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

通过体外实验和计算机模拟，人们在理解蛋白质如何获得其结构方面取得了重大进展，但对体内蛋白质折叠的了解却少得多。在体内共翻译折叠过程中，新生多肽链（NPC）以矢量方式从核糖体出口通道连续挤出，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。本研究的长期目标是通过以下方法来促进对蛋白质折叠的理解：a）通过基于原子力显微镜的单分子力光谱（AFM-SMFS）和转向分子动力学计算机模拟（SMD）研究一维约束下单个蛋白质的矢量折叠; B）使用AFM直接检查NPC本身的折叠行为。该项目将研究主要由α螺旋重复序列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样“矢量”结构，使其成为矢量折叠研究的理想模型系统。目的是a）设计重复蛋白用于矢量折叠研究; B）通过AFM检查在1D约束下重复蛋白的矢量折叠; c）使用SMD模拟检查重复蛋白的矢量折叠途径。将SMD导出的力-延伸关系与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期间的时间演变，重建和分析解折叠和重折叠轨迹; d）通过AFM检查新生多肽链的折叠行为。 将使用体外蛋白质表达系统产生停滞的核糖体-NPC复合物。这些停滞的NPC将被AFM针尖拾取并拉伸以检查其折叠状态。 该项目有望缩小体外和体内蛋白质折叠理解之间的差距。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。 从事该项目的研究生将参与独特的国际研究和教育交流体验。透过让K12学生、家长及教师参与外展活动，提高公众的科学素养。该项目由生物科学理事会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理事会物理学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.