EFFECTS OF TOM20 AND TOM22 ON THE STRUCTURE OF PROTEINS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。此子项目(SPID 0018)不再处于活动状态。恩根博士在获得R01奖学金后，已从NM-INBRE毕业。与蛋白质进入线粒体相关的构象变化正在研究中。了解进口过程中的个体相互作用将增加我们对这一基本细胞过程的理解。氢交换(HX)和高分辨率质谱学(MS)方法被用来观察在最初接收要输入到线粒体的蛋白质的蛋白质存在的情况下，重离子掺入蛋白质中的情况。在本报告所述期间，外膜复合体转运蛋白(Tom20)中的一种蛋白与测试蛋白(DHFR)进行了HX-MS分析。结果表明，Tom20能够扰乱测试蛋白质的动态和/或构象，可能是通过一种旨在使进口过程中更容易展开的机制。为了表征测试蛋白与活的、具有进口能力的线粒体的相互作用，开发了质谱学方法。结果表明，线粒体的外表面与蛋白质的结合是非特异性的。我们没有成功地克服这一实验困难，但我们认为加入MS友好洗涤剂可能是一个解决方案。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This subproject (SPID 0018) is no longer active. Dr. Engen has graduated from the NM-INBRE after receiving an R01 grant. Conformational changes associated with protein import into mitochondria are being investigated. Understanding the individual interactions during import will increase our understanding of this fundamental cellular process. Hydrogen exchange (HX) and high-resolution mass spectrometry (MS) methods are being used to observe the incorporation of deuterium into proteins in the presence of proteins that initially receive the protein that is to be imported into mitochondria. In this reporting period, one protein from the transporter of the outer membrane complex (Tom20) was incuabted with a test protein (DHFR) and HX MS analysis were carried out. The results indicate that Tom20 is capable of disrupting the dynamics and/or conformation of test proteins, perhaps in a mechanism designed to make unfolding easier during importation. To characterize the interaction of the test protein with live, import-competent mitochondria, mass spectrometry methods were developed. The results indicate that the outer surface of mitochondria binds quite non-specifically to proteins. We were unsuccessful in overcoming this experimental difficulty but feel that inclusion of MS friendly detergents may be a solution.