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生存的关键。包括人类病原体在内的细菌的生存能力与获取或竞争过渡金属的能力有关。若干人类疾病已被证明是由于细胞金属贩运中断造成的。在过渡金属的情况下，维持适当的金属稳态包括控制具有功能酶等所需的最佳水平与金属毒性浓度之间的微妙平衡。为了实现这种控制，生物体已经发展出机制来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蛋白质，蛋白质具有区分通常具有相似大小和电荷的金属离子的能力。所涉及的金属蛋白，统称为金属运输蛋白，控制摄取和外排（金属转运蛋白），将金属靶向递送到特定的酶（金属伴侣蛋白），并根据金属离子浓度调节其他蛋白质的表达（金属调节蛋白）等。虽然已经有许多例子证明了蛋白质对各种金属离子具有这些功能，但允许特定金属识别和金属特异性生物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质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，我们计划分别检测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。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，以及评估蛋白质之间的相互作用。为了做到这一点，利用诱变和几种结构技术，包括x射线吸收光谱（XAS），晶体学，核磁共振和基于质谱的技术。为了评估这种不同蛋白质组的功能，将采用针对每种蛋白质的分析；RcnR的转录报告试验，以及解决蛋白质将金属传递到金属伴侣靶酶的能力的试验。除了了解基本的生物化学外，对金属运输的分子机制的详细了解可能会导致治疗金属超载（包括中毒）或金属代谢缺陷导致的金属缺乏/过量患者的治疗方法的发展，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.