Single Molecule Tools for Probing Protein Aggregation

  • Anoop Rawat
  • Sudipta Maiti
Research Article


Protein aggregation poses a fundamental problem in biophysics, whose solutions have enormous potential for societal benefits. Many devastating and incurable diseases, such as Alzheimer’’s, Parkinson’’s and Type II diabetes, are strongly linked to the misfolding and aggregation of specific proteins. The links between misfolding, aggregation and toxicity, with clues spread across fields from physics and physical chemistry to clinical science and epidemiology, have remained difficult to decipher. In addition, the transience of the aggregation intermediates makes it a difficult challenge. Optical spectroscopy and microscopy, providing unparalleled sensitivity and resolution, are two of the few tools which have been able to provide some effective leads in understanding the process. Here we present a short summary of the applications of fluorescence correlation spectroscopy and total internal reflection fluorescence microscopy to this problem, primarily focusing on the progress made in our laboratory.


Single-molecule fluorescence Amyloids Protein aggregation FCS TIRF 


  1. 1.
    Chiti F, Dobson CM (2006) Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem 75:333––366CrossRefGoogle Scholar
  2. 2.
    Jahn TR, Radford SE (2008) Folding versus aggregation: polypeptide conformations on competing pathways. Arch Biochem Biophys 469:100––117CrossRefGoogle Scholar
  3. 3.
    Kim S, Kim JH, Lee JS, Park CB (2015) Beta-sheet-forming, self-assembled peptide nanomaterials towards optical, energy, and healthcare applications. Small 11:3623–3640CrossRefGoogle Scholar
  4. 4.
    Bemporad F, Chiti F (2012) Protein misfolded oligomers: experimental approaches, mechanism of formation, and structure-toxicity relationships. Chem Biol 19:315––327CrossRefGoogle Scholar
  5. 5.
    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
  6. 6.
    Johnson RD, Steel DG, Gafni A (2014) Structural evolution and membrane interactions of Alzheimer’s amyloid-beta peptide oligomers: new knowledge from single-molecule fluorescence studies. Protein Sci 23:869–883CrossRefGoogle Scholar
  7. 7.
    Sengupta P, Garai K, Sahoo B, Shi Y, Callaway DJ, Maiti S (2003) The amyloid beta peptide Aβ1–40 is thermodynamically soluble at physiological concentrations. Biochemistry 42:10506–10513CrossRefGoogle Scholar
  8. 8.
    Garai K, Sengupta P, Sahoo B, Maiti S (2006) Selective destabilization of soluble amyloid-β oligomers by divalent metal ions. Biochem Biophys Res Commun 345:210–215CrossRefGoogle Scholar
  9. 9.
    Garai K, Sureka R, Maiti S (2007) Detecting amyloid-β aggregation with fiber-based fluorescence correlation spectroscopy. Biophys J 92:L55–L57CrossRefGoogle Scholar
  10. 10.
    Garai K (2007) Zinc lowers amyloid-beta toxicity by selectively precipitating aggregation intermediates. Biochemistry 46:10655–10663CrossRefGoogle Scholar
  11. 11.
    Garai K, Sahoo B, Sengupta P, Maiti S (2008) Quasihomogeneous nucleation of amyloid beta yields numerical bounds for the critical radius, the surface tension, and the free energy barrier for nucleus formation. J Chem Phys 128:045102CrossRefADSGoogle Scholar
  12. 12.
    Sahoo B, Balaji J, Nag S, Kaushalya SK, Maiti S (2008) Protein aggregation probed by two-photon fluorescence correlation spectroscopy of native tryptophan. J Chem Phys 129:075103CrossRefADSGoogle Scholar
  13. 13.
    Sahoo B, Nag S, Sengupta P, Maiti S (2009) On the stability of the soluble amyloid aggregates. Biophys J 97:1454–1460CrossRefGoogle Scholar
  14. 14.
    Nag S, Chen J, Irudayaraj J, Maiti S (2010) Measurement of the attachment and assembly of small amyloid-β oligomers on live cell membranes at physiological concentrations using single-molecule tools. Biophys J 99:1969–1975CrossRefGoogle Scholar
  15. 15.
    Nag S, Sarkar B, Bandyopadhyay A, Sahoo B, Sreenivasan VK, Kombrabail M, Muralidharan C, Maiti S (2011) Nature of the amyloid-β monomer and the monomer-oligomer equilibrium. J Biol Chem 286:13827–13833CrossRefGoogle Scholar
  16. 16.
    Chandrakesan M, Sarkar B, Mithu VS, Abhyankar R, Bhowmik D, Nag S, Sahoo B, Shah R, Gurav S, Banerjee R, Dandekar S, Jose JC, Sengupta N, Madhu PK, Mait S (2013) The basic structural motif and major biophysical properties of amyloid-β are encoded in the fragment 18–35. Chem Phys 422:80–87CrossRefADSGoogle Scholar
  17. 17.
    Sarkar B, Das AK, Maiti S (2013) Thermodynamically stable amyloid-ß monomers have much lower membrane affinity than the small oligomers. Front Physiol 4:84CrossRefGoogle Scholar
  18. 18.
    Nag S, Sarkar B, Chandrakesan M, Abhyanakar R, Bhowmik D, Kombrabail M, Dandekar S, Lerner E, Haas E, Maiti S (2013) A folding transition underlies the emergence of membrane affinity in amyloid-β. Phys Chem Chem Phys 15:19129–19133CrossRefGoogle Scholar
  19. 19.
    Sarkar B, Mithu VS, Chandra B, Mandal A, Chandrakesan M, Bhowmik D, Madhu PK, Maiti S (2014) Significant structural differences between transient amyloid-β oligomers and less-toxic fibrils in regions known to harbor familial Alzheimer’’s mutations. Angew Chem Int Ed Engl 53:6888–6892CrossRefGoogle Scholar
  20. 20.
    Bhowmik D, Das AK, Maiti S (2015) Rapid, cell-free assay for membrane-active forms of amyloid-β. Langmuir 31:4049–4053CrossRefGoogle Scholar
  21. 21.
    Das AK, Rawat A, Bhowmik D, Pandit R, Huster D, Maiti S (2015) An early folding contact between Phe19 and Leu34 is critical for amyloid-β oligomer toxicity. ACS Chem Neurosci 6:1290–1295CrossRefGoogle Scholar
  22. 22.
    Matsumura S, Shinoda K, Yamada M, Yokojima S, Inoue M, Ohnishi T, Shimada T, Kikuchi K, Masui D, Hashimoto S, Sato M, Ito A, Akioka M, Takagi S, Nakamura Y, Nemoto K, Hasegawa Y, Takamoto H, Inoue H, Nakamura S, Nabeshima Y, Teplow DB, Kinjo M, Hoshi M (2011) Two distinct amyloid β-protein (Aβ) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses. J Biol Chem 286:11555–11562CrossRefGoogle Scholar
  23. 23.
    Mirbaha H, Holmes BB, Sanders DW, Bieschke J, Diamond MI (2015) Tau trimers are the minimal propagation unit spontaneously internalized to seed intracellular aggregation. J Biol Chem 290:14893–14903CrossRefGoogle Scholar
  24. 24.
    Bag N, Ali A, Chauhan VS, Wohland T, Mishra A (2013) Membrane destabilization by monomeric hIAPP observed by imaging fluorescence correlation spectroscopy. Chem Commun 49:9155–9157CrossRefGoogle Scholar
  25. 25.
    Magde D, Elson E, Webb WW (1972) Thermodynamic fluctuations in a reacting system-—measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29:705CrossRefADSGoogle Scholar
  26. 26.
    Sengupta P, Balaji J, Maiti S (2002) Measuring diffusion in cell membranes by fluorescence correlation spectroscopy. Methods 27:374–387CrossRefGoogle Scholar
  27. 27.
    Sengupta P, Garai K, Balaji J, Periasamy N, Maiti S (2003) Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscop. Biophys J 84:1977–1984CrossRefGoogle Scholar
  28. 28.
    Balaji J, Maiti S (2005) Quantitative measurement of the resolution and sensitivity of confocal microscopes using line-scanning fluorescence correlation spectroscopy. Micros Res Tech 66:198–202CrossRefGoogle Scholar
  29. 29.
    Kaushalya SK, Balaji J, Garai K, Maiti S (2005) Fluorescence correlation microscopy with real-time alignment readout. Appl Opt 44:3262–3265CrossRefADSGoogle Scholar
  30. 30.
    Garai K, Muralidhar M, Maiti S (2006) Fiber-optic fluorescence correlation spectrometer. Appl Opt 45:7538–7542CrossRefADSGoogle Scholar
  31. 31.
    Singh NK, Chacko JV, Sreenivasan VK, Nag S, Maiti S (2011) Ultracompact alignment-free single molecule fluorescence device with a foldable light path. J Biomed Opt 16:025004CrossRefGoogle Scholar
  32. 32.
    Abhyankar R, Sahoo B, Singh NK, Meijer LM, Sarkar B, Das AK, Nag S, Chandrakesan M, Bhowmik D, Dandekar S, Maiti S (2012) Amyloid diagnostics: probing protein aggregation and conformation with ultrasensitive fluorescence detection. Proc SPIE 8233:82330BCrossRefADSGoogle Scholar
  33. 33.
    Axelrod D (1981) Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol 89:141–145CrossRefGoogle Scholar
  34. 34.
    Ding H, Schauerte JA, Steel DG, Gafni A (2012) β-Amyloid (1–40) peptide interactions with supported phospholipid membranes: a single-molecule study. Biophys J 103:1500–1509CrossRefGoogle Scholar
  35. 35.
    Narayan P, Ganzinger KA, McColl J, Weimann L, Meehan S, Qamar S, Carver JA, Wilson MR, St George-Hyslop P, Dobson CM, Klenerman D (2013) Single molecule characterization of the interactions between amyloid-β peptides and the membranes of hippocampal cells. J Am Chem Soc 135:1491–1498CrossRefGoogle Scholar
  36. 36.
    Zijlstra N, Blum C, Segers-Nolten IM, Claessens MM, Subramaniam V (2012) Molecular composition of sub-stoichiometrically labeled α-synuclein oligomers determined by single-molecule photobleaching. Angew Chem Int Ed Engl 51:8821–8824CrossRefGoogle Scholar
  37. 37.
    Kaushalya SK, Desai R, Arumugam S, Ghosh H, Balaji J, Maiti S (2008) Three-photon microscopy shows that somatic release can be a quantitatively significant component of serotonergic neurotransmission in the mammalian brain. J Neurosci Res 86:3469–3480CrossRefGoogle Scholar
  38. 38.
    Bhowmik D, Mote KR, MacLaughlin CM, Biswas N, Chandra B, Basu JK, Walker GC, Madhu PK, Maiti S (2015) Cell-membrane-mimicking lipid-coated nanoparticles confer Raman enhancement to membrane proteins and reveal membrane-attached amyloid-β conformation. ACS Nano 9:9070–9077CrossRefGoogle Scholar

Copyright information

© The National Academy of Sciences, India 2015

Authors and Affiliations

  1. 1.Department of Chemical SciencesTata Institute of Fundamental ResearchMumbaiIndia

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