Skip to main content

Engineering Optogenetic Protein Analogs

Part of the Methods in Molecular Biology book series (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.

Key words

  • Optogenetics
  • Chemogenetics
  • LOV
  • RapR
  • LOVTRAP
  • Z-lock
  • Zdk
  • Engineered extrinsic disorder

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-0755-8_7
  • Chapter length: 14 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   109.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-0755-8
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   149.99
Price excludes VAT (USA)
Hardcover Book
USD   199.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Christie JM, Salomon M, Nozue K, Wada M, Briggs WR (1999) LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proc Natl Acad Sci U S A 96:8779–8783

    CAS  CrossRef  Google Scholar 

  2. Harper SM, Neil LC, Gardner KH (2003) Structural basis of a phototropin light switch. Science 301:1541–1544

    CAS  CrossRef  Google Scholar 

  3. Swartz TE, Corchnoy SB, Christie JM, Lewis JW, Szundi I, Briggs WR et al (2001) The photocycle of a flavin-binding domain of the blue light photoreceptor phototropin. J Biol Chem 276:36493–36500

    CAS  CrossRef  Google Scholar 

  4. Zayner JP, Antoniou C, Sosnick TR (2012) The amino-terminal helix modulates light-activated conformational changes in AsLOV2. J Mol Biol 419:61–74

    CAS  CrossRef  Google Scholar 

  5. Eitoku T, Nakasone Y, Matsuoka D, Tokutomi S, Terazima M (2005) Conformational dynamics of phototropin 2 LOV2 domain with the linker upon photoexcitation. J Am Chem Soc 127:13238–13244

    CAS  CrossRef  Google Scholar 

  6. Salomon M, Christie JM, Knieb E, Lempert U, Briggs WR (2000) Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. Biochemistry 39:9401–9410

    CAS  CrossRef  Google Scholar 

  7. Wang H, Hahn KM (2016) LOVTRAP: a versatile method to control protein function with light. Curr Protoc Cell Biol 73:21.10.1–21.10.14

    CrossRef  Google Scholar 

  8. Wang H, Vilela M, Winkler A, Tarnawski M, Schlichting I, Yumerefendi H et al (2016) LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat Methods 13:755–758

    CAS  CrossRef  Google Scholar 

  9. Toettcher JE, Gong D, Lim WA, Weiner OD (2011) Light-based feedback for controlling intracellular signaling dynamics. Nat Methods 8:837–839

    CAS  CrossRef  Google Scholar 

  10. Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B et al (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461:104–108

    CAS  CrossRef  Google Scholar 

  11. Wu YI, Wang X, He L, Montell D, Hahn KM (2011) Spatiotemporal control of small GTPases with light using the LOV domain. Methods Enzymol 497:393–407

    CAS  CrossRef  Google Scholar 

  12. Wang X, He L, Wu YI, Hahn KM, Montell DJ (2010) Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat Cell Biol 12:591–597

    CAS  CrossRef  Google Scholar 

  13. Yoo SK, Deng Q, Cavnar PJ, Wu YI, Hahn KM, Huttenlocher A (2010) Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev Cell 18:226–236

    CAS  CrossRef  Google Scholar 

  14. Hayashi-Takagi A, Yagishita S, Nakamura M, Shirai F, Wu YI, Loshbaugh AL et al (2015) Labelling and optical erasure of synaptic memory traces in the motor cortex. Nature 525:333–338

    CAS  CrossRef  Google Scholar 

  15. Dietz DM, Sun H, Lobo MK, Cahill ME, Chadwick B, Gao V et al (2012) Rac1 is essential in cocaine-induced structural plasticity of nucleus accumbens neurons. Nat Neurosci 15:891–896

    CAS  CrossRef  Google Scholar 

  16. Stone OJ, Pankow N, Liu B, Sharma VP, Eddy RJ, Wang H et al (2019) Optogenetic control of cofilin and αTAT in living cells using Z-lock. Nat Chem Bio 15(12):1183–1190

    Google Scholar 

  17. Takano T, Wu M, Nakamuta S, Naoki H, Ishizawa N, Namba T et al (2017) Discovery of long-range inhibitory signaling to ensure single axon formation. Nat Commun 8:33

    CrossRef  Google Scholar 

  18. Karginov AV, Ding F, Kota P, Dokholyan NV, Hahn KM (2010) Engineered allosteric activation of kinases in living cells. Nat Biotechnol 28:743–747

    CAS  CrossRef  Google Scholar 

  19. Karginov AV, Zou Y, Shirvanyants D, Kota P, Dokholyan NV, Young DD et al (2011) Light regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP. J Am Chem Soc 133:420–423

    CAS  CrossRef  Google Scholar 

  20. Dagliyan O, Tarnawski M, Chu PH, Shirvanyants D, Schlichting I, Dokholyan NV et al (2016) Engineering extrinsic disorder to control protein activity in living cells. Science 354:1441–1444

    CAS  CrossRef  Google Scholar 

  21. Klomp JE, Huyot V, Ray AM, Collins KB, Malik AB, Karginov AV (2016) Mimicking transient activation of protein kinases in living cells. Proc Natl Acad Sci U S A 113:14976–14981

    CAS  CrossRef  Google Scholar 

  22. Dagliyan O, Dokholyan NV, Hahn KM (2019) Engineering proteins for allosteric control by light or ligands. Nat Protoc 14:1863–1883

    CAS  CrossRef  Google Scholar 

  23. Karginov AV, Tsygankov D, Berginski M, Chu PH, Trudeau ED, Yi JJ et al (2014) Dissecting motility signaling through activation of specific Src-effector complexes. Nat Chem Biol 10:286–290

    CAS  CrossRef  Google Scholar 

  24. Dagliyan O, Shirvanyants D, Karginov AV, Ding F, Fee L, Chandrasekaran SN et al (2013) Rational design of a ligand-controlled protein conformational switch. Proc Natl Acad Sci 110:6800–6804

    CAS  CrossRef  Google Scholar 

  25. Dagliyan O, Krokhotin A, Ozkan-Dagliyan I, Deiters A, Der CJ, Hahn KM et al (2018) Computational design of chemogenetic and optogenetic split proteins. Nat Commun 9:4042

    CrossRef  Google Scholar 

  26. Chu PH, Tsygankov D, Berginski ME, Dagliyan O, Gomez SM, Elston TC et al (2014) Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms. Proc Natl Acad Sci U S A 111:12420–12425

    CAS  CrossRef  Google Scholar 

  27. Lungu Oana I, Hallett Ryan A, Choi Eun J, Aiken Mary J, Hahn Klaus M, Kuhlman B (2012) Designing photoswitchable peptides using the AsLOV2 domain. Chem Biol 19:507–517

    CAS  CrossRef  Google Scholar 

  28. Zimmerman SP, Kuhlman B, Yumerefendi H (2016) Engineering and application of LOV2-based photoswitches. Methods Enzymol 580:169–190

    CAS  CrossRef  Google Scholar 

  29. Guntas G, Hallett RA, Zimmerman SP, Williams T, Yumerefendi H, Bear JE et al (2015) Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc Natl Acad Sci USA 112:112–117

    CAS  CrossRef  Google Scholar 

  30. Hallett RA, Zimmerman SP, Yumerefendi H, Bear JE, Kuhlman B (2016) Correlating in vitro and in vivo activities of light-inducible dimers: a cellular optogenetics guide. ACS Synth Biol 5:53–64

    CAS  CrossRef  Google Scholar 

  31. Strickland D, Lin Y, Wagner E, Hope CM, Zayner J, Antoniou C et al (2012) TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods 9:379–384

    CAS  CrossRef  Google Scholar 

  32. Di Ventura B, Kuhlman B (2016) Go in! Go out! Inducible control of nuclear localization. Curr Opin Chem Biol 34:62–71

    CrossRef  Google Scholar 

  33. Niopek D, Wehler P, Roensch J, Eils R, Di Ventura B (2016) Optogenetic control of nuclear protein export. Nat Commun 7:10624

    CAS  CrossRef  Google Scholar 

  34. Niopek D, Benzinger D, Roensch J, Draebing T, Wehler P, Eils R et al (2014) Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nat Commun 5:4404

    CAS  CrossRef  Google Scholar 

  35. Lerner AM, Yumerefendi H, Goudy OJ, Strahl BD, Kuhlman B (2018) Engineering improved photoswitches for the control of nucleocytoplasmic distribution. ACS Synth Biol 7:2898–2907

    CAS  CrossRef  Google Scholar 

  36. Yumerefendi H, Dickinson DJ, Wang H, Zimmerman SP, Bear JE, Goldstein B et al (2015) Control of protein activity and cell fate specification via light-mediated nuclear translocation. PLoS One 10:e0128443

    CrossRef  Google Scholar 

  37. Yumerefendi H, Wang H, Dickinson DJ, Lerner AM, Malkus P, Goldstein B et al (2018) Light-dependent cytoplasmic recruitment enhances the dynamic range of a nuclear import photoswitch. Chembiochem 19:2898–2907

    CrossRef  Google Scholar 

  38. Yi JJ, Wang H, Vilela M, Danuser G, Hahn KM (2014) Manipulation of endogenous kinase activity in living cells using photoswitchable inhibitory peptides. ACS Synth Biol 3:788–795

    CAS  CrossRef  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Klaus M. Hahn .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

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

Download citation

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

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0754-1

  • Online ISBN: 978-1-0716-0755-8

  • eBook Packages: Springer Protocols