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  • Perspective
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Structure- and mechanism-guided design of single fluorescent protein-based biosensors

Nature Chemical Biology volume 17, pages 509–518 (2021)Cite this article

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Abstract

Intensiometric genetically encoded biosensors, based on allosteric modulation of the fluorescence of a single fluorescent protein, are powerful tools for enabling imaging of neural activities and other cellular biochemical events. The archetypical example of such biosensors is the GCaMP series of Ca2+ biosensors, which have been steadily improved over the past two decades and are now indispensable tools for neuroscience. However, no other biosensors have reached levels of performance, or had revolutionary impacts within specific disciplines, comparable to that of the Ca2+ biosensors. Of the many reasons why this has been the case, a critical one has been a general black-box view of biosensor structure and mechanism. With this Perspective, we aim to summarize what is known about biosensor structure and mechanisms and, based on this foundation, provide guidelines to accelerate the development of a broader range of biosensors with performance comparable to that of the GCaMP series.

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Fig. 1: Single FP-based biosensors and topologies used in biosensor creation.
Fig. 2: Representation of the gate post and bulge residues in FPs derived from different species.
Fig. 3: Gate posts, bulges and adjacent sequences in selected FPs.
Fig. 4: Representative X-ray crystal structures of biosensors.
Fig. 5: Conservation of bulge residue Glu144 in RFP- and cpRFP-based biosensors.
Fig. 6: GFP- and RFP-based biosensor structures provide insight into the mechanism of the fluorescence response.

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Acknowledgements

We thank F. Subach, K. Horikawa, Y. Yang, T. Nagai, Y. Qin, Y. Li, F. Sun and H. Ai for providing gene sequences, and G. Baird for comments. R.E.C. acknowledges the Japan Society for the Promotion of Science (JSPS), Natural Sciences and Engineering Research Council of Canada (NSERC) and Canadian Institutes of Health Research (CIHR) for funding support.

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  1. These authors contributed equally: Yusuke Nasu, Yi Shen.

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  1. Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan

    Yusuke Nasu, Luke Kramer & Robert E. Campbell

  2. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

    Yi Shen & Robert E. Campbell

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Y.N., Y.S. and R.E.C. wrote and edited the manuscript. Y.N., Y.S., L.K. and R.E.C. performed the literature survey that informed the conclusions and opinions expressed in this Perspective.

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Correspondence to Robert E. Campbell.

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R.E.C. is listed as an inventor on patents related to the development of various monomeric FPs. R.E.C. and Y.S. are listed as inventors on patent applications that describe single FP-based Ca2+ biosensors.

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Nasu, Y., Shen, Y., Kramer, L. et al. Structure- and mechanism-guided design of single fluorescent protein-based biosensors. Nat Chem Biol 17, 509–518 (2021). https://doi.org/10.1038/s41589-020-00718-x

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