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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_5, © Springer Science+Business Media LLC 2017
Chapter 5
Ancestral Protein Reconstruction and Circular Permutation
for Improving the Stability and Dynamic Range
of FRET Sensors
Ben E. Clifton*, Jason H. Whitfield*, Inmaculada Sanchez-Romero,
Michel K. Herde, Christian Henneberger, Harald Janovjak,
and Colin J. Jackson
Abstract
Small molecule biosensors based on Förster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET
sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such
as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be
unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude
imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here,
we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic
information to improve the thermostability of proteins, while circular permutation enables the termini of
an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide
a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and
discuss considerations for in situ characterization of the FRET sensors.
Key words Ancestral protein reconstruction, Phylogenetic analysis, Protein engineering,
Thermostability, Circular permutation, Förster resonance energy transfer, Fluorescence, Biosensor
1 Introduction
Optical biosensors, including those that rely on Förster resonance
energy transfer (FRET sensors), allow robust, noninvasive quantification of small molecule dynamics in biological systems [1].
FRET is a physical phenomenon whereby excitation of a donor
fluorophore results in nonradiative energy transfer to an acceptor
*These authors contributed equally.
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_5, © Springer Science+Business Media LLC 2017
Chapter 5
Ancestral Protein Reconstruction and Circular Permutation
for Improving the Stability and Dynamic Range
of FRET Sensors
Ben E. Clifton*, Jason H. Whitfield*, Inmaculada Sanchez-Romero,
Michel K. Herde, Christian Henneberger, Harald Janovjak,
and Colin J. Jackson
Abstract
Small molecule biosensors based on Förster resonance energy transfer (FRET) enable small molecule signaling to be monitored with high spatial and temporal resolution in complex cellular environments. FRET
sensors can be constructed by fusing a pair of fluorescent proteins to a suitable recognition domain, such
as a member of the solute-binding protein (SBP) superfamily. However, naturally occurring SBPs may be
unsuitable for incorporation into FRET sensors due to their low thermostability, which may preclude
imaging under physiological conditions, or because the positions of their N- and C-termini may be suboptimal for fusion of fluorescent proteins, which may limit the dynamic range of the resulting sensors. Here,
we show how these problems can be overcome using ancestral protein reconstruction and circular permutation. Ancestral protein reconstruction, used as a protein engineering strategy, leverages phylogenetic
information to improve the thermostability of proteins, while circular permutation enables the termini of
an SBP to be repositioned to maximize the dynamic range of the resulting FRET sensor. We also provide
a protocol for cloning the engineered SBPs into FRET sensor constructs using Golden Gate assembly and
discuss considerations for in situ characterization of the FRET sensors.
Key words Ancestral protein reconstruction, Phylogenetic analysis, Protein engineering,
Thermostability, Circular permutation, Förster resonance energy transfer, Fluorescence, Biosensor
1 Introduction
Optical biosensors, including those that rely on Förster resonance
energy transfer (FRET sensors), allow robust, noninvasive quantification of small molecule dynamics in biological systems [1].
FRET is a physical phenomenon whereby excitation of a donor
fluorophore results in nonradiative energy transfer to an acceptor
*These authors contributed equally.