Structural and Functional Studies for Mitochondrial Protein Translocations

线粒体蛋白质易位的结构和功能研究

• 批准号: 8018332
• 负责人: BINGDONG SHA
• 金额: $ 18.41万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-10 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8018332
• 关键词: Affinity; Amino Acids; Binding; Binding Sites; Biogenesis; Biological Assay; C-terminal; Calorimetry; Cells; Choline; Complex; Crystallization; Cytosol; Data; Detergents; Goals; In Vitro; Integral Membrane Protein; Lipids; Membrane; Membrane Proteins; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Mutagenesis; Mutate; Mutation; N-terminal; Peptide Phage Display Library; Peptides; Peripheral; Play; Protein translocation; Proteins; Resolution; Roentgen Rays; Role; Screening procedure; Side; Signal Transduction; Structure; Synchrotrons; Techniques; Testing; Titrations; Transmembrane Domain; Yeast Protein Complex; Yeasts; base; beamline; in vitro Assay; in vivo; insight; loss of function; member; mitochondrion intermembrane space; mutant; protein complex; protein transport; receptor; translocase

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）：线粒体膜的蛋白质易位在线粒体生物发生中起关键作用。蛋白质从细胞胞质到线粒体基质的转运是由外膜（TOM）复合物的易位酶和内膜（TIM）复合物的易位酶进行的。该提案的长期目标是对酵母汤姆和蒂姆复合物进行X射线晶体记录说唱研究，以揭示这些易位的基本机制，这些机制通过这些机制促进了外部和内部线粒体膜的前体。在Tom Clanserocon中，TOM70P充当带有内部靶向信号的线粒体前体的受体。 TIMSOP，TIM21P和TIM44P是TIM23 Clressocon中的重要成员。在膜间空间（IMS）中，Timsop充当N末端线粒体靶向序列的前体的受体。 TIM21P可以与Tom Complex成员Tom22p相互作用，以促进Tom Clanserocon的前体释放。 Tim44p是一种外围膜蛋白，与基质侧的线粒体内膜稳定相关。我们分别确定了酵母TOM/OP和TIM44P的晶体结构，分别为3.0a和3.2a分辨率。我们已经结晶的酵母菌和Tim21p以及Timsop晶体衍射为2.7a。通过使用噬菌体肽显示库筛选和等温滴定量热法（ITC）技术的组合，我们已经确定了TOM70P的肽底物。我们构成了TOM70P及其肽底物的蛋白质复合物。我们还构成了Timsop的复合物和靶向肽Cox4n的线粒体。 TIM21P和TOM22P C末端结构域的蛋白质复合物已成为结晶试验。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和清洁剂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，以测试我们针对Tom和Tim Clanserocons提出的模型。体外和体内测定都将用于诱变研究。总的来说，该提案的目的构成了综合研究，该研究旨在理解Tom和Tim复合物在蛋白质易位的基本机制中，从细胞胞质到线粒体。