Protein unfolding in a physiological system

描述（由申请人提供）：受监管的展开至关重要 在许多蛋白质的生命周期中很重要，例如那些易位的蛋白质 线粒体和叶绿体膜，以及那些被降解的膜 ATP 依赖性蛋白酶，例如蛋白酶体。约占所有蛋白质的一半 在真核细胞中合成的物质被转运入或跨过膜。 蛋白质易位机制在生物学上是明确定义的，但是 它们运输和展开蛋白质的方式尚不清楚 生化和生物物理水平。与蛋白质折叠相比， 活细胞中蛋白质展开的机制尚未研究 以前。 该提案的目的是了解所发生的结构性变化 易位前的未折叠蛋白及其分子机制 展开的机器。一系列模型的展开路径 跨膜易位的蛋白质将被确定并与 溶液中自发展开的途径。其机制为 解折叠酶将通过化学或通过抑制候选物来确定 突变并测量对展开的影响。进口的组成部分 有助于展开的机制将被识别，并以何种方式 它们之间的相互作用由底物蛋白决定。假设 该机器通过物理牵引机制解开蛋白质将是 已测试。 这些信息对于理解蛋白质折叠过程是必要的。 细胞并了解复杂蛋白质机器的功能。的 结论也将对蛋白质的理解产生广泛的影响 细胞内的易位过程，特别是以下机制： 为靶向膜的蛋白质提供特异性。有趣的是， 易位和降解过程中的展开过程共享机制 特点。最后，该主题与人类疾病直接相关。对于 例如，遗传性草酸中毒是由于酶的错误分类造成的 从过氧化物酶体到线粒体。由于导入不需要展开 进入过氧化物酶体，并且错误分选的蛋白质含有功能性过氧化物酶体 瞄准信息，防止展开应该纠正排序缺陷。

Description (provided by applicant): Regulated unfolding is critically important in the lifecycle of many proteins, such as those translocated across mitochondrial and chloroplast membranes, as well as those degraded by ATP-dependent proteases such as the proteasome. About half of all proteins synthesized in the eukaryotic cell are transported into or across a membrane. The protein translocation machineries are well defined biologically, but the means by which they transport and unfold proteins are not well understood at the biochemical and biophysical level. In contrast to protein folding, the mechanism of protein unfolding in the living cell has not been studied previously. The aims of this proposal are to understand the structural changes that occur in the unfolding protein prior to translocation and the molecular mechanisms of the unfolding machinery. The pathways of unfolding for a range of model proteins that translocate across membranes will be determined and compared with the pathway of spontaneous unfolding in solution. The mechanism of the unfoldase will be determined by inhibiting candidates either chemically or by mutation and measuring the effect on unfolding. The components of the import machinery that contribute to unfolding will be identified and the way in which they interact with each other the substrate protein determined. The hypothesis that the machinery unravels proteins by a physical pulling mechanism will be tested. This information is necessary to understand protein unfolding processes in the cell and to understand the function of a complex protein machine. The conclusions will also have broad implications for the understanding of protein translocation processes in the cell, in particular on the mechanisms that provide specificity to protein targeting to membranes. Interestingly, the unfolding processes during translocation and degradation share mechanistic features. Finally, the subject is directly relevant to human diseases. For example, an inherited form of oxalosis is due to the miss-sorting of an enzyme from peroxisomes to mitochondria. Since unfolding is not required for import into peroxisomes and the miss-sorted protein contains functional peroxisomal targeting information, preventing unfolding should correct the sorting defect.