PHOSPHATE TRANSPORT PATHS WITHIN HOMODIMERIC PTP

同二聚体 PTP 内的磷酸盐转运路径

• 批准号: 2910424
• 负责人: Hartmut none Wohlrab
• 金额: $ 34.83万
• 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/2910424
• 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）是一个关键环节 在细胞的能量代谢中起着非常重要的作用 蛋白质。 该蛋白质内的许多突变中的任何一个都会使其 哺乳动物细胞不可能存活。 PTP是一种优秀的蛋白质 研究代谢物的跨膜转运。 其作用机理为 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实体 磷酸盐 (Pi) 和质子。 先前的定点研究 诱变已经确定了蛋白质中对于 运输。 现在的目标是鉴定跨膜次级蛋白 通过 PTP 排列 Pi 传输路径的结构元素 证明它们经历 Pi 引起的运动作为 与传输相关的事件序列。 PTP Pi 结合位点 膜两侧将通过标记残留物进行识别 具有 PTP 的竞争性抑制剂，这是一种膜不可渗透的抑制剂 照片标签。这些地点对于交通来说是必不可少的，因此 预计在一些被失活的 PTP 突变体中被修饰 保守突变。 运输假说意味着 PTP 作为同源二聚体，具有交替的两条 Pi 传输路径 Pi 转运至线粒体基质。 PTP因此有四个 磷酸盐结合位点，膜两侧各有两个。 只有一个 Pi 可以访问膜两侧的两个位点 任何一次。 为了支持这一假设，将用 Pi 可访问站点所在的光亲和力标签的帮助 不同的亚基，并在自旋标签的帮助下，这两个位点 膜的同一侧具有不同的构象。 在 此外，旋转标签将贴在 PTP 上以识别 与相关的跨膜二级结构元件 光标记残留物，由于它们在运输中的作用，应该 显示 Pi 引起的运动。 将继续努力做好高 PTP 的质量晶体以确定其 3D 结构。 结构 将允许准确定位功能相关的 PTP 的残基和区域，应该能够很好地理解 跨膜代谢物运输。