Abstract
Fluorescence correlation spectroscopy (FCS) is a flexible and powerful technique to measure the diffusion of fluorescently labeled particles. It has been important in examining a range of biological processes, from intracellular transport, to DNA hybridization. It is particularly suited to measuring the assembly of peptides, since peptides are often too small to be detected by standard light scattering methods, or may not contain aromatic amino acid residues, which limits the use of other spectroscopic techniques. In this protocol, we describe state-of-the-art sample preparation for Aβ1–42 peptide solutions and the measurement and analysis of the self-assembly of the peptide to form fibrils via a number of intermediate states using FCS.
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References
Ries J, Schwille P (2012) Fluorescence correlation spectroscopy. BioEssays 34:361–368
Magde D, Elson EL, Webb WW (1972) Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29(11):705–708
Elson EL, Magde D (1974) Fluorescence correlation spectroscopy. I. Conceptual basis and theory. Biopolymers 13:1–27
Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13:29–61
Rigler R, Mets Ű, Widengren J, Kask P (1993) Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion. Eur Biophys J 22:169–175
Kinjo M, Rigler R (1995) Ultrasensitive hybridization analysis using fluorescence correlation spectroscopy. Nucl Acids Res 23(10):1795–1799
Fitzpatrick J, Lillemeier B (2011) Fluorescence correlation spectroscopy: linking molecular dynamics to biological function in vitro and in situ. Curr Opin Struct Biol 21(5):650–660
Schwille P, Haustein E (2001) Fluorescence correlation spectroscopy—an introduction to its concepts and applications. Biophys Textbook Online 1(3):1–33
Rusu L, Gambhir A, McLaughlin S, Rädler JO (2004) Fluorescence correlation spectroscopy studies of peptide and protein binding phospholipid vesicles. Biophys J 87(2):1044–1053
Comas-Garcia M, Garmann RF, Singaram SW, Ben-Shaul A, Knobler CM, Gelbart WM (2014) Characterisation of viral capsid protein self-assembly around short single-stranded RNA. J Phys Chem B 118(27):7510–7519
Tjernberg LO, Pramanik A, Björling S, Thyberg P, Thyberg J, Nordstedt C, Berndt KD, Terenius L, Rigler R (1999) Amyloid β-peptide polymerization studied using fluorescence correlation spectroscopy. Chem Biol 6(1):53–62
Sengupta P, Garai K, Balaji J, Periasamy N, Maiti S (2003) Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy. Biophys J 84(3):1977–1984
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(4):045102-1–045102-7
Pal N, Verma SD, Singh MK, Singh MK, Sobhan S (2011) Fluorescence correlation spectroscopy: an efficient tool for measuring size, size-distribution and polydispersity of microemulsion droplets in solution. Anal Chem 83(20):7736–7744
Mittag JJ, Milani S, Walsh DM, Rädler JO, McManus JJ (2014) Simultaneous measurement of a range of particle sizes during Aβ1-42 fibrillogenesis quantified using fluorescence correlation spectroscopy. Biochem Biophys Res Commun 448(2):195–199
Provencher SW (1982) Contin: a general purpose constrained regularization program for inverting noisy linear algebraic and integral equations. Comput Phys Commun 27:229–242
Tirado M, Lopez Martinez C, Garcia de la Torre J (1984) Comparison of theories for the translational and rotational diffusion coefficients for rod-like macromolecules. Application to short DNA fragments. J Chem Phys 81(4):2047–2052
Petrásěk Z, Schwille P (2008) Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy. Biophys J 94:1437–1448
Dertinger T, Loman A, Ewers B, Müller CB, Krämer B, Enderlein J (2008) The optics and performance of dual-focus fluorescence correlation spectroscopy. Opt Express 16(19):14353–14368
Kapusta P (2010) Absolute diffusion coefficients: compilation of reference data for FCS calibration. PicoQuant GmbH Application Note
Dertinger T, Pacheco V, von der Hocht I, Hartman RH, Gregor I, Enderlein J (2007) Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurement. Chem Phys Chem 8(3):433–443
Acknowledgements
This work was made possible by funding from Science Foundation Ireland Stokes Lectureship (to J. J.McM); European Science Foundation networking programme “epitopeMap” (grant to J.O. R. and J. J. McM); EU FP7 (NanoTransKinetics grant to JJM, JOR), Deutsche Forschungsgemeinschaft (JJM, travel grant).
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Mittag, J.J., Rädler, J.O., McManus, J.J. (2018). Peptide Self-Assembly Measured Using Fluorescence Correlation Spectroscopy. In: Nilsson, B., Doran, T. (eds) Peptide Self-Assembly. Methods in Molecular Biology, vol 1777. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7811-3_8
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DOI: https://doi.org/10.1007/978-1-4939-7811-3_8
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