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Journal Article
Research Support, N.I.H., Extramural
Tethering Complex Proteins and Protein Complexes for Optical Tweezers Experiments.
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.
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