Single Molecule Studies of Protein Folding

蛋白质折叠的单分子研究

• 批准号: 0446385
• 负责人: Susan Marqusee
• 金额: $ 97.14万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0446385&HistoricalAwards=false
• 关键词: Single; Molecule; Studies; Protein; Folding

The objective of this project, jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences and the Experimental Physical Chemistry Program in the Chemistry Division, is to investigate the protein-folding problem using single molecule studies and an optical trap (laser tweezers) methodology. Protein folding, the mechanism by which the amino acid sequence encodes the energy landscape of a protein, remains a major challenge for modern molecular biology. This project involves the use of a novel method to manipulate and monitor protein conformational changes at the single molecule level using the optical trap or laser tweezers. This apparatus allows a direct probe of the role of mechanical stress on protein structure and stability. Mechanically induced unfolding experiments have the additional advantage that, unlike bulk studies, the reaction coordinate is well defined: the end-to-end distance of the molecule. In contrast to the more common mechanical induced unfolding studies using the atomic force microscope, the softer spring constant of the optical trap offers the potential to observe protein refolding as well as unfolding. The proposed set-up consists of single proteins, or even small domains, connected to polystyrene beads via long dsDNA handles. The handles are tethered to the protein via unique disulfide bonds. One bead is held in place at the end of a pipette by suction, the other by the optical trap. Force is applied by pulling the beads away from each other. Initial studies on the well-characterized protein ribonuclease H demonstrate the ability to monitor the unfolding and refolding trajectory of a single molecule. The protein is observed to fold through a compact intermediate. The simplicity of the set-up allows for easy manipulation of proteins of varying sequence, and with various attachment points of the handles allowing the force to be applied along different regions of the structure. This project will train students at the interface of chemistry, physics, and biology. The work will require a combination of students and postdoctoral fellows with diverse backgrounds to work closely together. The project will also involve undergraduates with a background in engineering and physics, giving them direct exposure to the biological sciences.

这个项目由分子和细胞生物科学部的分子生物物理学和化学部的实验物理化学计划共同支持，目的是利用单分子研究和光学陷阱(激光镊子)方法研究蛋白质折叠问题。蛋白质折叠，即氨基酸序列编码蛋白质能量格局的机制，仍然是现代分子生物学面临的主要挑战。这个项目涉及使用一种新的方法来操纵和监测单分子水平上的蛋白质构象变化，使用光学陷阱或激光镊子。该仪器可以直接探测机械应力对蛋白质结构和稳定性的作用。与整体研究不同，机械诱导的展开实验还有一个额外的优势，那就是反应坐标定义得很好：分子的端到端距离。与使用原子力显微镜进行更常见的机械诱导去折叠研究相比，光学陷阱的较软弹性常数提供了观察蛋白质再折叠和去折叠的可能性。建议的体系由单个蛋白质，甚至是小结构域组成，通过长dsDNA手柄连接到聚苯乙烯珠子上。手柄通过独特的二硫键与蛋白质相连。一颗珠子通过吸力固定在移液管的末端，另一颗珠子通过光学陷阱固定。力是通过将珠子相互拉开来施加的。对具有良好特性的蛋白质核糖核酸酶H的初步研究表明，它能够监测单个分子的展开和再折叠轨迹。观察到这种蛋白质可以通过致密的中间体折叠。设置的简单性允许容易地操纵不同序列的蛋白质，并且手柄的不同附着点允许沿结构的不同区域施加力。这个项目将培养学生在化学、物理和生物方面的能力。这项工作将需要具有不同背景的学生和博士后研究员密切合作。该项目还将让具有工程和物理背景的本科生参与，让他们直接接触生物科学。