PROTEIN UNFOLDING/REFOLDING DURING MITOCHONDRIAL IMPORT

蛋白质必须在运输到大多数细胞器之前展开。在线粒体输入中 分子伴侣启动大多数蛋白质的去折叠过程，然后将部分变性的蛋白质传递给线粒体膜上的输入机器。目前尚不清楚线粒体外膜(TOM)运输机制中的蛋白质是否具有内在的去折叠活性，以及在多大程度上具有内在的去折叠活性，它们用来帮助解开输入的蛋白质。如果Tom蛋白确实包含去折叠活性，这将意味着线粒体本身在进口过程中参与去折叠，而不是将整个工作留给细胞质伴侣。为了确定TOMS是否具有展开活性，将测试人类TOM复合体的两个成员(Tom20和Tom22)解开测试蛋白二氢叶酸还原酶(DHFR)的能力。氢交换质谱仪是一种对蛋白质结构和展开非常敏感的方法，将被用来比较单独进入DHFR与与Tom20或Tom22孵育的DHFR的氢交换。这些结果有望证明Tom20和Tom22有多少展开活动，所产生的展开的大小和DHFR中的展开位置。有了这些数据，运输过程中几个单独步骤发生的展开将被阐明，这些信息是理解进口过程中展开的基本生物过程的关键。

Proteins must unfold prior to transport into most organelles. In the mitochondrial import mechanism, molecular chaperones begin the unfolding process for most proteins and then pass the partially denatured protein to the import machinery in the mitochondrial membrane. It is unclear if and to what extent proteins in the transport machinery of the mitochondrial outer membrane (TOM) posses intrinsic unfolding activity that they use to help unfold importing proteins. If TOM proteins do contain unfolding activity, it would mean that mitochondria themselves participate in unfolding during import rather than leaving the entire job to the cytoplasmic chaperones. To determine if TOMs posses unfolding activity, two members of the human TOM complex (Tom20 and Tom22) will be tested for their ability to unfold the test protein dihydrofolate reductase (DHFR). Hydrogen exchange mass spectrometry, a method very sensitive to changes in protein structure and unfolding, will be used to compare the deuterium exchange into DHFR alone versus DHFR incubated with either Tom20 or Tom22. The results are expected to demonstrate how much unfolding activity Tom20 and Tom22 have, the magnitude of resulting unfolding and the location of the unfolding in DHFR. With such data, the unfolding that occurs at several individual steps in the transport process will be elucidated, information that is key to understanding the fundamental biological process of unfolding during import.