Structural Parameters in Metal Recognition

描述（由申请人提供）：维持适当的细胞金属离子水平是所有生物体存活的关键。包括人类病原体在内的细菌的生存能力与获得或竞争过渡金属的能力有关。已经证明，几种人类疾病是由于细胞金属贩运的中断造成的。在过渡金属的情况下，维持适当的金属稳态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，生物体已经发展出机制，以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。这些金属运输系统依赖于能够区分通常具有相似大小和电荷的金属离子的蛋白质。所涉及的金属蛋白，统称为金属运输蛋白，控制摄取和流出（金属转运蛋白），靶向金属的特定酶的交付（金属伴侣）并响应金属离子浓度调节其他蛋白质的表达（金属调节剂）等。虽然已经表征了针对各种金属离子实现这些功能的蛋白质的许多实例，允许特异性金属识别和金属特异性生物反应的机制还不是众所周知的。拟议研究的总体目标是了解允许运输蛋白区分金属的结构参数，以及驱动金属特异性生物反应的相关蛋白质结构变化。为了实现这一目标，我们计划研究幽门螺杆菌和大肠杆菌中金属伴侣蛋白（HypA）和金属调节蛋白（RcnR）参与金属识别的结构参数。coli中表达。该方法包括克隆和表达蛋白质，表征其金属离子亲和力，阐明金属位点和蛋白质的结构，以及评估蛋白质-蛋白质相互作用。为了做到这一点，诱变和几种结构技术，包括X射线吸收光谱（XAS），晶体学，NMR和质谱技术，被利用。为了评估这组不同蛋白质的功能，将采用对每种蛋白质特异性的测定; RcnR的转录报告基因测定，以及解决蛋白质将金属递送至金属伴侣蛋白的靶酶的能力的测定。除了对基本生物化学的理解之外，对涉及金属贩运的分子机制的详细理解可能导致开发用于治疗金属过载（包括中毒）或由金属代谢缺陷引起的缺乏/过量的患者的疗法，以及设计干扰细菌金属代谢的新抗生素。

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.