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; Colin J. Jackson

doi:10.1007/978-1-4939-6940-1

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, Colin J. Jackson

Research output: Chapter in Book/Conference paper › Chapter › peer-review

7 Citations (Scopus)

Abstract

Small molecule biosensors based on Forster 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.

Original language	English
Title of host publication	Synthetic Protein Switches: Methods And Protocols
Editors	V Stein
Publisher	Springer Nature
Pages	71-87
Number of pages	17
Volume	1596
ISBN (Electronic)	978-1-4939-6940-1
ISBN (Print)	978-1-4939-6938-8
DOIs	https://doi.org/10.1007/978-1-4939-6940-1
Publication status	Published - 2017
Externally published	Yes

Publication series

Name	Methods in Molecular Biology
Volume	1596
ISSN (Print)	1064-3745

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10.1007/978-1-4939-6940-1

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Clifton, B. E., Whitfield, J. H., Sanchez-Romero, I., Herde, M. K., Henneberger, C., Janovjak, H., & Jackson, C. J. (2017). Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors. In V. Stein (Ed.), Synthetic Protein Switches: Methods And Protocols (Vol. 1596, pp. 71-87). (Methods in Molecular Biology; Vol. 1596). Springer Nature. https://doi.org/10.1007/978-1-4939-6940-1

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title = "Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors",

abstract = "Small molecule biosensors based on Forster 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.",

keywords = "Ancestral protein reconstruction, Biosensor, Circular permutation, Fluorescence, Forster resonance energy transfer, Phylogenetic analysis, Protein engineering, Thermostability",

author = "Clifton, {Ben E.} and Whitfield, {Jason H.} and Inmaculada Sanchez-Romero and Herde, {Michel K.} and Christian Henneberger and Harald Janovjak and Jackson, {Colin J.}",

year = "2017",

doi = "10.1007/978-1-4939-6940-1",

language = "English",

isbn = "978-1-4939-6938-8",

volume = "1596",

series = "Methods in Molecular Biology",

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Clifton, BE, Whitfield, JH, Sanchez-Romero, I, Herde, MK, Henneberger, C, Janovjak, H & Jackson, CJ 2017, Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors. in V Stein (ed.), Synthetic Protein Switches: Methods And Protocols. vol. 1596, Methods in Molecular Biology, vol. 1596, Springer Nature, pp. 71-87. https://doi.org/10.1007/978-1-4939-6940-1

Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors. / Clifton, Ben E.; Whitfield, Jason H.; Sanchez-Romero, Inmaculada et al.
Synthetic Protein Switches: Methods And Protocols. ed. / V Stein. Vol. 1596 Springer Nature, 2017. p. 71-87 (Methods in Molecular Biology; Vol. 1596).

Research output: Chapter in Book/Conference paper › Chapter › peer-review

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T1 - Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors

AU - Clifton, Ben E.

AU - Whitfield, Jason H.

AU - Sanchez-Romero, Inmaculada

AU - Herde, Michel K.

AU - Henneberger, Christian

AU - Janovjak, Harald

AU - Jackson, Colin J.

PY - 2017

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N2 - Small molecule biosensors based on Forster 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.

AB - Small molecule biosensors based on Forster 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.

KW - Ancestral protein reconstruction

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KW - Circular permutation

KW - Fluorescence

KW - Forster resonance energy transfer

KW - Phylogenetic analysis

KW - Protein engineering

KW - Thermostability

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U2 - 10.1007/978-1-4939-6940-1

DO - 10.1007/978-1-4939-6940-1

M3 - Chapter

C2 - 28293881

SN - 978-1-4939-6938-8

VL - 1596

T3 - Methods in Molecular Biology

SP - 71

EP - 87

BT - Synthetic Protein Switches: Methods And Protocols

A2 - Stein, V

PB - Springer Nature

ER -

Clifton BE, Whitfield JH, Sanchez-Romero I, Herde MK, Henneberger C, Janovjak H et al. Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors. In Stein V, editor, Synthetic Protein Switches: Methods And Protocols. Vol. 1596. Springer Nature. 2017. p. 71-87. (Methods in Molecular Biology). doi: 10.1007/978-1-4939-6940-1

Ancestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors

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