Department of Biology, Johns Hopkins University, Baltimore, MD, USA.
Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA.
Methods Mol Biol. 2022;2478:427-460. doi: 10.1007/978-1-0716-2229-2_16.
Tethering proteins to force probes, typically micrometer-sized beads, is a prerequisite for dissecting their properties with optical tweezers. DNA handles serve as spacers between the tethered protein of interest and the bead surface. Attachment sites of the DNA handles to both the surface of beads and to the protein of interest must be mechanically stable for optical tweezers experiments. The most prominent method for attaching DNA handles to proteins utilizes thiol chemistry, linking modified DNA to engineered cysteines in the target protein. This method, although experimentally straightforward, is impractical for the large number of proteins that endogenously contain multiple or essential cysteines at undesired positions. Here, we describe two alternative approaches that take advantage of genetically encoded tag sequences in the target protein. The first method uses the enzymes Sfp and BirA, and the second uses the more recently described SpyTag-SpyCatcher system. We outline the process of generating the DNA handles themselves, as well as how to make the DNA-protein chimeras for carrying out optical tweezers experiments. These methods have robustly worked for several diverse and complex proteins, including ones that are difficult to produce or purify, and for protein-containing complexes such as the ribosome. They will be useful in cases where chemistry-based approaches are impractical or not feasible.
将 tethering 蛋白与力探针（通常为微米大小的珠子）连接起来，是使用光学镊子对其性质进行剖析的前提条件。DNA 接头可作为连接感兴趣的 tethering 蛋白与珠子表面的间隔物。DNA 接头与珠子表面和感兴趣的蛋白的附着点必须在光学镊子实验中具有机械稳定性。将 DNA 接头连接到蛋白的最突出方法是利用硫醇化学，将修饰的 DNA 与目标蛋白中的工程化半胱氨酸连接。尽管这种方法在实验上很简单，但对于大量内源性含有多个或必需半胱氨酸的蛋白来说，在不想要的位置上，这种方法是不切实际的。在这里，我们描述了两种利用目标蛋白中遗传编码标签序列的替代方法。第一种方法使用 Sfp 和 BirA 酶，第二种方法使用最近描述的 SpyTag-SpyCatcher 系统。我们概述了生成 DNA 接头本身的过程，以及如何制备用于进行光学镊子实验的 DNA-蛋白嵌合体。这些方法已经成功地用于几种不同且复杂的蛋白，包括那些难以生产或纯化的蛋白，以及包含蛋白的复合物，如核糖体。在基于化学的方法不切实际或不可行的情况下，这些方法将非常有用。
