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Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

机译:蛋白质-DNA嵌合体,用于使用光学镊子进行单分子机械折叠研究

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摘要

Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasi-equilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060, 2005), appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.
机译:在这里,我们报告了一种方法,该方法将对单个蛋白质的机械行为的研究扩展到了光镊的低力状态。这种实验方法依赖于使用DNA手柄将蛋白质特异性附着到聚苯乙烯珠上,并最大程度地减少拴系表面之间的非特异性相互作用。手柄可以连接到任何暴露的半胱氨酸残基对上。使用不同长度的手柄来机械操纵单体和聚合蛋白。光镊的低弹簧常数使我们能够在准平衡条件下直接监测重折叠事件和不同分子结构之间的波动。这种方法已经在蛋白质RNase H的重折叠过程中产生了重要的结果(Cecconi等人,科学309:2057-2060,2005),这种方法似乎很健壮,并且广泛适用于任何工程化包含一对反应性半胱氨酸的蛋白质。残留物。它代表了一种研究单分子水平蛋白质折叠的新策略,应该适用于需要对蛋白质分子进行束缚的一系列问题。

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