Alternate frame folding (AFF) and protein/fragment exchange (FREX) are related technologies for engineering allosteric conformational changes into proteins that have no pre-existing allosteric properties. One of their chief purposes is to turn an ordinary protein into a biomolecular switch capable of transforming an input event into an optical or functional readout. Here, we present a guide for converting an arbitrary binding protein into a fluorescent biosensor with Förster resonance energy transfer output. Because the AFF and FREX mechanisms are founded on general principles of protein structure and stability rather than a property that is idiosyncratic to the target protein, the basic design steps—choice of permutation/cleavage sites, molecular biology, and construct optimization—remain the same for any target protein. We highlight effective strategies as well as common pitfalls based on our experience with multiple AFF and FREX constructs.
CITATION STYLE
Ha, J. H., & Loh, S. N. (2017). Construction of allosteric protein switches by alternate frame folding and intermolecular fragment exchange. In Methods in Molecular Biology (Vol. 1596, pp. 27–41). Humana Press Inc. https://doi.org/10.1007/978-1-4939-6940-1_2
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