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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. JacksonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1596)

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 

Notes

Acknowledgments

Research 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.), NRW-Rückkehrerprogramm (to C.H.), and German Research Foundation (DFG, SFB1089 B03, SPP1757 HE6949/1-1 and HE6949/3-1, all to C.H.).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Ben E. Clifton
    • 1
  • Jason H. Whitfield
    • 1
  • Inmaculada Sanchez-Romero
    • 2
  • Michel K. Herde
    • 3
  • Christian Henneberger
    • 3
    • 4
    • 5
  • Harald Janovjak
    • 2
  • Colin J. Jackson
    • 1
    Email author
  1. 1.Research School of ChemistryThe Australian National UniversityCanberraAustralia
  2. 2.Institute of Science and Technology Austria (IST Austria)KlosterneuburgAustria
  3. 3.Institute of Cellular NeurosciencesUniversity of BonnBonnGermany
  4. 4.German Centre for Neurodegenerative DiseasesBonnGermany
  5. 5.University College of LondonLondonUK

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