Vectorial Folding of Proteins and Nascent Polypeptide Chains by AFM and Computer Simulations

在了解蛋白质如何通过体外实验和计算机模拟获得其结构方面已经取得了重大进展，但对蛋白质在体内折叠的了解要少得多。在体内的共翻译折叠过程中，新生的多肽链(NPC)以载体的方式从核糖体出口隧道中依次挤出，并在严格的构象约束下开始折叠。目前尚不清楚这种一维(1D)约束如何影响折叠途径。这项研究的长期目标是通过以下方式促进对蛋白质折叠的理解：a)用基于原子力显微镜的单分子力谱(AFM-SMFS)和Steed分子动力学计算机模拟(SMD)研究单一蛋白质在一维约束下的矢量折叠；b)使用AFM直接研究NPC本身的折叠行为。这个项目将研究主要由α-螺旋重复组成的蛋白质的折叠行为，这些重复序列堆叠并形成延伸的螺线管状“矢量”结构，使其成为矢量折叠研究的理想模型系统。其目的是a)设计重复蛋白用于载体折叠研究；b)利用原子力显微镜检查一维约束下重复蛋白的载体折叠；c)使用SMD模拟来研究重复蛋白的载体折叠路径。SMD导出的力-伸长关系将与AFM数据进行比较。通过构建天然接触图，并监测其在拉伸和松弛过程中的时间演变，将重建和分析展开和重新折叠的轨迹；d)通过原子力显微镜检查新生多肽链的折叠行为。停滞不前的核糖体-NPC复合体将使用体外蛋白质表达系统来生产。这些停滞不前的NPC将被AFM尖端捡起并拉伸以检查它们的折叠状态。该项目承诺缩小对蛋白质折叠的理解在体外和体内之间的差距。该项目将为研究生和本科生提供跨学科的教育和研究机会。参与该项目的研究生将参与一次独特的国际研究和教育交流体验。通过让K12学生、他们的家长和老师参与的外展活动将提高公众的科学素养。该项目由生物科学局分子和细胞生物科学部的分子生物物理学和数学和物理科学局物理部的生命系统物理学项目共同支持。

Significant progress has been made toward understanding how proteins acquire their structure through in vitro experiments and computer simulations, but much less is known about protein folding in vivo. During co-translational folding in vivo, the nascent polypeptide chain (NPC) is extruded sequentially in a vectorial manner from the ribosome exit tunnel and starts folding under severe conformational constraints. It is presently unknown how such one dimensional (1D) constraints affect the folding pathway. The long-term objective of this research is to advance understanding of protein folding by: a) studying the vectorial folding of single proteins under 1D constraints by Atomic Force Microscopy-based single-molecule force spectroscopy (AFM-SMFS) and steered molecular dynamics computer simulations (SMD); b) directly examining the folding behavior of the NPC itself, using AFM. This project will examine folding behavior of proteins composed primarily of alpha-helical repeats that stack and form extended, solenoid-like "vectorial" structures making them ideal model systems for vectorial folding studies. The objectives are to a) engineer repeat proteins for vectorial folding studies; b) examine by AFM vectorial folding of repeat proteins under 1D constraints; c) use SMD simulations to examine vectorial folding pathways of repeat proteins. SMD-derived force-extension relationships will be compared with the AFM data. Unfolding and refolding trajectories will be reconstructed and analyzed by building a native contact map, and monitoring its time evolution during stretching and relaxing; d) examine the folding behavior of the nascent polypeptide chain by AFM. Stalled ribosome-NPC complexes will be produced using an in vitro protein expression system. These stalled NPCs will be picked up by the AFM tip and stretched to examine their folding status. The project promises to narrow the gap between an understanding of protein folding in vitro and in vivo. This project will provide interdisciplinary education and research opportunities for graduate and undergraduate students. The graduate students working on this project will participate in a unique international research and educational exchange experience. Outreach activities by involving K12 students, their parents and teachers will raise the scientific literacy of the public. This project is jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Physics of Living Systems Program in the Division of Physics in the Mathematical and Physical Sciences Directorate.