Advertisement

Journal of Biosciences

, Volume 43, Issue 3, pp 421–430 | Cite as

GFP fluorescence: A few lesser-known nuggets that make it work

  • Parijat Sarkar
  • Amitabha Chattopadhyay
Commentary
  • 136 Downloads

Abstract

Use of Green Fluorescent Protein (GFP) as a marker has revolutionized biological research in the last few decades. In this brief commentary, we reflect upon the success story of GFP and highlight a few lesser-known facets about GFP that add up to its usefulness.

Keywords

GFP REES FRAP lateral diffusion 

Abbreviations

AOT

sodium bis(2-ethylhexyl)sulfosuccinate

EGFP

enhanced green fluorescent protein

EYFP

enhanced yellow fluorescent protein

FRAP

fluorescence recovery after photobleaching

GFP

green fluorescent protein

GPCR

G protein-coupled receptor

REES

red edge excitation shift

Notes

Acknowledgments

AC gratefully acknowledges support from SERB Distinguished Fellowship (Department of Science and Technology, India). PS thanks the Council of Scientific and Industrial Research for the award of a Shyama Prasad Mukherjee Fellowship. AC is a Distinguished Visiting Professor at Indian Institute of Technology (Bombay), and Adjunct Professor at Tata Institute of Fundamental Research (Mumbai), RMIT University (Melbourne, Australia) and Indian Institute of Science Education and Research (Kolkata). Some of the work described in this article was carried out by former members of AC’s research group whose contributions are gratefully acknowledged. We thank members of the Chattopadhyay laboratory for their comments and discussions.

References

  1. Barak LS, Zhang J, Ferguson SSG, Laporte SA and Caron MG 1999 Signaling, desensitization, and trafficking of G protein-coupled receptors revealed by green fluorescent protein conjugates. Methods Enzymol. 302 153–171CrossRefPubMedGoogle Scholar
  2. Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormö M and Remington SJ 1997 Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc. Natl. Acad. Sci. USA 94 2306–2311CrossRefPubMedGoogle Scholar
  3. Carayon K, Moulédous L, Combedazou A, Mazères S, Haanappel E, Salomé L and Mollereau C 2014 Heterologous regulation of mu-opioid (MOP) receptor mobility in the membrane of SH-SY5Y cells. J. Biol. Chem. 289 28697–28706CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chakraborty H and Chattopadhyay A 2017 Sensing tryptophan microenvironment of amyloid protein utilizing wavelength-selective fluorescence approach. J. Fluoresc. 27 1995–2000CrossRefPubMedGoogle Scholar
  5. Chalfie M 2009 GFP: Lighting up life. Proc. Natl. Acad. Sci. USA 106 10073–10080CrossRefPubMedGoogle Scholar
  6. Chalfie M and Kain SR 2006 Green fluorescent protein: properties, applications, and protocols (New Jersey: Wiley)Google Scholar
  7. Chalfie M, Tu Y, Euskirchen G, Ward WW and Prasher DC 1994 Green fluorescent protein as a marker for gene expression. Science 263 802–805CrossRefPubMedGoogle Scholar
  8. Chattopadhyay A 1990 Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)–labeled lipids: fluorescent probes of biological and model membranes. Chem. Phys. Lipids 53 1–15CrossRefPubMedGoogle Scholar
  9. Chattopadhyay A 2014 GPCRs: lipid-dependent membrane receptors that act as drug targets. Adv. Biol. 2014 143023CrossRefGoogle Scholar
  10. Chattopadhyay A and Haldar S 2014 Dynamic insight into protein structure utilizing red edge excitation shift. Acc. Chem. Res. 47 12–19CrossRefPubMedGoogle Scholar
  11. Chattopadhyay A and Jafurulla M 2015 Novel insights in membrane biology utilizing fluoresence recovery after photobleaching. Adv. Exp. Med. Biol. 842 27–40CrossRefPubMedGoogle Scholar
  12. Chattopadhyay A and Mukherjee S 1993 Fluorophore environments in membrane–bound probes: a red edge excitation shift study. Biochemistry 32 3804–3811CrossRefPubMedGoogle Scholar
  13. Chattopadhyay A and Mukherjee S 1999 Depth-dependent solvent relaxation in membranes: wavelength-selective fluorescence as a membrane dipstick. Langmuir 15 2142–2148CrossRefGoogle Scholar
  14. Chattopadhyay A, Rawat SS, Kelkar DA, Ray S and Chakrabarti A 2003 Organization and dynamics of tryptophan residues in erythroid spectrin: novel structural features of denatured spectrin revealed by the wavelength-selective fluorescence approach. Protein Sci. 12 2389–2403CrossRefPubMedPubMedCentralGoogle Scholar
  15. Demchenko AP 2008 Site-selective red-edge effects. Methods Enzymol. 450 59–78CrossRefPubMedGoogle Scholar
  16. Dickson RM, Cubitt AB, Tsien RY and Moerner WE 1997 On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388 355–358CrossRefPubMedGoogle Scholar
  17. Dix JA and Verkman AS 2008 Crowding effects on diffusion in solutions and cells. Annu. Rev. Biophys. 37 247–263CrossRefPubMedGoogle Scholar
  18. Edidin M 1994 Fluorescence photobleaching and recovery, FPR, in the analysis of membrane structure and dynamics; in Mobility and Proximity in Biological Membranes (eds) S Damjanovich, M Edidin, J Szöllõsi and L Trón (Boca Raton FL: CRC) pp 109–135Google Scholar
  19. Fiorino F, Severino B, Magli E, Ciano A, Caliendo G, Santagada V, Frecentese F and Perissutti E 2014 5-HT1A receptor: an old target as a new attractive tool in drug discovery from central nervous system to cancer. J. Med. Chem. 57 4407–4426CrossRefPubMedGoogle Scholar
  20. Fucile S, Palma E, Martínez-Torres A, Miledi R and Eusebi F 2002 The single-channel properties of human acetylcholine α7 receptors are altered by fusing α7 to the green fluorescent protein. Proc. Natl. Acad. Sci. USA 99 3956–3961CrossRefPubMedGoogle Scholar
  21. Ganim Z and Rief M 2017 Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding. Proc. Natl. Acad. Sci. USA 114 11052–11056CrossRefPubMedGoogle Scholar
  22. Guha S, Rawat SS, Chattopadhyay A and Bhattacharyya B 1996 Tubulin conformation and dynamics: a red edge excitation shift study. Biochemistry 35 13426–13433CrossRefPubMedGoogle Scholar
  23. Haldar S and Chattopadhyay A 2007 Dipolar relaxation within the protein matrix of the green fluorescent protein: a red edge excitation shift study. J. Phys. Chem. B 111 14436–14439CrossRefPubMedGoogle Scholar
  24. Haldar S and Chattopadhyay A 2009a The green journey. J. Fluoresc. 19 1–2CrossRefPubMedGoogle Scholar
  25. Haldar S and Chattopadhyay A 2009b Green fluorescent protein: a molecular lantern that illuminates the cellular interior. J. Biosci. 34 169–172CrossRefPubMedGoogle Scholar
  26. Haldar S and Chattopadhyay A 2013 Application of NBD-labeled lipids in membrane and cell biology; in Springer Series on Fluorescence (eds) Y Mely, G Duportail (Heidelberg: Springer) vol. 13, pp 37–50Google Scholar
  27. Haldar S, Chaudhuri A and Chattopadhyay A 2011 Organization and dynamics of membrane probes and proteins utilizing the red edge excitation shift. J. Phys. Chem. B 115 5693–5706CrossRefPubMedGoogle Scholar
  28. Herschel JFW 1845 On a case of superficial colour presented by a homogeneous liquid internally colourless. Philos. Trans. R. Soc. (London) 135 143–145CrossRefGoogle Scholar
  29. Jain N, Bhasne K, Hemaswasthi M and Mukhopadhyay S 2013 Structural and dynamical insights into the membrane-bound α-synuclein. PLoS ONE 8 e83752CrossRefPubMedPubMedCentralGoogle Scholar
  30. Jameson DM 2014 Introduction to fluorescence (Boca Ratan, FL: CRC Press)CrossRefGoogle Scholar
  31. Kalipatnapu S and Chattopadhyay A 2004 A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin1A receptor. FEBS Lett. 576 455–460CrossRefPubMedGoogle Scholar
  32. Kalipatnapu S and Chattopadhyay A 2005 Membrane organization of the human serotonin1A receptor monitored by detergent insolubility using GFP fluorescence. Mol. Membr. Biol. 22 539–547CrossRefPubMedGoogle Scholar
  33. Kallal L and Benovic JL 2000 Using green fluorescent proteins to study G-protein-coupled receptor localization and trafficking. Trends Pharmacol. Sci. 21 175–180CrossRefPubMedGoogle Scholar
  34. Lacivita E, Leopoldo M, Berardi F and Perrone R 2008 5-HT1A receptor, an old target for new therapeutic agents. Curr. Top. Med. Chem. 8 1024–1034CrossRefPubMedGoogle Scholar
  35. Lippincott-Schwartz J, Snapp E and Kenworthy A 2001 Studying protein dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2 444–456CrossRefPubMedGoogle Scholar
  36. Lo C-A, Kays I, Emran F, Lin T-J, Cvetkovska V and Chen BE 2015 Quantification of protein levels in single living cells. Cell Rep. 13 2634–2644CrossRefPubMedGoogle Scholar
  37. Milligan G 1999 Exploring the dynamics of regulation of G protein-coupled receptors using green fluorescent protein. Br. J. Pharmacol. 128 501–510CrossRefPubMedPubMedCentralGoogle Scholar
  38. Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY and Remington SJ 1996 Crystal structure of the Aequorea victoria green fluorescent protein. Science 273 1392–1395CrossRefPubMedGoogle Scholar
  39. Parasassi T and Gratton E 1995 Membrane lipid domains and dynamics as detected by laurdan fluorescence. J. Fluoresc. 5 59–69CrossRefPubMedGoogle Scholar
  40. Patterson GH 2004 A new harvest of fluorescent proteins. Nat. Biotechnol. 22 1524–1525CrossRefPubMedGoogle Scholar
  41. Patterson GH, Knobel SM, Sharif WD, Kain SR and Piston DW 1997 Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73 2782–2790CrossRefPubMedPubMedCentralGoogle Scholar
  42. Pierce KL, Premont RT and Lefkowitz RJ 2002 Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3 639–650CrossRefPubMedGoogle Scholar
  43. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG and Cormier MJ 1992 Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111 229–233CrossRefPubMedGoogle Scholar
  44. Prendergast FG 1999 Biophysics of the green fluorescent protein. Methods Cell Biol. 58 1- 18PubMedGoogle Scholar
  45. Pucadyil TJ and Chattopadhyay A 2007a The human serotonin1A receptor exhibits G-protein-dependent cell surface dynamics. Glycoconj. J. 24 25–31CrossRefPubMedGoogle Scholar
  46. Pucadyil TJ and Chattopadhyay A 2007b Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin1A receptor in the plasma membrane of living cells. Biochim. Biophys. Acta 1768 655–668CrossRefPubMedGoogle Scholar
  47. Pucadyil TJ, Kalipatnapu S and Chattopadhyay A 2005 The serotonin1A receptor: a representative member of the serotonin receptor family. Cell. Mol. Neurobiol. 25 553–580CrossRefPubMedGoogle Scholar
  48. Pucadyil TJ, Kalipatnapu S, Harikumar KG, Rangaraj N, Karnik SS and Chattopadhyay A 2004 G-protein-dependent cell surface dynamics of the human serotonin1A receptor tagged to yellow fluorescent protein. Biochemistry 43 15852–15862CrossRefPubMedGoogle Scholar
  49. Raja SM, Rawat SS, Chattopadhyay A and Lala AK 1999 Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus. Biophys. J. 76 1469–1479CrossRefPubMedPubMedCentralGoogle Scholar
  50. Rawat SS, Kelkar DA and Chattopadhyay A 2004 Monitoring gramicidin conformations in membranes: a fluorescence approach. Biophys. J. 87 831–843CrossRefPubMedPubMedCentralGoogle Scholar
  51. Ries J, Kaplan C, Platonova E, Eghlidi H and Ewers H 2012 A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9 582–584CrossRefPubMedGoogle Scholar
  52. Saffman PG and Delbrück M 1975 Brownian motion in biological membranes. Proc. Natl. Acad. Sci. USA 72 3111–3113CrossRefPubMedGoogle Scholar
  53. Shimomura O, Johnson FH and Saiga Y 1962 Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59 223–239CrossRefPubMedGoogle Scholar
  54. Soboleski MR, Oaks J and Halford WP 2005 Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. FASEB J. 19 440–442CrossRefPubMedPubMedCentralGoogle Scholar
  55. Suhling K, Siegel J, Phillips D, French PMW, Lévêque-Fort S, Webb SED and Davis DM 2002 Imaging the environment of green fluorescent protein. Biophys. J. 83 3589–3595CrossRefPubMedPubMedCentralGoogle Scholar
  56. Tsien RY 1998 The green fluorescent protein. Annu. Rev. Biochem. 67 509–544CrossRefPubMedGoogle Scholar
  57. Tsien RY 2009 Constructing and exploiting the fluorescent protein paintbox (Nobel lecture). Angew. Chem. Int. Ed. 48 5612–5626CrossRefGoogle Scholar
  58. Uskova MA, Borst J-W, Hink MA, van Hoek A, Schots A, Klyachko NL and Visser AJWG 2000 Fluorescence dynamics of green fluorescent protein in AOT reversed micelles. Biophys. Chem. 87 73–84CrossRefPubMedGoogle Scholar
  59. Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF and Babu MM 2013 Molecular signatures of G-protein-coupled receptors. Nature 494 185–194CrossRefPubMedGoogle Scholar
  60. White J and Stelzer E 1999 Photobleaching GFP reveals protein dynamics inside live cells. Trends Cell Biol. 9 61–65CrossRefPubMedGoogle Scholar
  61. Wilson DW, Crabb BS and Beeson JG 2010 Development of fluorescent Plasmodium falciparum for in vitro growth inhibition assays. Malar. J. 9 152CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Indian Academy of Sciences 2018

Authors and Affiliations

  1. 1.CSIR-Centre for Cellular and Molecular BiologyHyderabadIndia

Personalised recommendations