PROTEIN UNFOLDING/REFOLDING DURING MITOCHONDRIAL IMPORT

线粒体导入过程中蛋白质的解折叠/重折叠

• 批准号: 6972152
• 负责人: JOHN R ENGEN
• 金额: $ 13.77万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-05 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6972152
• 关键词: animal tissue; conformation; deuterium; dihydrofolate reductase; enzyme activity; intracellular transport; mass spectrometry; membrane activity; mitochondrial membrane; molecular chaperones; protein folding; protein structure function; protein transport

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.

蛋白质在进入大多数细胞器之前必须解折叠。在线粒体输入中， 分子伴侣开始大部分蛋白质的解折叠过程，然后将部分变性的蛋白质传递到线粒体膜上的输入机构。目前尚不清楚线粒体外膜（TOM）转运机制中的蛋白质是否以及在多大程度上具有内在的解折叠活性，它们用于帮助解折叠输入蛋白。如果TOM蛋白确实含有解折叠活性，这将意味着线粒体本身在输入过程中参与解折叠，而不是将整个工作交给细胞质伴侣。为了确定TOM是否具有解折叠活性，将测试人TOM复合物的两个成员（Tom20和Tom22）解折叠测试蛋白二氢叶酸还原酶（DHFR）的能力。氢交换质谱法是一种对蛋白质结构和解折叠的变化非常敏感的方法，将用于比较氘交换到单独的DHFR与与Tom20或Tom22孵育的DHFR中。这些结果有望证明Tom20和Tom22具有多少去折叠活性，所产生的去折叠的幅度以及去折叠在DHFR中的位置。有了这些数据，在运输过程中的几个单独的步骤发生的展开将得到阐明，信息是理解在进口过程中展开的基本生物过程的关键。