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: 7238991
• 负责人: BINGDONG SHA
• 金额: $ 27.55万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/7238991
• 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; Research Personnel; 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; programs; protein structure; protein transport; receptor; size; 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）：线粒体膜上的蛋白质易位在线粒体生物发生中起着关键作用。蛋白质从细胞质溶胶到线粒体基质的转运是由外膜（TOM）复合体的转座酶和内膜（TIM）复合体的转座酶完成的。本研究的长期目标是对酵母TOM和TIM复合物进行x射线晶体测井研究，以揭示这些转位子促进前体穿过线粒体内外膜的基本机制。在TOM转座子中，Tom70p作为线粒体前体的受体，具有内部靶向信号。TimSOp、Tim21p和Tim44p是TIM23易位中的重要成员。在膜间空间（IMS）中，TimSOp作为具有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，促进TOM转座子前体的释放。Tim44p是一种外周膜蛋白，稳定地与线粒体内膜在基质侧相关。我们测定了酵母Tom/Op和Tim44p的晶体结构，分别达到3.0A和3.2A的分辨率。我们对酵母TimSOp和Tim21p进行了结晶，并对TimSOp晶体进行了2.7A的x射线衍射。通过噬菌体肽展示文库筛选和等温滴定量热法（ITC）技术的结合，我们鉴定出了Tom70p的肽底物。我们构建了Tom70p及其肽底物的蛋白复合物。我们还构建了TimSOp与线粒体靶向肽Cox4N的复合物。构建了Tim21p和Tom22p c端结构域的蛋白复合物进行结晶试验。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-CHOLINE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转座子模型。体外和体内实验都将用于诱变研究。总的来说，本提案的目的构成了综合性研究，旨在了解TOM和TIM复合物在细胞细胞质到线粒体的蛋白质易位中的基本机制。