STRUCTURE/FUNCTION OF MITOCHONDRIAL CITRATE CARRIER

线粒体柠檬酸盐载体的结构/功能

• 批准号: 6386540
• 负责人: Ronald Sloan Kaplan
• 金额: $ 27.11万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386540
• 关键词: X ray crystallography; citrates; conformation; crosslink; crystallization; cysteine; liposomes; membrane transport proteins; mitochondrial membrane; mutant; protein engineering; protein structure function; site directed mutagenesis; thiols; yeasts

The long-term objectives of this project are to elucidate the molecular structure of the mitochondrial citrate transport protein (CTP) at high resolution and to define the functions of specific amino acid residues in substrate recognition and translocation. This transporter catalyzes the exchange of tricarboxylates, dicarboxylates, and phosphoenolpyruvate across the inner mitochondrial membrane, land as such is essential to the energy metabolism of eukaryotic cells. Recently, we have: i) constructed a Cys-less CTP which displays functional properties that are nearly identical to the wild-type transporter; and ii) developed procedures enabling the purification of abundant quantities of the wild-type and Cys-less CTPs, as well as CTP mutants containing site-specifically engineered single Cys residues. From this foundation, we propose to initiate studies that will fundamentally advance our understanding of how this metabolically important transport protein functions at the molecular, chemical, and the atomic levels. Specifically, experiments will be conducted to: i) identify residues that comprise the substrate translocation pathway and/or are essential for CTP function using cysteine-substitution mutagenesis in combination with chemical modification of the resulting single Cys mutants; ii) determine the helix-helix and helix-loop proximities, tilt angles of helices, and ligand-induced conformational changes utilizing Cys-scanning mutagenesis in conjunction with site-directed thiol cross-linking and site- directed spin labeling; and iii) identify conditions enabling the growth of X-ray diffraction quality crystals and to subsequently determine the structure of the CTP. In combination, these studies will provide information essential to an understanding of the chemical and structural bases for Mitochondrial CTP function, and will provide the first high-resolution structure of a metabolite transport protein. The health relatedness of this project concerns the central role of the CTP in the bioenergetics of eukaryotic cells. Consequently, altered CTP function in disease (e.g., diabetes, cancer) is an important aspect of the aberrant intermediary metabolism that characterizes these pathologies. Thus, an elucidation of the chemical and structural bases for substrate translocation through the CTP is essential to an understanding of the role of the CTP in energy production in both normal physiological and pathological states.

该项目的长期目标是在高分辨率下阐明线粒体柠檬酸转运蛋白（CTP）的分子结构，并确定特定氨基酸残基在底物识别和转运中的功能。 这种转运蛋白催化三羧酸、二羧酸和磷酸烯醇丙酮酸穿过线粒体内膜的交换，因此对真核细胞的能量代谢至关重要。 最近，我们有：i）构建了显示出与野生型转运蛋白几乎相同的功能特性的无Cys CTP;和ii）开发了能够纯化大量野生型和无Cys CTP以及含有位点特异性工程化的单个Cys残基的CTP突变体的方法。 从这个基础上，我们建议启动研究，从根本上推进我们对这种代谢重要的转运蛋白如何在分子，化学和原子水平上发挥作用的理解。 i）使用半胱氨酸取代诱变结合所得单Cys突变体的化学修饰来鉴定包含底物易位途径和/或CTP功能所必需的残基; ii）确定螺旋-螺旋和螺旋-环的接近度，螺旋的倾斜角，以及利用Cys扫描诱变结合定点巯基交联和定点自旋标记的配体诱导的构象变化;和iii）确定能够生长X射线衍射质量晶体的条件并随后确定CTP的结构。 这些研究将为理解线粒体CTP功能的化学和结构基础提供必要的信息，并将提供代谢物转运蛋白的第一个高分辨率结构。 该项目的健康相关性涉及CTP在真核细胞生物能量学中的核心作用。 因此，疾病中CTP功能的改变（例如，糖尿病、癌症）是表征这些病理的异常中间代谢的重要方面。 因此，阐明通过CTP的底物易位的化学和结构基础对于理解CTP在正常生理和病理状态下的能量产生中的作用是必不可少的。