Structural Parameters in Metal Recognition

金属识别中的结构参数

• 批准号: 8991995
• 负责人: MICHAEL J MARONEY
• 金额: $ 3.53万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-15 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8991995
• 关键词: Acidity; Acids; Active Sites; Address; Affinity; Antibiotics; Bacteria; Binding; Biochemistry; Biological; Biological Assay; Charge; Cloning; Collaborations; Complex; Crystallography; DNA; DNA Binding; DNA-Binding Proteins; Defect; Dependence; Ensure; Environment; Enzymes; Equilibrium; Escherichia coli; Family; Genetic Transcription; Goals; Growth; Health; Helicobacter pylori; Homeostasis; Homology Modeling; Human; Hydrogenase; Ions; Lead; Ligands; Link; Mass Spectrum Analysis; Metal Binding Site; Metalloproteins; Metals; Molecular; Mutagenesis; Mutate; Mutation; Nickel; Organism; Organized by Structure Protein; Patients; Physiological; Poisoning; Positioning Attribute; Proteins; Reporter; Research; Resistance; Roentgen Rays; Role; Site; Specificity; Spectrum Analysis; Stomach; Structural Protein; Structure; System; Techniques; Testing; Transition Elements; Urease; absorption; base; design; dimer; flexibility; glutamylalanine; human disease; member; metal metabolism; mutant; pathogen; protein protein interaction; protein structure; protein transport; response; restraint; targeted delivery; therapy development; toxic metal; trafficking; uptake

DESCRIPTION (provided by applicant): Maintaining proper cellular levels of metal ions is key to the survival of all organisms. The viability of bacteria, including human pathogens, has been linked to the ability to acquire or compete for transition metals. Several human diseases have been shown to result from a breakdown in cellular metal trafficking. In the case of transition metals, maintaining proper metal homeostasis involves controlling a delicate balance between optimal levels required to have functional enzymes, etc., and the concentration at which the metals become toxic. To achieve this control, organisms have developed mechanisms to ensure the acquisition of specific metals necessary for growth, exclude toxic metals, and control their intercellular levels. These metal trafficking systems rely on proteins that have the ability to distinguish between metal ions that often have similar sizes and charges. The metalloproteins involved, collectively known as metal trafficking proteins, control uptake and efflux (metallotransporters), target the delivery of metals to specific enzymes (metallochaperones) and regulate the expression of the other proteins in response to metal ion concentration (metalloregulators), etc. Although many examples of proteins that achieve these functions for various metal ions have been characterized, the mechanisms that allow for specific metal recognition and metal-specific biological responses are not well known. The overall objective of the proposed research is to understand the structural parameters that that allow trafficking proteins to distinguish between metals, and the related protein structural changes that drive metal specific biological responses. Toward this goal, we plan to examine structural parameters that are involved in metal-recognition by a metallochaperone (HypA) and a metalloregulator (RcnR) in Helicobacter pylori and E. coli, respectively. The approach involves cloning and expressing the proteins, characterizing their metal ion affinities, elucidating the structure of th metal sites and proteins, and assessing protein-protein interactions. To do this, mutagenesis and several structural techniques including X-ray absorption spectroscopy (XAS), crystallography, NMR, and mass-spectrometry-based techniques, are utilized. To assess function in this diverse group of proteins, assays specific to each protein will be employed; a transcription reporter assay for RcnR, and assays that address the ability of the protein to deliver metals to the target enzymes for the metallochaperone. In addition to the understanding of the basic biochemistry, a detailed understanding of the molecular mechanisms involved in metal trafficking may lead to the development of therapies for the treatment of patients with metal overloads (including poisoning) or deficiencies/excesses resulting from defects in metal metabolism, and to the design of new antibiotics that interfere with bacterial metal metabolism.

描述（由申请人提供）：维持适当的金属离子水平是所有生物生存的关键。细菌的生存能力，包括人类病原体，与获得或竞争过渡金属的能力有关。已经证明了几种人类疾病是由于细胞金属运输的破坏而引起的。在过渡金属的情况下，保持适当的金属稳态涉及控制具有功能性酶等所需的最佳水平之间的微妙平衡，以及金属变得有毒的浓度。为了实现这一控制，生物体已经开发了确保获得生长所需的特定金属，排除有毒金属并控制其细胞间水平的机制。这些金属运输系统依赖于具有区分通常具有相似尺寸和电荷的金属离子的蛋白质。涉及的金属蛋白（统称为金属运输蛋白）控制吸收和排出（金属脱孢子蛋白），将金属递送至特定酶（金属蛋白）的表达，并调节其他蛋白质的表达，并响应于金属离子浓度（金属调节剂）的特征，以供应金属的特征。识别和金属特异性生物学反应尚不清楚。拟议研究的总体目标是了解允许运输蛋白可以区分金属的结构参数以及驱动金属特异性生物学反应的相关蛋白质结构变化。为了实现这一目标，我们计划分别检查金属伴侣（HYPA）和金属聚合物（RCNR）分别在幽门螺杆菌和大肠杆菌中参与金属识别的结构参数。该方法涉及克隆和表达蛋白质，表征其金属离子亲和力，阐明金属位点和蛋白质的结构，并评估蛋白质 - 蛋白质相互作用。为此，使用了诱变和几种结构技术，包括X射线吸收光谱（XAS），晶体学，NMR和基于质谱的技术。为了评估这种多种蛋白质的功能，将采用特定于每种蛋白质的测定；用于RCNR的转录报告基因测定法，以及解决蛋白质将金属递送到靶酶的能力的测定法。除了了解基本的生物化学外，对金属运输涉及的分子机制的详细了解可能会导致用于治疗金属过载（包括中毒）或过量的治疗金属代谢缺陷而导致金属代谢缺陷引起的疗法，并导致金属代谢中的缺陷，以及新的抗生素与新抗生素的设计，使金属含有物质含量的新抗生素。