Structural and Functional Studies for Mitochondrial Protein Translocations

线粒体蛋白质易位的结构和功能研究

基本信息

批准号：
7810635
负责人：
BINGDONG SHA
金额：
$ 27.27万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2011-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Protein translocations across mitochondria membranes play critical roles in mitochondria biogenesis. The protein transports from the cell cytosol to the mitochondria matrix are carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complex. The long-term goal of this proposal is to carry out X-ray crystal log rap hie studies on yeast TOM and TIM complexes to uncover the basic mechanisms by which these translocons facilitate the precursors across the outer and inner mitochondria membranes. In the TOM translocon, Tom70p functions as the receptor for mitochondria precursors with internal targeting signals. TimSOp, Tim21p and Tim44p are important members in TIM23 translocon. In the intermembrane space (IMS), TimSOp functions as the receptor for the precursor with the N-terminal mitochondrion targeting sequence. Tim21p can interact with TOM complex member Tom22p to facilitate the release of the precursor from the TOM translocon. Tim44p is a peripheral membrane protein and is stably associated with the mitochondria inner membrane at the matrix side. We have determined the crystal structure of yeast Tom/Op and Tim44p to 3.0A and 3.2A resolution, respectively. We have crystallized yeast TimSOp and Tim21p and the TimSOp crystals diffracted X-ray to 2.7A. By use of the combination of phage peptide display library screening and Isothermal Titration Calorimetry (ITC) technique, we have identified a peptide substrate for Tom70p. We have constituted the protein complex of Tom70p and its peptide substrate. We have also constituted the complex of TimSOp and the mitochondrion targeting peptide Cox4N. The protein complex of Tim21p and Tom22p C-terminal domain has been constituted for crystallization trials. We propose to determine the crystal structures of the Tom70p-peptide substrate complex. We intend to crystallize and determine the crystal structures of the TimSOp-targeting peptide complex and Tim21p-Tom22p protein complex. We also plan to solve the crystal structure of Tim44p and detergent FOS-CHOLINE complex/finally, we will conduct the structure-based mutagenesis studies to test our proposed models for TOM and TIM translocons. Both in vitro and in vivo assays will be utilized in the mutagenesis studies. Collectively, the aims of this proposal constitute the comprehensive studies that seek to understand the basic mechanisms via which the TOM and TIM complexes function in protein translocations from cell cytosol to mitochondrion.

描述（由申请人提供）：跨线粒体膜的蛋白质易位在线粒体生物发生中发挥关键作用。蛋白质从细胞质到线粒体基质的转运是通过外膜转位酶（TOM）复合物和内膜转位酶（TIM）复合物进行的。该提案的长期目标是对酵母 TOM 和 TIM 复合物进行 X 射线晶体对数研究，以揭示这些易位子促进前体穿过线粒体外膜和内膜的基本机制。在 TOM 易位子中，Tom70p 充当具有内部靶向信号的线粒体前体的受体。 TimSOp、Tim21p 和 Tim44p 是 TIM23 易位子的重要成员。在膜间隙 (IMS) 中，TimSOp 充当具有 N 端线粒体靶向序列的前体的受体。 Tim21p 可以与 TOM 复合体成员 Tom22p 相互作用，以促进前体从 TOM 易位子中释放。 Tim44p 是一种外周膜蛋白，与基质侧的线粒体内膜稳定结合。我们已分别以 3.0A 和 3.2A 分辨率测定了酵母 Tom/Op 和 Tim44p 的晶体结构。我们结晶了酵母 TimSOp 和 Tim21p，TimSOp 晶体的 X 射线衍射达到 2.7A。通过结合噬菌体肽展示库筛选和等温滴定量热法（ITC）技术，我们鉴定了Tom70p的肽底物。我们构建了Tom70p及其肽底物的蛋白质复合物。我们还构建了 TimSOp 和线粒体靶向肽 Cox4N 的复合物。 Tim21p 和 Tom22p C 端结构域的蛋白质复合物已构建用于结晶试验。我们建议确定 Tom70p-肽底物复合物的晶体结构。我们打算结晶并确定 TimSOp 靶向肽复合物和 Tim21p-Tom22p 蛋白复合物的晶体结构。我们还计划解决 Tim44p 和洗涤剂 FOS-胆碱复合物的晶体结构/最后，我们将进行基于结构的诱变研究，以测试我们提出的 TOM 和 TIM 易位子模型。体外和体内测定都将用于诱变研究。总的来说，该提案的目标是进行全面的研究，旨在了解 TOM 和 TIM 复合物在蛋白质从细胞质到线粒体易位中发挥作用的基本机制。