Ancestral protein reconstruction and circular permutation for Improving the stability and dynamic range of FRET sensors Book Section

Author(s): Clifton, Ben E; Whitfield, Jason H; Sanchez-Romero, Inmaculada; Herde, Michel K; Henneberger, Christian; Janovjak, Harald; Jackson, Colin J
Article/Chapter Title: Ancestral protein reconstruction and circular permutation for Improving the stability and dynamic range of FRET sensors
Title Series: Methods in Molecular Biology
Affiliation Research School of Chemistry, The Australian National University, Building 137, Sullivans Creek Road, Canberra, ACT, 2601, Australia.
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: Protein Engineering; Fluorescence; ancestral protein reconstruction; biosensor; F├Ârster resonance energy transfer; phylogenetic analysis; thermostability; circular permutation
Book Title: Synthetic Protein Switches
Volume: 1596
ISBN: 978-1-4939-6940-1
Publisher: Springer  
Date Published: 2017-03-15
Start Page: 71
End Page: 87
DOI: 10.1007/978-1-4939-6940-1_5
Notes: 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 R├╝ckkehrerprogramm (to C.H.), and German Research Foundation (DFG, SFB1089 B03, SPP1757 HE6949/1-1 and HE6949/3-1, all to C.H.).
Open access: no