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Photoswitching Proteins pp 113–126Cite as

Engineering Optogenetic Protein Analogs

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2173))

Abstract

This chapter provides an overview of the technologies we have developed to control proteins with light. First, we focus on the LOV domain, a versatile building block with reversible photo-response, kinetics tunable through mutagenesis, and ready expression in a broad range of cells and animals. Incorporation of LOV into proteins produced a variety of approaches: simple steric block of the active site released when irradiation lengthened a linker (PA-GTPases), reversible release from sequestration at mitochondria (LOVTRAP), and Z-lock, a method in which a light-cleavable bridge is placed where it occludes the active site. The latter two methods make use of Zdk, small engineered proteins that bind selectively to the dark state of LOV. In order to control endogenous proteins, inhibitory peptides are embedded in the LOV domain where they are exposed only upon irradiation (PKA and MLCK inhibition). Similarly, controlled exposure of a nuclear localization sequence and nuclear export sequence is used to reversibly send proteins into the nucleus. Another avenue of engineering makes use of the heterodimerization of FKBP and FRB proteins, induced by the small molecule rapamycin. We control rapamycin with light or simply add it to target cells. Incorporation of fused FKBP-FRB into kinases, guanine exchange factors, or GTPases leads to rapamycin-induced protein activation. Kinases are engineered so that they can interact with only a specific substrate upon activation. Recombination of split proteins using rapamycin-induced conformational changes minimizes spontaneous reassembly. Finally, we explore the insertion of LOV or rapamycin-responsive domains into proteins such that light-induced conformational changes exert allosteric control of the active site. We hope these design ideas will inspire new applications and broaden our reach towards dynamic biological processes that unfold when studied in vivo.

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Acknowledgments

We are grateful to the National Institutes of Health for funding (GM-R35GM122596 to K.M.H.) and thank Andrei Karginov, Onur Dagliyan, and Brian Kuhlman for helpful suggestions.

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Authors and Affiliations

  1. Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA

    Bei Liu, Daniel J. Marston & Klaus M. Hahn

  2. Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Klaus M. Hahn

Authors
  1. Bei Liu
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  2. Daniel J. Marston
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  3. Klaus M. Hahn
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Correspondence to Klaus M. Hahn .

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Editors and Affiliations

  1. IPMB and BioQuant, Heidelberg University, Heidelberg, Baden-Württemberg, Germany

    Dominik Niopek

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Liu, B., Marston, D.J., Hahn, K.M. (2020). Engineering Optogenetic Protein Analogs. In: Niopek, D. (eds) Photoswitching Proteins . Methods in Molecular Biology, vol 2173. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0755-8_7

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  • DOI: https://doi.org/10.1007/978-1-0716-0755-8_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0754-1

  • Online ISBN: 978-1-0716-0755-8

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