Cotranslational Events and Folding Pathways of an Alpha-Helical Model Protein

蛋白质折叠是一种多方面的、鲜为人知的现象，具有重要的生物学意义。大多数已发表的文献涉及纯化的全长变性多肽的体外复性。相反，这个项目解决了共翻译蛋白质折叠和错误折叠，因为它发生在核糖体机制中。这项研究的主要目的是开发一种新的方法学来研究原核生物翻译装置中蛋白质折叠的机制和构象方面。新生多肽链的折叠动力学将在大肠杆菌翻译机器的背景下进行研究。这些研究将在无细胞系统中通过氢/氢交换脉冲标记来进行。采用高效液相色谱-飞行时间电喷雾质谱仪和飞行时间-MALDI质谱仪进行检测。载脂蛋白(ApoMb)是一种具有单一结构域、包含所有α-螺旋片段的细胞质蛋白，是进行这些研究的很好的模型系统。将使用具有大肠杆菌优化密码子使用的apoMb基因。氯霉素乙酰转移酶的对照实验也将平行进行。在这个项目中开发的方法学将为未来解决与生物相关的蛋白质折叠问题的重要方面的研究奠定基础。这些研究包括：(A)研究小分子单域蛋白在表达过程中是否发生共翻译折叠；(B)绘制新生多肽构象图，并识别哪些特定氨基酸在链延长的不同阶段发生折叠或错误折叠。人们对蛋白质在细胞中如何折叠或错误折叠的结构方面知之甚少。该项目旨在制定填补这一空白的初步步骤。

Protein folding is a multifaceted poorly understood phenomenon of great biological significance. Most of the published literature on the subject deals with the in vitro refolding of purified full-length denatured polypeptides. In contrast, this project addresses cotranslational protein folding and misfolding, as it occurs within the ribosomal machinery. The main goal of this research is to develop a novel methodology to study the mechanistic and conformational aspects of protein folding within the prokaryotic translation apparatus. The folding kinetics of nascent polypeptide chains will be investigated in the context of the Escherichia coli translation machinery. These studies will be performed by hydrogen/deuterium exchange pulse labeling in cell-free systems. Liquid chromatography-coupled time-of-flight electrospray mass spectrometry and time-of-flight MALDI mass spectrometry will be employed for detection. Apomyoglobin (apoMb) serves as a good model system for these studies since it is a well-characterized cytoplasmic protein with single domain containing all alpha-helical segments. An apoMb gene with E. coli-optimized codon usage will be employed. Control experiments will also be performed in parallel on chloramphenicol acetyltransferase. The methodology developed in this project will set the stage to enable future investigations addressing important aspects of biologically relevant protein folding questions. These include (a) studying whether cotranslational folding takes place during expression of small single domain proteins; and (b) mapping nascent polypeptide conformation, and identifying which specific amino acids become folded or misfolded at different stages of chain elongation. Very little is known about the structural aspects of how proteins fold or misfold in the cell. This project aims at devising initial steps towards filling this gap.