Protein unfolding in a physiological system

描述（由申请人提供）：调节性解折叠是关键性的 在许多蛋白质的生命周期中很重要，例如那些跨 线粒体和叶绿体膜，以及那些降解的 ATP依赖性蛋白酶，如蛋白酶体。大约一半的蛋白质 在真核细胞中合成的蛋白质被转运进入或穿过膜。 蛋白质易位机制在生物学上是明确的，但 它们运输和展开蛋白质的方式尚不清楚， 生物化学和生物物理水平。与蛋白质折叠相反， 蛋白质在活细胞中的解折叠机制尚未被研究 以前。 本提案的目的是了解发生的结构性变化， 在易位前的解折叠蛋白中， 展开的机器一系列模型的解折叠路径 跨膜转运的蛋白质将被确定并与 在溶液中自发展开的途径。的机理 解折叠酶将通过化学地或通过 突变和测量对展开的影响。进口的组成部分 将确定有助于展开的机制，以及 它们相互作用的底物蛋白质确定。的假设 这台机器通过物理拉动机制分解蛋白质， 测试. 这些信息对于理解蛋白质在细胞中的解折叠过程是必要的。 细胞和理解复杂蛋白质机器的功能。的 这些结论也将对理解蛋白质 在细胞中的易位过程，特别是对机制， 为蛋白质靶向膜提供特异性。有趣的是 在易位和降解过程中的解折叠过程共享机制 功能.最后，该主题与人类疾病直接相关。为 例如，一种遗传形式的草酸盐中毒是由于一种酶的错误分选 从过氧化物酶体到线粒体。由于导入不需要展开 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.