title: Construction of Allosteric Protein Switches by Alternate Frame Folding and Intermolecular Fragment Exchange.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NY, USA.
authors.forename: Jeung-Hoi
authors.initials: JH
authors.lastname: Ha
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NY, USA. lohs@upstate.edu.
authors.forename: Stewart N
authors.initials: SN
authors.lastname: Loh
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D016364:Reading Frames / Q000235:genetics*
chemical_list: D011506:Proteins
keywords: AFF; Biosensor; FRET; FREX; Fluorescence; Protein design; Protein engineering