PHOSPHATE TRANSPORT PATHS WITHIN HOMODIMERIC PTP

同二聚体 PTP 内的磷酸盐转运路径

• 批准号: 6181142
• 负责人: Hartmut none Wohlrab
• 金额: $ 36.92万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181142
• 关键词: X ray crystallography; affinity labeling; chemical binding; crystallization; cytoplasm; dimer; extracellular matrix; ion transport; membrane proteins; membrane transport proteins; mitochondrial membrane; molecular site; mutant; phosphates; protein binding; protein purification; protein structure function; transport inhibitor

The mitochondrial phosphate transport protein (PTP) is a critical link in the energy metabolism of the cell and is thus a very important protein. Any one of many mutations within this protein makes it impossible for a mammalian cell to survive. PTP is an excellent protein to study transmembrane transport of metabolites. Its mechanism of transport is easier to understand than that of other metabolite transport proteins because it transports the simple entities inorganic phosphate (Pi) and proton. Previous studies with site-directed mutagenesis have identified regions in the protein that are critical for transport. The aim now is to identify transmembrane secondary protein structure elements that line the Pi transport path through PTP and to demonstrate that they undergo Pi-induced movements as part of the sequence of events associated with the transport. PTP Pi binding sites on both sides of the membrane will be identified by labeling residues with a competitive inhibitor of PTP, which is a membrane-impermeable photolabel. These sites should be essential for transport and are thus expected to be modified in some PTP mutants inactivated by a conservative mutation. The transport hypothesis implies that PTP functions as a homodimer with two Pi transport paths alternating in the transport of Pi into the mitochondrial matrix. PTP has thus four phosphate binding sites, two on either side of the membrane. Only one of the two sites on either side of the membrane is accessible to Pi at any one time. To support this hypothesis, it will be shown with the help of the photoaffinity label that the Pi-accessible sites are on different subunits and with the help of spin labels that the two sites on the same side of the membrane have different conformations. In addition, spin labels will be attached to PTP to identify the transmembrane secondary structure elements that are associated with the photolabeled residues and that, due to their role in transport, should show Pi-induced movements. Efforts will continue to prepare high quality crystals of PTP to determine its 3D structure. The structure will permit the accurate localization of the functionally relevant residues and regions of PTP and should lead to a good understanding of transmembrane metabolite transport.

线粒体磷酸转运蛋白(PTP)是一个重要的环节 在细胞的能量新陈代谢中，因此是非常重要的 蛋白。这种蛋白质中的任何一个突变都会使它 哺乳动物细胞是不可能存活的。PTP是一种极好的蛋白质 目的：研究代谢产物的跨膜转运。它的作用机理是 运输比其他代谢物更容易理解 运输蛋白质，因为它运输简单的无机物 磷酸盐(PI)和质子。以前对站点定向的研究 突变已经确定了蛋白质中对 运输。现在的目标是鉴定跨膜次级蛋白 通过PTP和TO排列PI传输路径的结构元素 证明它们经历PI诱导的运动作为 与传输关联的事件序列。PTP PI结合位点 在膜的两侧将通过标记残留物进行鉴定 带有竞争性的PTP抑制剂，它是一种膜不渗透的 照片上的标签。这些地点对于交通来说应该是必不可少的，因此 预计会在一些由一种 保守突变。运输假说暗示PTP 起到同源二聚体的作用，两条PI运输路径在 将PI转运到线粒体基质中。因此，PTP有四个 磷酸盐结合部位，膜两边各有两个。只有一个 在膜的两侧的两个位置中的两个位置可由PI在 任何一次。为了支持这一假设，将使用 PI可访问站点所在的光亲和标签的帮助 不同的亚基和借助自旋标记的两个位点 在膜的同一侧有不同的构象。在……里面 此外，自旋标签将贴在PTP上，以识别 跨膜二级结构元件，与 光标记残留物，由于它们在运输中的作用，应该 显示PI诱导的运动。将继续努力准备高 高质量的PTP晶体以确定其3D结构。该结构 将允许准确定位与功能相关的 PTP的残留物和区域，并应导致对 代谢物的跨膜转运。