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_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, Colin J. Jackson

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

7 Citations (Scopus)

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.

Original language	English
Title of host publication	Synthetic Protein Switches
Subtitle of host publication	Methods and Protocols
Editors	Victor Stein
Place of Publication	New York
Publisher	Humana Press
Chapter	5
Pages	71-87
Number of pages	17
ISBN (Electronic)	9781493969401
ISBN (Print)	9781493969388
DOIs	https://doi.org/10.1007/978-1-4939-6940-1_5
Publication status	Published - 2017
Externally published	Yes

Publication series

Name	Methods in Molecular Biology
Volume	1596
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

Ancestral protein reconstruction
Biosensor
Circular permutation
Fluorescence
Förster resonance energy transfer
Phylogenetic analysis
Protein engineering
Thermostability

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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 (pp. 71-87). (Methods in Molecular Biology; Vol. 1596). Humana Press. https://doi.org/10.1007/978-1-4939-6940-1_5

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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 F{\"o}rster 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, F{\"o}rster 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_5",

language = "English",

isbn = "9781493969388",

series = "Methods in Molecular Biology",

publisher = "Humana Press",

pages = "71--87",

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booktitle = "Synthetic Protein Switches",

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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. Methods in Molecular Biology, vol. 1596, Humana Press, New York, pp. 71-87. https://doi.org/10.1007/978-1-4939-6940-1_5

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. / Victor Stein. New York: Humana Press, 2017. p. 71-87 (Methods in Molecular Biology; Vol. 1596).

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

TY - CHAP

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

Y1 - 2017

N2 - 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.

AB - 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.

KW - Ancestral protein reconstruction

KW - Biosensor

KW - Circular permutation

KW - Fluorescence

KW - Förster resonance energy transfer

KW - Phylogenetic analysis

KW - Protein engineering

KW - Thermostability

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U2 - 10.1007/978-1-4939-6940-1_5

DO - 10.1007/978-1-4939-6940-1_5

M3 - Chapter

C2 - 28293881

AN - SCOPUS:85015627399

SN - 9781493969388

T3 - Methods in Molecular Biology

SP - 71

EP - 87

BT - Synthetic Protein Switches

A2 - Stein, Victor

PB - Humana Press

CY - New York

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. New York: Humana Press. 2017. p. 71-87. (Methods in Molecular Biology). doi: 10.1007/978-1-4939-6940-1_5

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

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