Parameters Involved in Metal Recognition

金属识别涉及的参数

• 批准号: 8225281
• 负责人: MICHAEL J MARONEY
• 金额: $ 27.86万
• 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 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8225281
• 关键词: Address; Affinity; Antibiotics; Bacteria; Binding; Binding Sites; Biochemistry; Biological; Biological Assay; Charge; Cloning; Complex; Crystallography; Cysteine; DNA; Defect; Ensure; Enzymes; Equilibrium; Escherichia coli; Goals; Growth; Health; Helicobacter pylori; Histidine; Human; In Vitro; Ions; Lead; Ligands; Link; Metal Binding Site; Metalloproteins; Metals; Molecular; Motivation; Nickel; Organism; Patients; Periplasmic Binding Proteins; Physiological; Poisoning; Proteins; Research; Roentgen Rays; Role; Site; Site-Directed Mutagenesis; Specificity; Spectrum Analysis; Structural Protein; Structure; System; Transition Elements; Work; absorption; base; design; electronic structure; human disease; in vivo; metal metabolism; mutant; novel; pathogen; permease; prevent; protein structure; protein transport; response; stoichiometry; targeted delivery; therapy development; toxic metal; trafficking; transcription factor; 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 concentrations 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 cellular concentration. 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). Although many examples of proteins that achieve these functions for various metal ions have been characterized, the mechanisms that allow for 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. Metal recognition is essential in many trafficking proteins in order to generate biological responses geared to one, or a small group of, metals and avoid crosstalk with other metal ions. Toward this goal, we plan to examine structural parameters that are involved in metal-recognition in a metallotransporter (NikA), a metallochaperone (HypA) and metalloregulators (NikR and RcnR) in E. coli and Helicobacter pylori. The approach involves cloning and expressing trafficking proteins, characterizing their metal ion affinities, and elucidating the structure of the metal sites, employing X-ray absorption spectroscopy (XAS) as a probe of metal site structure and crystallography and NMR to examine protein structure. Alterations in the metal binding site and other critical protein features can be produced by site-directed mutagenesis, and the effect on metal site structure and/or protein structure addressed. Strategies involving both in vitro and in vivo assays are employed to assess the effect on function in mutant trafficking proteins. 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 resulting from defects in metal metabolism, to the design of new antibiotics that interfere with bacterial metal metabolism. PUBLIC HEALTH RELEVANCE: The research proposed seeks to provide a detailed understanding of the molecular mechanisms involved in cellular metal trafficking. This information will be useful in the development of therapies for the treatment of patients with metal overloads (including poisoning) or deficiencies resulting from defects in metal metabolism, and in 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，保持适当的金属浓度涉及控制具有功能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浓度的机制。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（共同称为金属运输蛋白）控制摄取和外排（金属抗碳酸盐），将金属递送至特定酶（金属蛋白蛋白），并调节其他蛋白质的表达，以响应金属离子浓度（MetalOloreReRators）。尽管已经表征了许多为各种金属离子实现这些功能的蛋白质的例子，但允许金属识别和金属特异性生物学反应的机制尚不清楚。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。在许多运输蛋白中，金属识别是必不可少的，以产生针对一种或一小组金属的生物反应，并避免与其他金属离子串扰。为了实现这一目标，我们计划检查金属抗植物（NIKA）中涉及金属识别的结构参数，金属伴侣（HYPA）和冶金基团（NIKR和RCNR）在大肠杆菌和幽门螺杆菌中。该方法涉及克隆和表达运输蛋白，表征其金属离子亲和力，并阐明金属位点的结构，采用X射线吸收光谱（XAS）作为金属位点结构和晶体学和NMR检查蛋白质结构的探针。金属结合位点和其他关键蛋白质特征的改变可以通过定向的诱变产生，以及对金属位点结构和/或蛋白质结构的影响。采用涉及体外和体内测定的策略来评估对突变体运输蛋白功能的影响。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理解可能会导致发展用于治疗金属过载的患者（包括中毒）或因金属代谢缺陷而导致的金属代谢缺陷引起的疗法，从而导致与细菌型金属代谢的新抗生素的设计。公共卫生相关性：拟议的研究旨在详细了解细胞金属贩运所涉及的分子机制。该信息将有助于开发用于治疗金属过载（包括中毒）或因金属代谢缺陷而导致的缺乏的治疗疗法，并在干扰细菌金属代谢的新抗生素的设计中。