Single Molecule Studies of Protein Folding

该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项目共同支持，目的是利用单分子研究和光阱（激光镊子）方法研究蛋白质折叠问题。蛋白质折叠，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的主要挑战。该项目涉及使用一种新方法，利用光学陷阱或激光镊子在单分子水平上操纵和监测蛋白质构象的变化。这种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。机械诱导展开实验还有一个额外的优势，那就是，与体研究不同，反应坐标是明确的：分子的端到端距离。与使用原子力显微镜进行的更常见的机械诱导展开研究相比，光学陷阱的更软的弹簧常数提供了观察蛋白质折叠和展开的潜力。所提出的装置由单个蛋白质，甚至小结构域组成，通过长dsDNA手柄连接到聚苯乙烯珠上。这些把手通过独特的二硫键与蛋白质相连。一个头通过吸力固定在移液器的末端，另一个头通过光学捕集器固定。力是通过将珠子相互拉开来施加的。对具有良好特征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。观察到蛋白质通过紧密的中间体折叠。设置的简单性允许易于操作不同序列的蛋白质，并且手柄的各种附着点允许沿着结构的不同区域施加力。这个项目将训练学生掌握化学、物理和生物的交叉知识。这项工作需要不同背景的学生和博士后紧密合作。该项目还将涉及具有工程和物理背景的本科生，让他们直接接触生物科学。

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.