MAGNESIUM TRANSPORT IN MICROORGANISMS

最丰富的细胞内二价阳离子，细胞内镁 浓度受到严格控制。在此和其他基础上工作 实验室表明，镁离子在人体内起着重要的调节作用 细胞新陈代谢和生长。因此，所有细胞都可能拥有 感知镁离子并控制其通过的有趣机制 通过细胞膜。此外，镁离子在生物中是独一无二的 水合半径最大、原子最小的阳离子 半径。因此，在水环境中，其体积会发生变化 从原子到水合阳离子几乎是400倍，20倍于 任何其他常见的生物阳离子。因为它是原子阳离子 它穿过双层，水的独特的化学性质 镁离子转运蛋白的独特分子性质可能反映了镁离子的存在。 鼠伤寒沙门氏菌CORA转运蛋白代表一个新的家族 与其他已知蛋白质缺乏显著相似性的转运蛋白， 然而，在革兰氏阴性细菌中广泛存在。虽然毫不含糊地 完整的膜蛋白，CORA的316个氨基酸中有29%含有Frank 充电。CORA的膜拓扑结构表明，该蛋白由 两个基本独立的结构域：N-末端的周质结构域 约230个氨基酸和一个80个氨基酸的C-末端结构域包括 只有三个跨膜片段。此外，我们最近还克隆了 第二种新的、也是非常不寻常的镁离子转运体 在革兰氏阴性和革兰氏阳性细菌中都是如此。 目的我将使用分子和化学交联法来 确定CORA是否起同源齐聚物的作用。AIMS II和III治疗 CORA的周质结构域和膜域是独立的结构。一个 膜结构域的模型将由半胱氨酸构建 取代和交联实验以及来自定点的 突变研究，以确定对阳离子结合重要的残基。 周质结构域的结构将由遗传学研究 突变体的选择和有限的定点突变。这个 结构域将被提纯，用于初步的核磁共振和结晶研究。 AIMS II和AIMS III中的数据加在一起，将能够建造一个完整的 独特的Cora镁离子转运蛋白的三维模型。目标 IV将进一步确定新的镁离子转运体类别，表征 它们的特性并确定它们的系统发育分布。总括 这个项目应该会让我们更好地理解镁离子的基础 转运和细胞内镁离子的动态平衡。

The most abundant intracellular divalent cation, intracellular Mg concentration is tightly controlled. Work from this and other laboratories suggests Mg2+ plays a fundamental regulatory role in cellular metabolism and growth. Thus, all cells likely possess interesting mechanisms for sensing Mg2+ and for controlling its passage through the cell membrane. Moreover, Mg2+ is unique among biological cations in possessing the largest hydrated radius and the smallest atomic radius. As a consequence, in an aqueous environment, its volume change from atomic to hydrated cation is almost 400-fold, 20 times larger than that of any other common biological cation. Since it is the atomic cation that traverses the bilayer, the unique chemical properties of aqueous Mg2+ may be mirrored by unique molecular properties of Mg2+ transporters. The CorA transporter of Salmonella typhimurium represents a new family of transporters lacking significant similarity to other known proteins, yet widespread among Gram-negative bacteria. While unambiguously an integral membrane protein, 29% of CorA's 316 amino acids bear frank charge. The membrane topology of CorA shows that the protein consists of two essentially independent domains: an N-terminal periplasmic domain of about 230 amino acids and an 80 amino acid C-terminal domain comprised of only three transmembrane segments. Further, we have recently cloned a second new and again, highly unusual class of Mg2+ transporter present in both Gram-negative and Gram-positive bacteria. Aim I will use both molecular and chemical crosslinking methods to determine if CorA functions as a homo-oligomer. Aims II and III treat the periplasmic and membrane domains of CorA as independent structures. A model of the membrane domain will be constructed from cysteine substitution and crosslinking experiments and from site-directed mutagenesis studies to determine residues important for cation binding. The structure of the periplasmic domain will be investigated by genetic selection for mutants and by limited site-directed mutagenesis. The domain will be purified for preliminary NMR and crystallization studies. Together, data in Aims II and III will enable construction of a complete three-dimensional model of the unique CorA Mg2+ transport protein. Aim IV will identify further new classes of Mg2+ transporters, characterize their properties and determine their phylogenetic distribution. Overall this project should lead to a greater understanding of the basis of Mg2+ transport and cellular Mg2+ homeostasis.