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

蛋白质折叠是一个多方面的知之甚少的现象，具有重要的生物学意义。关于该主题的大多数已发表文献涉及纯化的全长变性多肽的体外重折叠。相反，这个项目致力于共翻译蛋白质折叠和错误折叠，因为它发生在核糖体机器内。本研究的主要目的是发展一种新的方法来研究蛋白质折叠的机制和构象方面的原核翻译装置。新生多肽链的折叠动力学将在大肠杆菌翻译机制的背景下进行研究。这些研究将在无细胞系统中通过氢/氘交换脉冲标记进行。将采用液相色谱-飞行时间电喷雾质谱法和飞行时间MALDI质谱法进行检测。脱辅基肌红蛋白（apoMb）作为一个很好的模型系统，这些研究，因为它是一个很好的特点，细胞质蛋白与单域包含所有的α-螺旋片段。apoMb基因与E.将使用大肠杆菌优化的密码子选择。还将对氯霉素乙酰转移酶平行进行对照实验。该项目中开发的方法将为未来解决生物相关蛋白质折叠问题的重要方面的研究奠定基础。这些包括（a）研究在小的单结构域蛋白质的表达过程中是否发生共翻译折叠;和（B）绘制新生多肽构象，并鉴定哪些特定氨基酸在链延伸的不同阶段被折叠或错误折叠。关于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差距的初步步骤。

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.