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

Clifton, Benjamin; Whitfield, Jason; Sanchez-Romero, Immaculada; Herde, Michel; Henneberger, Christian; Janovjak, Harald; Jackson, Colin

Description

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,...[Show more]

dc.contributor.authorClifton, Benjamin
dc.contributor.authorWhitfield, Jason
dc.contributor.authorSanchez-Romero, Immaculada
dc.contributor.authorHerde, Michel
dc.contributor.authorHenneberger, Christian
dc.contributor.authorJanovjak, Harald
dc.contributor.authorJackson, Colin
dc.contributor.editorStein, Viktor
dc.date.accessioned2021-05-09T23:43:54Z
dc.identifier.isbn978-1-4939-6938-8
dc.identifier.urihttp://hdl.handle.net/1885/232547
dc.description.abstractSmall 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.
dc.description.sponsorshipResearch was funded by Human Frontiers Science Program Young Investigator Award (HFSP to H.J., C.H., and C.J.J., grant number: RGY0084/2012), German Academic Exchange Service (DAAD-Go8) Travel Fellowship (to C.H. and C.J.J.), NRWRückkehrerprogramm (to C.H.), and German Research Foundation (DFG, SFB1089 B03, SPP1757 HE6949/1-1 and HE6949/3-1, all to C.H.).
dc.format.mimetypeapplication/pdf
dc.language.isoen_AU
dc.publisherHumana Press
dc.relation.ispartofSynthetic Protein Switches : Methods and Protocols
dc.relation.isversionof1 Edition
dc.rights© Springer Science+Business Media LLC 2017
dc.source.urihttps://link.springer.com/book/10.1007/978-1-4939-6940-1
dc.subjectAncestral protein reconstruction
dc.subjectPhylogenetic analysis
dc.subjectProtein engineering
dc.subjectThermostability
dc.subjectCircular permutation
dc.subjectFörster resonance energy transfer
dc.subjectFluorescence
dc.subjectBiosensor
dc.titleAncestral Protein Reconstruction and Circular Permutation for Improving the Stability and Dynamic Range of FRET Sensors
dc.typeBook chapter
local.description.notesImported from ARIES
local.description.refereedYes
dc.date.issued2017
local.identifier.absfor030406 - Proteins and Peptides
local.identifier.ariespublicationU4217927xPUB887
local.publisher.urlhttps://link.springer.com/
local.type.statusPublished Version
local.contributor.affiliationClifton, Benjamin, College of Science, ANU
local.contributor.affiliationWhitfield, Jason, College of Science, ANU
local.contributor.affiliationSanchez-Romero, Immaculada, Institute of Science and Technology Austria
local.contributor.affiliationHerde, Michel, University of Bonn
local.contributor.affiliationHenneberger, Christian, University of Bonn Medical School
local.contributor.affiliationJanovjak, Harald, IST Austria
local.contributor.affiliationJackson, Colin, College of Science, ANU
local.description.embargo2099-12-31
local.bibliographicCitation.startpage71
local.bibliographicCitation.lastpage88
local.identifier.doi10.1007/978-1-4939-6940-1_5
local.identifier.absseo970103 - Expanding Knowledge in the Chemical Sciences
dc.date.updated2020-11-23T10:11:25Z
local.bibliographicCitation.placeofpublicationNew York
local.identifier.scopusID2-s2.0-85015627399
CollectionsANU Research Publications

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