Encyclopedia of Biophysics

Living Edition
| Editors: Gordon Roberts, Anthony Watts, European Biophysical Societies

Kinetics: Single-Molecule Techniques

  • Clive R. BagshawEmail author
Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-35943-9_57-1

Synonyms

Introduction

Although single molecules have been imaged by electron microscopy for many years, their detection by optical methods since the 1990s has allowed their kinetic, as well as structural, properties to be investigated (Makarov 2015; Weiss 2000). This advance has given new insights into mechanism by revealing details that are masked by traditional ensemble measurements. Furthermore, there is no need to initiate the reaction by mixing or perturbing the environment because an individual molecule can be observed as it spontaneously switches between states under equilibrium or steady-state conditions (Stone et al. 2007). This approach also has good dynamic range; for example, if a molecule exists in a particular state for just 1% of its time, on average, then it may readily be observed at the single-molecule level but masked at the ensemble level because of the contribution of 99% of molecules in other states. However, technical challenges remain...
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Supplementary material

Video 1

Single-turnover assay of Cy3-EDA-ATP by skeletal muscle myosin subfragment 1 (MPG 6376 kb)

References

  1. Bagshaw CR, Cherny D (2006) Blinking fluorophores: what do they tell us about protein dynamics? Biochem Soc Trans 34:979–982CrossRefGoogle Scholar
  2. Bagshaw CR, Conibear PB (2000) Single molecule enzymology: critical aspects exemplified by myosin ATPase activity. Single Mol 1:269–275CrossRefGoogle Scholar
  3. Chen Q, Groote R, Schonherr H, Vancso GJ (2009) Probing single enzyme kinetics in real-time. Chem Soc Rev 38:2671–2683CrossRefGoogle Scholar
  4. Conibear PB, Kuhlman PA, Bagshaw CR (1998) Measurement of ATPase activities of myosin at the level of tracks and single molecules. Adv Exp Med Biol 453:15–26CrossRefGoogle Scholar
  5. Elenko MP, Szostak JW, van Oijen AM (2009) Single-molecule imaging of an in vitro-evolved RNA aptamer reveals homogeneous ligand binding kinetics. J Am Chem Soc 131:9866–9867CrossRefGoogle Scholar
  6. English BP, Min W, van Oijen AM, Lee KT, Luo G, Sun H, Cherayil BJ, Kou SC, Xie XS (2006) Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited. Nat Chem Biol 2:87–94CrossRefGoogle Scholar
  7. Funatsu T, Harada Y, Tokunaga M, Saito K, Yanagida T (1995) Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374:555–559CrossRefGoogle Scholar
  8. Gell C, Brockwell D, Smith A (2006) Handbook of single molecule fluorescence spectroscopy. Oxford University Press, OxfordGoogle Scholar
  9. Hamada M, Nakanishi S, Itoh T, Ishikawa M, Biju V (2010) Blinking suppression in CdSe/ZnS single quantum dots by TiO2 nanoparticles. ACS Nano 4:4445–4454CrossRefGoogle Scholar
  10. Makarov DE (2015) Single Molecule Science: physical principles and models. CRC Press, Boca RatonCrossRefGoogle Scholar
  11. McAnaney TB, Zeng W, Doe CF, Bhanji N, Wakelin S, Pearson DS, Abbyad P, Shi X, Boxer SG, Bagshaw CR (2005) Protonation, photobleaching, and photoactivation of yellow fluorescent protein (YFP 10 C): a unifying mechanism. Biochemistry 44:5510–5524CrossRefGoogle Scholar
  12. Selvin PR, Ha T (eds) (2008) Single-molecule techniques: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  13. Stone MD, Mihalusova M, O’Connor CM, Prathapam R, Collins K, Zhuang X (2007) Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein. Nature 446:458–461CrossRefGoogle Scholar
  14. Weiss S (2000) Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy. Nat Struct Biol 7:724–729CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association (EBSA) 2018

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of California at Santa CruzSanta CruzUSA

Section editors and affiliations

  • Clive R. Bagshaw
    • 1
  1. 1.Department of Chemistry and BiochemistryUniversity of California at Santa CruzSanta CruzUSA