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The Design and Application of Genetically Encodable Biosensors Based on Fluorescent Proteins

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Fluorescent Protein-Based Biosensors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1071))

Abstract

To track the activity of cellular signaling molecules within the endogenous cellular environment, researchers have developed a diverse set of genetically encodable fluorescent biosensors. These sensors, which can be targeted to specific subcellular regions to monitor specific pools of a given signaling molecule in real time, rely upon conformational changes in a sensor domain to alter the photophysical properties of green fluorescent protein (GFP) family members. In this introductory chapter, we first discuss the properties of GFP family members before turning our attention to the design and application of genetically encodable fluorescent biosensors to live cell imaging.

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References

  1. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544

    Article  PubMed  CAS  Google Scholar 

  2. Zimmer M (2002) Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 102:759–781

    Article  PubMed  CAS  Google Scholar 

  3. Remington SJ (2006) Fluorescent proteins: maturation, photochemistry and photophysics. Curr Opin Struct Biol 16:714–721

    Article  PubMed  CAS  Google Scholar 

  4. Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182

    Article  PubMed  CAS  Google Scholar 

  5. Miyawaki A, Griesbeck O, Heim R, Tsien RY (1999) Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci U S A 96:2135–2140

    Article  PubMed  CAS  Google Scholar 

  6. Davidson MW, Campbell RE (2009) Engineered fluorescent proteins: innovations and applications. Nat Methods 6:713–717

    Article  PubMed  CAS  Google Scholar 

  7. Shaner NC, Patterson GH, Davidson MW (2007) Advances in fluorescent protein technology. J Cell Sci 120:4247–4260

    Article  PubMed  CAS  Google Scholar 

  8. Newman RH, Fosbrink MD, Zhang J (2011) Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem Rev 111:3614–3666

    Article  PubMed  CAS  Google Scholar 

  9. Sample V, Newman RH, Zhang J (2009) The structure and function of fluorescent proteins. Chem Soc Rev 38:2852–2864

    Article  PubMed  CAS  Google Scholar 

  10. Day RN, Davidson MW (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38:2887–2921

    Article  PubMed  CAS  Google Scholar 

  11. Pakhomov AA, Martynov VI (2008) GFP family: structural insights into spectral tuning. Chem Biol 15:755–764

    Article  PubMed  CAS  Google Scholar 

  12. Chattoraj M, King BA, Bublitz GU, Boxer SG (1996) Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci U S A 93:8362–8367

    Article  PubMed  CAS  Google Scholar 

  13. Brejc K, Sixma TK, Kitts PA, Kain SR et al (1997) Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci U S A 94:2306–2311

    Article  PubMed  CAS  Google Scholar 

  14. Ormo M, Cubitt AB, Kallio K, Gross LA et al (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395

    Article  PubMed  CAS  Google Scholar 

  15. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909

    Article  PubMed  CAS  Google Scholar 

  16. Abad MF, Di Benedetto G, Magalhaes PJ, Filippin L et al (2004) Mitochondrial pH monitored by a new engineered green fluorescent protein mutant. J Biol Chem 279:11521–11529

    Article  PubMed  CAS  Google Scholar 

  17. Kneen M, Farinas J, Li Y, Verkman AS (1998) Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys J 74:1591–1599

    Article  PubMed  CAS  Google Scholar 

  18. Llopis J, McCaffery JM, Miyawaki A, Farquhar MG et al (1998) Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci U S A 95:6803–6808

    Article  PubMed  CAS  Google Scholar 

  19. Griesbeck O, Baird GS, Campbell RE, Zacharias DA et al (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem 276:29188–29194

    Article  PubMed  CAS  Google Scholar 

  20. Nagai T, Ibata K, Park ES, Kubota M et al (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87–90

    Article  PubMed  CAS  Google Scholar 

  21. Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci U S A 91:12501–12504

    Article  PubMed  CAS  Google Scholar 

  22. Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22:445–449

    Article  PubMed  CAS  Google Scholar 

  23. Goedhart J, van Weeren L, Hink MA, Vischer NO et al (2010) Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Methods 7:137–139

    Article  PubMed  CAS  Google Scholar 

  24. Klarenbeek JB, Goedhart J, Hink MA, Gadella TW et al (2011) A mTurquoise-based cAMP sensor for both FLIM and ratiometric read-out has improved dynamic range. PLoS One 6:e19170

    Article  PubMed  CAS  Google Scholar 

  25. Verkhusha VV, Lukyanov KA (2004) The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat Biotechnol 22:289–296

    Article  PubMed  CAS  Google Scholar 

  26. Shu X, Shaner NC, Yarbrough CA, Tsien RY et al (2006) Novel chromophores and buried charges control color in mFruits. Biochemistry 45:9639–9647

    Article  PubMed  CAS  Google Scholar 

  27. Chudakov DM, Lukyanov S, Lukyanov KA (2005) Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol 23:605–613

    Article  PubMed  CAS  Google Scholar 

  28. Wachter RM, Watkins JL, Kim H (2010) Mechanistic diversity of red fluorescence acquisition by GFP-like proteins. Biochemistry 49:7417–7427

    Article  PubMed  CAS  Google Scholar 

  29. Merzlyak EM, Goedhart J, Shcherbo D, Bulina ME et al (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods 4:555–557

    Article  PubMed  CAS  Google Scholar 

  30. Shaner NC, Lin MZ, McKeown MR, Steinbach PA et al (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5:545–551

    Article  PubMed  CAS  Google Scholar 

  31. Shcherbo D, Merzlyak EM, Chepurnykh TV, Fradkov AF et al (2007) Bright far-red fluorescent protein for whole-body imaging. Nat Methods 4:741–746

    Article  PubMed  CAS  Google Scholar 

  32. Shcherbo D, Murphy CS, Ermakova GV, Solovieva EA et al (2009) Far-red fluorescent tags for protein imaging in living tissues. Biochem J 418:567–574

    Article  PubMed  CAS  Google Scholar 

  33. Lin MZ, McKeown MR, Ng HL, Aguilera TA et al (2009) Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals. Chem Biol 16:1169–1179

    Article  PubMed  CAS  Google Scholar 

  34. Lam AJ, St Pierre F, Gong Y, Marshall JD et al (2012) Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9:1005–1012

    Article  PubMed  CAS  Google Scholar 

  35. Meyer AJ, Dick TP (2010) Fluorescent protein-based redox probes. Antioxid Redox Signal 13:621–650

    Article  PubMed  CAS  Google Scholar 

  36. Hanson GT, Aggeler R, Oglesbee D, Cannon M et al (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279:13044–13053

    Article  PubMed  CAS  Google Scholar 

  37. Hung YP, Albeck JG, Tantama M, Yellen G (2011) Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor. Cell Metab 14:545–554

    Article  PubMed  CAS  Google Scholar 

  38. Dittmer PJ, Miranda JG, Gorski JA, Palmer AE (2009) Genetically encoded sensors to elucidate spatial distribution of cellular zinc. J Biol Chem 284:16289–16297

    Article  PubMed  CAS  Google Scholar 

  39. Park JG, Qin Y, Galati DF, Palmer AE (2012) New sensors for quantitative measurement of mitochondrial Zn(2+). ACS Chem Biol 7:1636–1640

    Article  PubMed  CAS  Google Scholar 

  40. Qin Y, Dittmer PJ, Park JG, Jansen KB et al (2011) Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn2+ with genetically encoded sensors. Proc Natl Acad Sci U S A 108:7351–7356

    Article  PubMed  CAS  Google Scholar 

  41. Miyawaki A, Llopis J, Heim R, McCaffery JM et al (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887

    Article  PubMed  CAS  Google Scholar 

  42. Nagai T, Sawano A, Park ES, Miyawaki A (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci U S A 98:3197–3202

    Article  PubMed  CAS  Google Scholar 

  43. Nagai T, Yamada S, Tominaga T, Ichikawa M et al (2004) Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A 101:10554–10559

    Article  PubMed  CAS  Google Scholar 

  44. Ohkura M, Matsuzaki M, Kasai H, Imoto K et al (2005) Genetically encoded bright Ca2+ probe applicable for dynamic Ca2+ imaging of dendritic spines. Anal Chem 77:5861–5869

    Article  PubMed  CAS  Google Scholar 

  45. Palmer AE, Giacomello M, Kortemme T, Hires SA et al (2006) Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem Biol 13:521–530

    Article  PubMed  CAS  Google Scholar 

  46. Tallini YN, Ohkura M, Choi BR, Ji G et al (2006) Imaging cellular signals in the heart in vivo: cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proc Natl Acad Sci U S A 103:4753–4758

    Article  PubMed  CAS  Google Scholar 

  47. Zhang J, Allen MD (2007) FRET-based biosensors for protein kinases: illuminating the kinome. Mol Biosyst 3:759–765

    Article  PubMed  CAS  Google Scholar 

  48. Zhang J, Ma Y, Taylor SS, Tsien RY (2001) Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering. Proc Natl Acad Sci U S A 98:14997–15002

    Article  PubMed  CAS  Google Scholar 

  49. Allen MD, Zhang J (2006) Subcellular dynamics of protein kinase A activity visualized by FRET-based reporters. Biochem Biophys Res Commun 348:716–721

    Article  PubMed  CAS  Google Scholar 

  50. Depry C, Allen MD, Zhang J (2011) Visualization of PKA activity in plasma membrane microdomains. Mol Biosyst 7:52–58

    Article  PubMed  CAS  Google Scholar 

  51. Zhang J, Hupfeld CJ, Taylor SS, Olefsky JM et al (2005) Insulin disrupts beta-adrenergic signalling to protein kinase A in adipocytes. Nature 437:569–573

    Article  PubMed  CAS  Google Scholar 

  52. Zhou X, Herbst-Robinson KJ, Zhang J (2012) Visualizing dynamic activities of signaling enzymes using genetically encodable FRET-based biosensors from designs to applications. Methods Enzymol 504:317–340

    Article  PubMed  CAS  Google Scholar 

  53. Knopfel T, Tomita K, Shimazaki R, Sakai R (2003) Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins. Methods 30:42–48

    Article  PubMed  CAS  Google Scholar 

  54. Lundby A, Mutoh H, Dimitrov D, Akemann W et al (2008) Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements. PLoS One 3:e2514

    Article  PubMed  Google Scholar 

  55. Zhang J, Campbell RE, Ting AY, Tsien RY (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3:906–918

    Article  PubMed  CAS  Google Scholar 

  56. Komatsu N, Aoki K, Yamada M, Yukinaga H et al (2011) Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol Biol Cell 22:4647–4656

    Article  PubMed  CAS  Google Scholar 

  57. Yang F, Moss LG, Phillips GN Jr (1996) The molecular structure of green fluorescent protein. Nat Biotechnol 14:1246–1251

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Robert H. Newman & Jin Zhang

  2. Department of Biology, North Carolina A&T State University, Greensboro, NC, USA

    Robert H. Newman

Authors
  1. Robert H. Newman
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  2. Jin Zhang
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Editor information

Editors and Affiliations

  1. Dept. of Pharmacology & Molecular Scienc, Johns Hopkins University, Baltimore, Maryland, USA

    Jin Zhang

  2. Dept. of Pharmacology & Molecular Scienc, Johns Hopkins University, Baltimore, Maryland, USA

    Qiang Ni

  3. Department of Biology, North Caroline A&T State Univ, Greensboro, North Carolina, USA

    Robert H Newman

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Newman, R.H., Zhang, J. (2014). The Design and Application of Genetically Encodable Biosensors Based on Fluorescent Proteins. In: Zhang, J., Ni, Q., Newman, R. (eds) Fluorescent Protein-Based Biosensors. Methods in Molecular Biology, vol 1071. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-622-1_1

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  • DOI: https://doi.org/10.1007/978-1-62703-622-1_1

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-621-4

  • Online ISBN: 978-1-62703-622-1

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