Structure and Function of Copper Transport ATPases

金属微量营养素，铁，铜，锌等，在蛋白质中发挥着基本的催化和结构功能。适当的金属水平是通过特定的分子伴侣和跨膜转运蛋白的协调作用来实现的。该项目的目标是了解无处不在的P1 B型ATP酶跨膜重金属转运所涉及的基本生物化学。 碱金属（Na+、K+、Ca 2+）在细胞环境中是游离的（水合的）。因此，它们与通道、共转运蛋白和泵可逆地相互作用并通过通道、共转运蛋白和泵转运。相比之下，重金属被结合到具有高缔合常数的分子伴侣/螯合分子。该项目旨在了解重金属是如何传递给它们的转运蛋白的，它们如何与可能高于其伴侣的亲和力协调，以及它们随后如何从转运蛋白中释放。在这些研究中，闪烁古生球菌CopA将被用作模型Cu+-ATP酶。实验将测试CopZ、A.闪电Cu+分子伴侣与CopA相互作用，将Cu+递送至跨膜金属结合位点。金属与跨膜位点结合的化学计量将随着原子配位和配位氨基酸侧链的排列而沿着建立。该项目还将研究金属结合到细胞质结构域的变构调节的结构机制。最后，该项目的第四个目标是建立一个完整的铜+转运ATP酶的原子分辨率结构。这将大大提高对重金属迁移机制的理解。更广泛的影响：该项目将有助于理解金属转运蛋白的金属结合，转运和释放机制的原理。这项研究将有助于正在进行的教育活动，包括本科教学，课程开发和夏季研究。研究生和本科生形成实验室的核心，并将执行本项目的主要部分。这项工作也将影响我们机构与K-12计划的密切合作。这包括指导高中学生在实验室工作，参加高中教师培训。

Metal micronutrients, Fe, Cu, Zn, etc., play fundamental catalytic and structural functions in proteins. Proper metal levels are achieved by the coordinate action of specific chaperones and transmembrane transporters. The goal of this project is to understand the fundamental biochemistry involved in transmembrane heavy metal transport by ubiquitous P1B-type ATPases. Alkali metals (Na+, K+, Ca2+) are free (hydrated) in the cellular milieus. As such, they reversibly interact with and are transported by channels, co-transporters, and pumps. In contrast, heavy metals are bound to chaperones/chelating molecules with high association constants. This project aims to understand how heavy metals are delivered to their transporters, how they are coordinated with affinities likely higher than that of their chaperones, and how they are subsequently released from the transporter. In these studies, Archaeoglobus fulgidus CopA will be used as a model Cu+-ATPase. Experiments will be performed to test whether CopZ, the A. fulgidus Cu+ chaperone, interacts with CopA delivering Cu+ to transmembrane metal binding sites. The stoichiometry of metal binding to transmembrane sites will be established along with the atomic coordination and the arrangement of coordinating amino acid side chains. The project will also examine the structural mechanism of allosteric regulation by metal binding to cytoplasmic domains. Finally, the fourth goal of the project is to establish the complete atomic resolution structure of a Cu+ transporting ATPase. This would greatly enhance understanding the mechanism of heavy metal transport. Broader Impact: The project will contribute to understanding the principles governing metal binding, translocation and release mechanisms of metal transporters. The research will contribute to ongoing educational activities including undergraduate teaching, curriculum development and summer research. Graduate and undergraduate students form the core of the laboratory and will perform major parts of this project. This work will also impact a close collaboration with K-12 programs at our institution. This ranges from guiding high school students working in the lab to participation in high school teachers training.