STRUCTURE/FUNCTION OF MITOCHONDRIAL CITRATE CARRIER

该项目的长期目标是高分辨率地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易位中的功能。这种转运体催化三羧酸盐、二羧酸盐和磷酸烯醇丙酮酸盐在线粒体内膜上的交换，对真核细胞的能量代谢至关重要。最近，我们已经：i)构建了一个无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工程单个Cys残基的CTP突变体的程序。在此基础上，我们建议开展研究，从根本上提高我们对这种重要代谢转运蛋白在分子、化学和原子水平上的功能的理解。具体来说，将进行以下实验：i)使用半胱氨酸替代诱变技术结合对单个Cys突变体进行化学修饰，确定构成底物易位途径和/或CTP功能所必需的残基；ii)利用cys扫描诱变技术结合位点定向巯基交联和位点定向自旋标记，确定螺旋-螺旋和螺旋-环接近度、螺旋倾斜角度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。综上所述，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（如糖尿病、癌症）中CTP功能的改变是表征这些病理的异常中间代谢的一个重要方面。因此，阐明通过CTP进行底物转运的化学和结构基础，对于理解正常生理和病理状态下CTP在能量产生中的作用至关重要。

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.