Abstract
Single-molecule fluorescence spectroscopy, and particularly its Förster resonance energy transfer implementation (SM-FRET), provides the opportunity to resolve the stochastic conformational fluctuations undergone by individual protein molecules while they fold–unfold, bind to their partners, or carry out catalysis. Such information is key to resolve the microscopic pathways and mechanisms underlying such processes, and cannot be obtained from bulk experiments. To fully resolve protein conformational dynamics, SM-FRET experiments need to reach microsecond, and even sub-microsecond, time resolutions. The key to reach such resolution lies in increasing the efficiency at which photons emitted by a single molecule are collected and detected by the instrument (photon count rates). In this chapter, we describe basic procedures that an end user can follow to optimize the confocal microscope optics in order to maximize the photon count rates. We also discuss the use of photoprotection cocktails specifically designed to reduce fluorophore triplet buildup at high irradiance (the major cause of limiting photon emission rates) while improving the mid-term photostability of the fluorophores. Complementary strategies based on the data analysis are discussed in depth by other authors in Chap. 14.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Wang Z, Campos LA, Muñoz V (2016) Chapter fourteen—Single-molecule fluorescence studies of fast protein folding. Methods Enzymol 581:417–459
Weiss S (1999) Fluorescence spectroscopy of single biomolecules. Science 283(5408):1676–1683
Stryer L, Haugland RP (1967) Energy transfer: a spectroscopic ruler. Proc Natl Acad Sci U S A 58(2):719–726
Seidel R, Dekker C (2007) Single-molecule studies of nucleic acid motors. Curr Opin Struct Biol 17(1):80–86
Zhuang X (2005) Single-Molecule RNA Science. Annu Rev Biophys Biomol Struct 34(1):399–414
Goodson KA, Wang Z, Haeusler AR, Kahn JD, English DS (2013) LacI-DNA-IPTG loops: equilibria among conformations by single-molecule FRET. J Phys Chem B 117(16):4713–4722
Chris A, Ferreon M, Deniz AA (2012) Protein folding at single-molecule resolution. Biochim Biophys Acta 1814(8):1021–1029
Haran G (2012) How, when and why proteins collapse: the relation to folding. Curr Opin Struct Biol 22(1):14–20
Schuler B, Eaton WA (2008) Protein folding studied by single molecule FRET. Curr Opin Struct Biol 18(1):16–26
Michalet X, Weiss S, Jäger M (2006) Single-molecule fluorescence studies of protein folding and conformational dynamics. Chem Rev 106:1785–1813
Aznauryan M, Nettels D, Holla A, Hofmann H, Schuler B (2013) Single-molecule spectroscopy of cold denaturation and the temperature-induced collapse of unfolded proteins. J Am Chem Soc 135(38):14040–14043
Schuler B, Soranno A, Hofmann H, Nettels D (2016) Single-molecule FRET spectroscopy and the polymer physics of unfolded and intrinsically disordered proteins. Annu Rev Biophys 45(1):207–231
Hofmann H (2014) Single-molecule spectroscopy of unfolded proteins and chaperonin action. Biol Chem 395(7–8):689–698
Smiley RD, Hammes GG (2006) Single molecule studies of enzyme mechanisms. Chem Rev 106(8):3080–3094
Chung HS, Eaton WA (2013) Single-molecule fluorescence probes dynamics of barrier crossing. Nature 502(7473):685–688
Chung HS, Louis JM, Eaton WA (2010) Distinguishing between protein dynamics and dye photophysics in single-molecule FRET experiments. Biophys J 98(4):696–706
Campos LA, Sadqi M, Liu J, Wang X, English DS, Muñoz V (2013) Gradual disordering of the native state on a slow two-state folding protein monitored by single-molecule fluorescence spectroscopy and NMR. J Phys Chem B 117(42):13120–13131
Michalet X et al (2014) Silicon Photon-Counting Avalanche Diodes for Single-Molecule Fluorescence Spectroscopy. IEEE J Sel Top Quantum Electron 20(6):248–267
Grewer C, Brauer H-D (1994) Mechanism of the triplet-state quenching by molecular oxygen in solution. J Phys Chem 98(16):4230–4235
Campos LA, Liu J, Wang X, Ramanathan R, English DS, Muñoz V (2011) A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy. Nat Methods 8(2):143–146
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Wang, Z., Mothi, N., Muñoz, V. (2022). Single-Molecule Fluorescence Spectroscopy Approaches for Probing Fast Biomolecular Dynamics and Interactions. In: Muñoz, V. (eds) Protein Folding. Methods in Molecular Biology, vol 2376. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1716-8_13
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1716-8_13
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1715-1
Online ISBN: 978-1-0716-1716-8
eBook Packages: Springer Protocols