Cecconi Ciro, Shank Elizabeth A, Marqusee Susan, Bustamante Carlos
CNR-Istituto Nanoscienze S3, Department of Physics, University of Modena e Reggio Emilia, Modena, Italy.
Methods Mol Biol. 2011;749:255-71. doi: 10.1007/978-1-61779-142-0_18.
In this chapter, we describe a method that extends the use of optical tweezers to the study of the folding mechanism of single protein molecules. This method entails the use of DNA molecules as molecular handles to manipulate individual proteins between two polystyrene beads. The DNA molecules function as spacers between the protein and the beads, and keep the interactions between the tethering surfaces to a minimum. The handles can have different lengths, be attached to any pair of exposed cysteine residues, and be used to manipulate both monomeric and polymeric proteins. By changing the position of the cysteine residues on the protein surface, it is possible to apply the force to different portions of the protein and along different molecular axes. Circular dichroism and enzymatic activity studies have revealed that for many proteins, the handles do not significantly affect the folding behavior and the structure of the tethered protein. This method makes it possible to study protein folding in the physiologically relevant low-force regime of optical tweezers and enables us to monitor processes - such as refolding events and fluctuations between different molecular conformations - that could not be detected in previous force spectroscopy experiments.
在本章中，我们描述了一种将光镊的应用扩展到研究单个蛋白质分子折叠机制的方法。该方法需要使用DNA分子作为分子手柄，以操纵两个聚苯乙烯珠之间的单个蛋白质。DNA分子充当蛋白质与珠子之间的间隔物，并将连接表面之间的相互作用保持在最低限度。手柄可以有不同的长度，连接到任何一对暴露的半胱氨酸残基上，并用于操纵单体和聚合蛋白质。通过改变蛋白质表面半胱氨酸残基的位置，可以将力施加到蛋白质的不同部分并沿着不同的分子轴。圆二色性和酶活性研究表明，对于许多蛋白质来说，手柄不会显著影响连接的蛋白质的折叠行为和结构。这种方法使得在光镊生理相关的低力状态下研究蛋白质折叠成为可能，并使我们能够监测以前的力谱实验中无法检测到的过程，如重折叠事件和不同分子构象之间的波动。
