Applications of Fluorescence Anisotropy in Understanding Protein Conformational Disorder and Aggregation

  • Neha Jain
  • Samrat Mukhopadhyay
Part of the Progress in Optical Science and Photonics book series (POSP, volume 2)


Fluorescence spectroscopy is an ultra-sensitive multiparametric technique that provides key insights into protein conformational dynamics and size changes simultaneously. Fluorescence polarization (anisotropy) is one of the parameters related to the rotational dynamics of a fluorophore either intrinsic to the molecule or attached to a biomolecule. The anisotropy measurements can be utilized to unravel the structural and dynamical properties of biomolecules. The advantage of fluorescence anisotropy measurements is that it is a concentration-independent parameter; it can be measured either in the steady-state or in the time-resolved format. Steady-state fluorescence anisotropy provides important information about the overall size/dynamics of biomolecules, whereas the time-resolved fluorescence anisotropy can distinguish between the local and the global dynamics of a fluorophore. Therefore, the time-resolved anisotropy measurements allow one to determine the conformational flexibility as well as the size of biomolecules and assemblies. In recent years, it has been demonstrated that fluorescence anisotropy can be effectively utilized to obtain structural and dynamical information of protein-based assemblies such as aggregates, protein–lipid complexes etc. This chapter provides an overview of the applications of fluorescence anisotropy to study protein conformational disorder, misfolding and aggregation, leading to the formation of nanoscopic amyloid fibrils that are implicated in a range of human diseases.


Fluorescence spectroscopy Fluorescence polarization Rotational dynamics Fluorescence anisotropy Time-resolved measurements Protein conformation Dynamics Disorder Protein misfolding Aggregation Protein–lipid complexes Amyloid fibrils 



We thank Dr. Mily Bhattacharya for critically reviewing and for making extremely valuable suggestions on this manuscript and the members of the Mukhopadhyay laboratory of Amyloid Biology for the research contributions described in this book chapter. Research grant from CSIR (to SM) and financial support from IISER Mohali is gratefully acknowledged.


  1. 1.
    Steiner RF (1991) Fluorescence anisotropy: theory and applications. In: Lakowicz JR (ed) Topics in fluorescence spectroscopy, vol 2: principles. Plenum Press, New YorkGoogle Scholar
  2. 2.
    Kawski A (1993) Fluorescence anisotropy: theory and applications of rotational depolarization. Crit Rev Anal Chem 6:459–529CrossRefGoogle Scholar
  3. 3.
    Munishkina LA, Fink AL (2007) Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins. Biochim Biophys Acta 1768:1862–1885CrossRefGoogle Scholar
  4. 4.
    Jha A, Narayan S, Udgaonkar JB, Krishnamoorthy G (2012) Solvent-induced tuning of internal structure in a protein amyloid protofibril. Biophys J 103:797–806CrossRefGoogle Scholar
  5. 5.
    Sheynis T, Friediger A, Xue W-F, Hellewell AL, Tipping KW, Hewitt EW, Radford S, Jelinek R (2013) Aggregation modulators interfere with membrane interactions of β2-microglobulin fibrils. Biophys J 105:745–755CrossRefGoogle Scholar
  6. 6.
    Yan Y, Marriott G (2003) Analysis of protein interactions using fluorescence technologies. Curr Opin Chem Biol 7:635–640CrossRefGoogle Scholar
  7. 7.
    Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Kluwer Academic, New YorkCrossRefGoogle Scholar
  8. 8.
    Valeur B (2001) Molecular fluorescence principles and applications. WILEY-VCH Verlag GmBH, WeinheimGoogle Scholar
  9. 9.
    Brown MP, Royer C (1997) Fluorescence spectroscopy as a tool to investigate protein interactions. Curr Opin Biotechnol 8:45–49CrossRefGoogle Scholar
  10. 10.
    Dobson CM (1999) Protein misfolding, evolution and disease. Trends Biochem Sci 24:329–332CrossRefGoogle Scholar
  11. 11.
    Luheshi LM, Crowther DC, Dobson CM (2008) Protein misfolding and disease: from the test tube to the organism. Curr Opin Chem Biol 12:25–31CrossRefGoogle Scholar
  12. 12.
    Kumar S, Udgaonkar JB (2010) Mechanisms of amyloid fibril formation by proteins. Curr Sci 98:639–656Google Scholar
  13. 13.
    Gradinaru CC, Marushchak DO, Samim M, Krull UJ (2010) Fluorescence anisotropy: from single molecules to live cells. Analyst 135:452–459CrossRefGoogle Scholar
  14. 14.
    Jameson DM, Ross JA (2010) Fluorescence polarization/anisotropy in diagnostics and imaging. Chem Rev 110:2685–2708CrossRefGoogle Scholar
  15. 15.
    Jameson DM, Sawyer WH (1995) Fluorescence anisotropy applied to biomolecular interactions. Methods Enzymol 246:283–300CrossRefGoogle Scholar
  16. 16.
    Saxena A, Udgaonkar JB, Krishnamoorthy G (2005) In applications of fluorescence spectroscopy. In: Hof M, Hutterer R, Fidler V (eds). Springer, New YorkGoogle Scholar
  17. 17.
    Bright FV, Munson CA (2003) Time-resolved fluorescence spectroscopy for illuminating complex systems. Anal Chim Acta 500:71–104CrossRefGoogle Scholar
  18. 18.
    Krishnamoorthy G (2012) Motional dynamics in proteins and nucleic acids control their function: revelation by time-domain fluorescence. Curr Sci 102:266–276Google Scholar
  19. 19.
    Vogel SS, Thaler C, Blank PS, Koushik SV (2009) Time-resolved fluorescence Anisotropy. In: Periasamy A, Clegg RM (eds) FLIM microscopy in biology and medicine. Taylor & Francis, Boca RatonGoogle Scholar
  20. 20.
    Sabaté R, Saupe SJ (2007) Thioflavin T fluorescence anisotropy: an alternative technique for the study of amyloid aggregation. Biochem Biophys Res Comm 360:135–138CrossRefGoogle Scholar
  21. 21.
    Matveeva EG, Rudolph A, Moll JR, Thompson RB (2012) Structure-selective anisotropy assay for amyloid beta oligomers. ACS Chem Neurosci 3:982–987CrossRefGoogle Scholar
  22. 22.
    Deprez E, Tauc P, Leh H, Mouscadet J-F, Auclair C, Brochon J-C (2000) Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy. Biochemistry 39:9275–9284CrossRefGoogle Scholar
  23. 23.
    Wang Y, Goodson T III (2007) Early aggregation in prion peptide nanostructures investigated by nonlinear and ultrafast time-resolved fluorescence spectroscopy. J Phys Chem B 111:327–330CrossRefGoogle Scholar
  24. 24.
    Zorrilla S, Rivas G, Lillo MP (2004) Fluorescence anisotropy as a probe to study tracer proteins in crowded solutions. J Mol Recognit 17:408–416CrossRefGoogle Scholar
  25. 25.
    Otosu T, Nishimoto E, Yamashita S (2010) Multiple conformational state of human serum albumin around single tryptophan residue at various pH revealed by time-resolved fluorescence spectroscopy. J Biochem 14:191–200CrossRefGoogle Scholar
  26. 26.
    Jain N, Bhattacharya M, Mukhopadhyay S (2011) Chain collapse of an amyloidogenic intrinsically disordered protein. Biophys J 101:1720–1729CrossRefGoogle Scholar
  27. 27.
    Bhattacharya M, Mukhopadhyay S (2012) Structural and dynamical insights into the molten-globule form of ovalbumin. J Phys Chem B 116:520–531CrossRefGoogle Scholar
  28. 28.
    Narang D, Sharma PK, Mukhopadhyay S (2013) Dynamics and dimension of an amyloidogenic disordered state of human β2-microglobulin. Eur Biophys J 42:767–776CrossRefGoogle Scholar
  29. 29.
    Allsop D, Swanson L, Moore S, Davies Y, York A, El-Agnaf OMA, Soutar I (2001) Fluorescence anisotropy: a method for early detection of Alzheimer and β-peptide (Aβ) aggregation. Biochem Biophys Res Comm 285:58–63CrossRefGoogle Scholar
  30. 30.
    Mukhopadhyay S, Nayak PK, Udgaonkar JB, Krishnamoorthy G (2006) Characterization of the formation of amyloid protofibrils from barstar by mapping residue-specific fluorescence dynamics. J Mol Biol 358:935–942CrossRefGoogle Scholar
  31. 31.
    Ludescher RD, Peting L, Hudson S, Hudson B (1987) Time-resolved fluorescence anisotropy for systems with lifetime and dynamic heterogeneity. Biophys Chem 28:59–75CrossRefGoogle Scholar
  32. 32.
    Bhattacharya M, Jain N, Mukhopadhyay S (2011) Insights into the mechanism of aggregation and fibril formation from bovine serum albumin. J Phys Chem B 115:4195–4205CrossRefGoogle Scholar
  33. 33.
    Jain N, Bhattacharya M, Mukhopadhyay S (2011) Kinetics of surfactant-induced aggregation of lysozyme studied by fluorescence spectroscopy. J Fluoresc 21:615–625CrossRefGoogle Scholar
  34. 34.
    Jain N, Bhasne K, Hemaswasthi M, Mukhopadhyay S (2013) Structural and dynamical insights into the membrane-bound α-synuclein. PLoS ONE 8(12):e83752. doi: 10.1371/journal.pone.0083752 CrossRefGoogle Scholar
  35. 35.
    Ghosh S, Saha S, Goswami D, Bilgrami S, Mayor S (2012) Dynamic imaging of homo-FRET in live cells by fluorescence anisotropy microscopy. Methods Enzymol 505:291–327CrossRefGoogle Scholar
  36. 36.
    Roberti MJ, Jovin TM, Jares-Erijman E (2011) Confocal fluorescence anisotropy and FRAP imaging of α-synuclein amyloid aggregates in living cells. PLoS ONE 6(8):e23338. doi: 10.1371/journal.pone.0023338 CrossRefGoogle Scholar
  37. 37.
    Ganguly S, Clayton AHA, Chattopadhyay A (2011) Organization of higher-order oligomers of the serotonin1A receptor explored utilizing homo-FRET in live cells. Biophys J 100:361–368CrossRefGoogle Scholar
  38. 38.
    Devauges V, Marquer C, Lécart S, Cossec J-C, Potier M-C, Fort E, Suhling K, Lévêque-Fort S (2012) Homodimerization of amyloid precursor protein at the plasma membrane: a homoFRET study by time-resolved fluorescence anisotropy imaging. PLoS ONE 7(9):e44434. doi: 10.1371/journal.pone.0044434 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Singapore 2015

Authors and Affiliations

  1. 1.Department of Biological Sciences and Centre for Protein Science, Design and EngineeringIndian Institute of Science Education and Research (IISER)MohaliIndia
  2. 2.Department of Chemical SciencesIndian Institute of Science Education and Research (IISER)MohaliIndia
  3. 3.Department of Molecular, Cellular and Developmental BiologyUniversity of MichiganAnn ArborUSA

Personalised recommendations