Abstract
Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET studies. The protocol describes two cell-free approaches to achieve a selective label scheme that allows the highest possible accuracy in inter‐dye distance determination.
Keywords: Cell-free protein synthesis, Selective double labeling, Unnatural amino acids, Precharged tRNA, Single-molecule FRET
Background
Single-molecule FRET (smFRET) is one of the most prominent tools in structural biology, in particular for analyzing structures and functional conformational changes of proteins (Michalet et al., 2006; Roy et al., 2008; Sustarsic and Kapanidis, 2015). However, an extensive application of smFRET is in many cases limited by the elaborative production of suitable protein samples. These proteins need to be equipped with two fluorophores, site-specifically attached at different positions within the protein structure. The classical cell-based production of proteins requires a sequence of time-consuming steps that can be overcome by employing cell-free protein synthesis (CFPS) systems, allowing a much faster and straightforward production and selection of proper double-labeled proteins. Moreover, another advantage of CFPS is given by the fact that several classes of proteins like proteases or membrane proteins, which are toxic to living cells or for other reasons are difficult to express in cells, can be synthesized successfully in CFPS systems. Finally, CFPS is an ideal tool for labs focusing on spectroscopic techniques like smFRET, since cell cultures are not necessary and safety regulations for recombinant organisms do not have to be considered. Despite the inherently low sample amounts required for smFRET, CFPS was so far not standardly employed for the production of samples in smFRET studies. This was mostly due to the much lower protein yields as compared to cell-based systems and the lack of an appropriate cell-free approach that allows for a convenient synthesis of adequate amounts of double-labeled protein. However, as demonstrated by our group, thanks to either a much more efficient orthogonal label scheme (Sadoine et al., 2017) or a direct incorporation of dyes with superior photophysical properties (Sadoine et al., 2018), the obtained protein yield from our two cell-free approaches perfectly meets the requirement of related measurements. Combined with state-of-the-art procedures in smFRET studies, the obtained efficiency histograms allow for the highest possible accuracy in inter-dye distance determination the method can deliver today. Here we present a general combined protocol to be used as a guide for preparing double-labeled proteins suitable for smFRET studies, using either one of the two cell-free methods or a combination of both. CFPS and smFRET represent a perfect combination to achieve the full potential of analyzing protein structures with fluorescence-based techniques and the two presented approaches have the potential to become the standard method to produce protein samples for smFRET studies.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This work was supported by Forschungszentrum Jülich resources. The protocol was adapted from procedures published in Sadoine et al. (2017) and Sadoine et al. (2018).
Competing interests
The authors declare no competing financial interests.
References
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