STRUCTURE/FUNCTION OF MITOCHONDRIAL CITRATE CARRIER

线粒体柠檬酸盐载体的结构/功能

• 批准号: 6195750
• 负责人: Ronald Sloan Kaplan
• 金额: $ 27.87万
• 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/6195750
• 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以高分辨率阐明线粒体柠檬酸转运蛋白（CTP）的分子结构，并定义特定氨基酸残基在底物识别和易位中的功能。 该转运蛋白催化三羧酸盐、二羧酸盐和磷酸烯醇丙酮酸穿过线粒体内膜的交换，因此对于真核细胞的能量代谢至关重要。 最近，我们： i) 构建了一种不含 Cys 的 CTP，其功能特性与野生型转运蛋白几乎相同； ii) 开发了能够纯化大量野生型和不含 Cys 的 CTP 以及含有位点特异性改造的单个 Cys 残基的 CTP 突变体的程序。 在此基础上，我们建议开展研究，从根本上增进我们对这种代谢重要转运蛋白如何在分子、化学和原子水平上发挥作用的理解。 具体而言，将进行实验以：i)使用半胱氨酸取代诱变结合所得单个Cys突变体的化学修饰来鉴定构成底物易位途径和/或对于CTP功能必需的残基； ii)利用Cys扫描诱变结合定点硫醇交联和定点自旋标记确定螺旋-螺旋和螺旋-环邻近度、螺旋的倾斜角和配体诱导的构象变化； 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。 结合起来，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 在真核细胞生物能学中的核心作用。 因此，疾病（例如糖尿病、癌症）中 CTP 功能的改变是表征这些病理学的异常中间代谢的一个重要方面。 因此，阐明通过 CTP 进行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能量产生中的作用至关重要。