Anion Exchange Mechanisms in Bacteria

该项目涉及OxlT，一种新的转运蛋白， 产草酸杆菌，一种革兰氏阴性厌氧菌。 OxlT 催化外部二价草酸盐交换为内部二价草酸盐， 一价甲酸盐。 这种生电交换，沿着质子 在细胞内脱羧过程中的消耗，构成了 间接质子泵，其产生质子动力， 细胞从草酸盐中提取“代谢能量”。 似乎 这种模式会被其他细胞中的其他反应复制，并且 间接质子泵在微生物学中很常见。 到 为了在分子水平上理解这一事件，我们建议研究 OxlT反向转运蛋白 目前，我们有办法净化 并重组OxlT，我们已经提出了抗OxlT多克隆 能帮助我们克隆oxlT基因的抗体 与 纯化的蛋白质和克隆的基因，我们将提高肽特异性 抗体和构建基因融合体以帮助确定 运输蛋白质的结构。 定点诱变将是 用于进一步表征的结构/功能特性 酶与生物化学和生物物理方法一起， 确定最小功能单元（单体或二聚体）。 螺旋含量和蛋白质的结合特性。 %%% 我们已经证明，微生物Oxalciformigenes具有 一种从环境中获取能量的新机制。 这 细胞使用一种特殊的膜蛋白，称为OxlT， 二价草酸分子进入细胞，以换取单价草酸分子。 甲酸盐，其同时被泵出细胞。 的 完成交换循环所需的甲酸盐， 在分子被带到 进入细胞。 负离子的不对称交换 导致跨细胞膜电位的形成 膜，然后细胞可以用来制造额外的能量， 成长 这种新的能量形成机制的核心是 膜蛋白OxlT。 我们将使用分子工具 生物学（克隆，诱变），免疫学（特异性抗体）。 生物化学（纯化和重组）和生物物理学 （光谱学）研究的结构和功能特性 帮助我们理解这种不寻常的细胞膜 “泵”蛋白质，如这一个，运作提供额外的能量 对于细胞。

This project concerns OxlT, a novel transport protein in Oxalobacter formigenes, a gram-negative anaerobic bacterium. OxlT catalyzes the exchange of external divalent oxalate for internal monovalent formate. This electrogenic exchange, along with proton consumption during intracellular decarboxylation, constitutes an indirect proton pump that generates a proton-motive force as the cell extracts "metabolic energy" from oxalate. It appears that this pattern is replicated by other reactions in other cells, and that indirect proton pumps may be common in microbiology. To understand this event at the molecular level, we propose to study OxlT, the antiport protein. Presently, we have methods to purify and reconstitute OxlT, and we have raised anti-OxlT polyclonal antibodies that will aid us in cloning the oxlT gene. With purified protein and a cloned gene, we will raise peptide-specific antibodies and construct gene fusions to aid in the determination of transport protein structure. Site-directed mutagenesis will be used to further characterize the structure/function properties of the enzyme together with biochemical and biophysical methods to determine the minimal functional unit (monomeric or dimeric), .- helical content and the binding properties of the protein. %%% We have shown that the microorganism, Oxalobacter formigenes, has a novel mechanism to harvest energy from its environment. This cell uses a specific membrane protein, termed OxlT, to bring divalent oxalate molecules into the cell in exchange for monovalent formate, which is simultaneously pumped out of the cell. The formate, which is required to complete the exchange cycle is generated by degradation of oxalate after the molecule is brought into the cell. The asymmetric exchange of negatively charged ions results in the formation of a membrane potential across the cell membrane, that the cell can then use to make additional energy and grow. At the heart of this new mechanism of energy formation is the membrane protein, OxlT. We will use the tools of molecular biology (cloning, mutagenesis), immunology (specific antibodies). biochemistry (purification and reconstitution) and biophysics (spectroscopy) to study the structural and functional properties of the enzyme to help us understand how such an unusual membrane "pump" protein, such as this one, operates to provide extra energy for the cells.