Project 1

我们将开发用于上述图3中提出的管道的<$Structural Restraints <$$>和<$Structure Calculation <$组件的技术，并将其应用于确定线粒体转位器的结构。 除了它们的ATP合成的主要作用之外，线粒体还容纳几个基本的生物过程，例如活性氧物质的产生、细胞凋亡、产热以及胆固醇和类固醇激素的生物合成。代谢产物和离子通过膜包埋的转位器选择性地进出线粒体。正如预期的那样，这些蛋白质必须在多种构象状态之间不断切换，这可能是为什么它们是难以结晶的目标。我们将解决三个重要的膜转运蛋白的结构：30 kDa的GDP/GTP载体（GGC）和解偶联蛋白2（UCP 2）的线粒体内膜，和19 kDa的外周苯二氮卓类受体（PBR）的外膜。GGC和UCP 2是大线粒体载体家族的成员，其分别使嘌呤核苷酸和质子跨内膜易位。PBR参与类固醇生成限制胆固醇穿过外膜的输入。 这些蛋白质都是多面体螺旋MP，其结构尚不清楚。 多聚螺旋MP提出了不同的问题，分配远程NOE比水溶性蛋白质，因为这两种类型的蛋白质具有不同的折叠性质的包装的氨基酸（AA）的蛋白质核心。前者还具有在主链和侧链共振中的强共振重叠的问题。我们将在两个技术领域开发方法和协议来解决这些问题。首先，我们将测试各种同位素标记方案，并通过使用瓦格纳开发的高分辨率4D NOE实验来评估其测量螺旋间NOE的有效性（项目2）。其次，我们将建立强大的协议，从剩余偶极耦合（RDC）获得全球方向约束。我们将 核心C将新技术集成到拟议的NMR管道中。

We will develop technologies for the ¿Structural Restraints¿ and ¿Structure Calculation¿ components of the pipeline proposed in Fig. 3 above, and apply them to determine the structures of mitochondrial translocators. In addition to their primary role of ATP synthesis, mitochondria house several basic biological processes, such as generation of reactive oxygen species, apoptosis, thermogenesis, and biosynthesis of cholesterol and steroid hormones. Metabolites and ions move selectively in and out of mitochondria through membrane-embedded translocators. As expected from their roles of translocating substrates, these proteins must constantly switch between multiple conformational states, which is probably why they are difficult targets to crystallize. We will solve the structures of three important membrane translocators: the 30 kDa GDP/GTP carrier (GGC) and uncoupling protein 2 (UCP2) of the inner mitochondrial membrane, and the 19 kDa peripheral benzodiazepine receptor (PBR) of the outer membrane. GGC and UCP2 are members of the large mitochondrial carrier family that translocates purine nucleotides and protons across the inner membrane, respectively. PBR is involved in the steroidogenesis-limiting import of cholesterol across the outer membrane. These proteins are all polytopic helical MPs for which structures are not known. Polytopic helical MPs pose different problems of assigning long-range NOEs than water-soluble proteins because the two types of proteins have different folding properties in terms of packing of amino acids (AA) in the protein core. The former also has the problem of strong resonance overlap in both backbone and sidechain resonances. We will develop methods and protocols in two technical areas to solve these problems. First, we will test various isotope labeling schemes and evaluate their effectiveness for measuring inter-helical NOEs by using high resolution 4D NOE experiments to be developed by Wagner (Project 2). Second, we will establish robust protocols for obtaining global orientation restraints from residual dipolar couplings (RDCs). We will work with Core C to integrate the new technologies into the proposed NMR pipeline.