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Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_2, © Springer Science+Business Media LLC 2017
Chapter 2
Construction of Allosteric Protein Switches by Alternate
Frame Folding and Intermolecular Fragment Exchange
Jeung-Hoi Ha and Stewart N. Loh
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.
Key words AFF, Biosensor, Fluorescence, FRET, FREX, Protein design, Protein engineering
Abbreviations
AFF
Alternate frame folding
CP
Circular permutant
Fn3
Fibronectin 3
FP
Fluorescent protein
FRET Förster resonance energy transfer
FREX Protein/fragment exchange
N
Normal fold of protein
N′
Alternate fold of protein
POI
Protein of interest
RBP
Ribose-binding protein
WT
Wild-type
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_2, © Springer Science+Business Media LLC 2017
Chapter 2
Construction of Allosteric Protein Switches by Alternate
Frame Folding and Intermolecular Fragment Exchange
Jeung-Hoi Ha and Stewart N. Loh
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.
Key words AFF, Biosensor, Fluorescence, FRET, FREX, Protein design, Protein engineering
Abbreviations
AFF
Alternate frame folding
CP
Circular permutant
Fn3
Fibronectin 3
FP
Fluorescent protein
FRET Förster resonance energy transfer
FREX Protein/fragment exchange
N
Normal fold of protein
N′
Alternate fold of protein
POI
Protein of interest
RBP
Ribose-binding protein
WT
Wild-type