Anion Exchange Mechanisms in Bacteria

9603997 Maloney这个项目与Oxlt有关，Oxlt是在革兰氏阴性厌氧菌中发现的一种新的运输蛋白，即形成草酸杆菌。OxlT催化外部二价草酸盐与内部单价甲酸盐的交换。这种生电交换，以及细胞内脱羧化过程中的质子消耗，构成了一个间接的质子泵，它产生了一种质子动力，允许产甲氧菌从草酸中提取能量。现在人们认为这种质子动力代谢周期在微生物学中广泛存在，为了在分子水平上理解这些事件，该项目集中在OxlT的研究上，OxlT是OflT这一过程的中心反转运蛋白。获得了针对OxlT N末端的抗体，克隆了该基因，并在大肠杆菌中表达后纯化了功能蛋白。因此，可以使用工具在四个逐步提高分辨率的领域研究结构-功能关系：(1)初步研究将集中在OXlT拓扑结构的研究上，使用多肽特异性抗体、基因融合和SH-定向探针，并以氨基酸序列疏水分析得出的预测为指导。(2)点突变将被用来产生一组单半胱氨酸衍生物，用无效的SH活性试剂进行探测，以定位OxlT易位途径。(3)突变也将用于鉴定重要的功能残基，重点是K355，它可能是OxlT疏水核心中唯一的带电残基；K355可能形成阴离子结合中心的一部分。(4)同时，利用OxlT/底物复合体的稳定性和最近在蛋白质纯化方面的成功，将进行二维结晶试验。总之，这项工作将把对OxlT的理解从细胞生理学层面扩展到分子生物学和生物物理学层面。这个项目是关于OxlT的，它是在土壤细菌Ox albacter formiges中发现的一种新的运输蛋白。OxlT催化两个净电荷不同的阴离子进行跨膜交换，因此当一个阴离子(二价草酸盐)向内移动而另一个(单价甲酸阴离子)向外移动时，膜电位就会上升。同样，当在细胞内时，草酸在消耗一个质子的反应中转化为甲酸盐。总体而言，草酸的新陈代谢产生了电和化学(Ph)梯度，这些梯度构成了一种“质子力”，福尔马氏菌用它来驱动其他与膜相关的事件。这种以质子为动力的代谢循环似乎在微生物中广泛传播。为了在分子水平上理解这些事件，该项目集中研究了OxlT，这是福尔马氏菌中处于该过程中心的反转运蛋白。最初的研究将集中在细胞膜上OxlT的整体组织，使用生化、遗传和免疫学工具。然后，突变将被用来将分子改变为适合用试剂探测的分子，这些试剂可能定位底物运输途径并识别重要的功能残基。最后，将进行二维结晶试验，以利用OxlT底物复合体的稳定性。总之，这些实验应该将对OxlT的理解从细胞生理学层面扩展到分子生物学和生物物理学层面。

9603997 Maloney This project concerns Oxlt, a novel transport protein found in the Gram-negative anaerobe, Oxalobacter formigenes. OxlT catalyzed 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 allowing O. formigenes to extract energy from oxalate. It is now thought such protonmotive metabolic cycles are widely spread in microbiology, and to understand these events at a molecular level this project concentrates on studies on OxlT, the antiport protein at the center of this process in O. formigenes. Antibodies against the OxlT N-terminus are available, the gene is cloned, and functional protein is purified after expression in Escherichia coli. Therefore, tools are available to study structure-function relationships in four areas of progressively increasing resolution: (1) Initial studies will focus on studies of OxlT topology, using peptidespecific antibodies, gene fusions and SH-directed probes, guided by predictions derived from hydropathy analysis of the amino acid sequence. (2) Site-directed mutagenesis will then be used to generate a panel of single-cysteine derivatives to be probed with impermeant SH-active reagents in order to localize the OxlT translocation pathway. (3) Mutagenesis will also be used to identify functionally important residues, with emphasis on K355, which may be the lone charged residue in the OxlT hydrophobic core; K355 may form part of an anion binding center. (4) Concurrently, capitalizing on the stability of the OxlT/substrate complex and on recent success in protein purification, trials of 2-dimensional crystallization will be conducted. Together, this work will extend an understanding of OxlT from the level of cell physiology to that of molecular biology and biophysics. This project is about OxlT, a novel transport protein found in the soil bacterium, Ox alobacter formigenes. OxlT catalyzes the transmembrane exchange of two anions that differ in net charge, so that a membrane potential arises as one (divalent oxalate) moves inward while the other (monovalent formate) moves outward. Similarly, when inside the cell, oxalate is transformed into formate in a reaction that consumes a single proton. Overall, then, the metabolism of oxalate generates electrical and chemical (ph) gradients that comprise a "protonmotive force" which O. formigenes uses to power other membrane-associated events. Such proton-motive metabolic cycles appear to be widely spread in microorganisms. To understand these events at a molecular level this project concentrates on studies of OxlT, the antiport protein at the center of the process in O. formigenes. Initial studies will focus on the overall organization of OxlT in the cell membrane, using biochemical, genetic and immunological tools. Mutagenesis will then be used to change the molecule into one suitable for probing with reagents that may localize the substrate transport pathway and identify functionally important residues. Finally, trials of 2-dimensional crystallization will be conducted to capitalize on the stability of the OxlT-substrate complex. Together, such experiments should experiments should extend an understanding of OxlT from the level of cell physiology to that of molecular biology and biophysics.