STRUCTURE/FUNCTION OF MITOCHONDRIAL CITRATE CARRIER

线粒体柠檬酸盐载体的结构/功能

• 批准号: 2734806
• 负责人: Ronald Sloan Kaplan
• 金额: $ 27.41万
• 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 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734806
• 关键词: Escherichia coli; X ray crystallography; citrates; crystallization; cysteine; membrane transport proteins; mitochondrial membrane; mutant; protein engineering; protein structure function; site directed mutagenesis; yeasts

DESCRIPTION: The long-term objective of this project is 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 the translocation process. This transporter catalyzes the exchange of tricarboxylates, dicarboxylates, and phosphoenolpyruvate across the inner mitochondrial membrane, and as such is essential to the energy metabolism of eukaryotic cells. Recently, we have developed a procedure enabling the expression and purification of abundant quantities (i.e., 25 mg of purified CTP/liter E. coli culture) of functionally competent yeast mitochondrial CTP. From this foundation, we propose to initiate studies which will fundamentally advance our understanding of how this metabolically important transport protein functions at the molecular, chemical, and ultimately the atomic levels. Specifically, experiments will be carried out to: i) identify residues that are mechanistically essential and/or otherwise important for function via cysteine-scanning mutagenesis of conserved residues, followed by overexpression, purification, and functional characterization of the mutated CTPs; ii) identify amino acids that comprise the substrate translocation pathway through the CTP via probing the accessibility of cysteines (engineered into specific locations within transmembrane domains of a Cys-less CTP construct) to covalent modifying reagents; and iii) identify conditions enabling the growth of X-ray quality crystals 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 set the stage for obtaining 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. Furthermore, altered CTP function in certain disease states (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.

描述：本项目的长期目标是阐明 线粒体柠檬酸转运蛋白（CTP）的分子结构， 高分辨率和确定特定氨基酸残基的功能 in the translocation易位process处理. 这种转运蛋白催化 三羧酸盐、二羧酸盐和磷酸烯醇式丙酮酸盐穿过内层 线粒体膜，并因此是必不可少的能量代谢， 真核细胞 最近，我们开发了一种程序， 表达和纯化丰富的量（即，25 mg纯化 CTP/升E.大肠杆菌培养）的功能感受态酵母线粒体 CTP。 在此基础上，我们建议开展研究， 从根本上推进了我们对这一代谢重要性的理解 转运蛋白在分子、化学和最终 原子水平。 具体而言，将进行实验以：i） 鉴定机械上必需和/或其它的残基 通过半胱氨酸扫描诱变保守的 残基，然后过表达，纯化和功能性 ii）鉴定包含以下的氨基酸： 通过探测CTP的底物易位途径， 半胱氨酸的可接近性（工程化到体内的特定位置） 无Cys CTP构建体的跨膜结构域）共价修饰 试剂;和iii）确定能够提高X射线质量的条件 CTP的晶体。 这些研究将提供信息， 对于了解化学和结构基础至关重要 线粒体CTP功能，并将为获得第一个 代谢物转运蛋白高分辨率结构。 健康 该项目的相关性涉及现金转拨方案在 真核细胞的生物能量学 此外，CTP功能的改变， 某些疾病状态（例如，糖尿病，癌症）是一个重要方面， 这些病理特征的异常中间代谢。 因此，阐明了底物的化学和结构基础， 通过CTP的易位是必不可少的作用的理解 CTP在正常生理和 病理状态。