptor proteins that control nitrogen metabolism. The signal transduction mechanism within GlnB that controls this process is poorly understood and we will gain information about this process through the biochemical and physiological analysis of some striking GlnB variants that we have obtained. These GlnB variants include (i) variants that are shifted toward the form that signals nitrogen deficiency; (ii) variants that are shifted toward the form that signals nitrogen sufficiency; and (iii) variants that are differentially altered in their affinities for ATP and 2- ketoglutarate. These variants will be examined by in vitro assays for small molecule binding, interaction with receptors and conformational changes. The variants will be examined in vivo for to determine the precise physiological impact of the biochemical properties measured in vitro. All of these effects will be interpreted in light of existing and proposed analysis of GlnB conformation and structure. The approach has a very high likelihood of success because the interesting variants are already in hand and the biochemical and genetic tools are available. We will also examine a related aspect of GlnB regulation, namely its removal from the cytoplasm by interaction with the membrane-associated AmtB. We have developed the tools for the direct in vitro analysis of this interaction and the information will be integrated with the above data on GlnB itself. The result of the proposed experiments will be a dramatically improved description of how GlnB homologs in all organisms integrate signals of nitrogen, carbon and energy status at the molecular level. The information is broadly important because such signal integration is critical for both beneficial (antibiotic production) and harmful (toxin production) microbial processes. We will therefore understand better how both beneficial and pathogenic organisms sense and respond to their environment.

GlnB及其同源物是几乎所有原核细胞中氮和碳状态信号的整合子， 包括所有主要病原体。GlnB可以以两种极端形式存在，一种是氮充足的信号， 一种是氮缺乏的信号这些形式之间的平衡受到结合（富碳）的影响。 2-酮戊二酸和ATP，并通过共价修饰GlnB响应于低水平的（富氮） 谷氨酰胺。这些不同形式的GlnB然后与一组关键的受体蛋白质差异地相互作用， 控制氮代谢。GlnB内控制这一过程的信号转导机制是 我们对这一过程知之甚少，我们将通过生物化学和生理学方法获得有关这一过程的信息。 分析了我们获得的一些引人注目的GlnB变体。这些GlnB变体包括（i） 向信号氮缺乏的形式转变;（ii）向信号氮缺乏的形式转变的变体， 信号氮充足;和（iii）在其对ATP和2-氨基-3-甲基-N 酮戊二酸。这些变体将通过体外测定来检查小分子结合、与 受体和构象变化。将在体内检查变体，以确定精确的 在体外测量的生化特性的生理影响。所有这些影响都将在 根据现有的和提出的GlnB构象和结构的分析。该方法具有很高的 成功的可能性，因为有趣的变异已经在手，生物化学和遗传学 工具可用。我们还将研究GlnB监管的一个相关方面，即将其从 通过与膜相关的AmtB相互作用在细胞质中表达。我们已经开发出了直接在 这种相互作用的体外分析和信息将与GlnB本身的上述数据相结合。 所提出的实验的结果将是对GlnB同系物如何在细胞中表达的显著改进的描述。 所有生物体都在分子水平上整合氮、碳和能量状态的信号。的信息 是广泛重要的，因为这样的信号整合对于有益的（抗生素生产）和 有害（毒素产生）微生物过程。因此，我们将更好地了解如何既有益， 病原生物感知并响应其环境。