STRUCTURE/FUNCTION OF MITOCHONDRIAL CITRATE CARRIER

该项目的长期目标是在高分辨率下阐明线粒体柠檬酸盐运输蛋白(CTP)的分子结构，并确定特定氨基酸残基在底物识别和转位中的功能。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在线粒体膜上的交换，因此对真核细胞的能量代谢是必不可少的。最近，我们已经：i)构建了一个无半胱氨酸的CTP，其功能性质与野生型转运蛋白几乎相同；以及ii)开发了大量的纯化程序，使大量的野生型和无半胱氨酸的CTP以及含有位点特异性工程的单个半胱氨酸残基的CTP突变体得以纯化。在此基础上，我们建议开始研究，从根本上提高我们对这种代谢重要的运输蛋白如何在分子、化学和原子水平上发挥功能的理解。具体地说，将进行以下实验：i)利用半胱氨酸取代突变结合对产生的单个Cys突变体的化学修饰，确定包含底物易位途径和/或对CTP功能必不可少的残基；ii)利用Cys扫描突变结合定点定向硫醇交联和定点自旋标记，确定螺旋和螺旋环的邻近位置、螺旋倾斜角和配体诱导的构象变化；以及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。总而言之，这些研究将为了解线粒体CTP功能的化学和结构基础提供必要的信息，并将提供第一个代谢产物运输蛋白的高分辨率结构。这个项目的健康相关性涉及到CTP在真核细胞生物能量学中的核心作用。因此，疾病(如糖尿病、癌症)中CTP功能的改变是这些病理特征的异常中间代谢的一个重要方面。因此，阐明通过CTP转运底物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至关重要的。

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.