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Fluorescent Nucleoside Triphosphates for Single-Molecule Enzymology

  • Christopher P. ToselandEmail author
  • Martin R. Webb
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 778)

Abstract

The interconversion of nucleoside triphosphate (NTP) and diphosphate occurs in some of the most ­important cellular reactions. It is catalyzed by diverse classes of enzymes, such as nucleoside triphosphatases, kinases, and ATP synthases. Triphosphatases include helicases, myosins, and G-proteins, as well as many other energy-transducing enzymes. The transfer of phosphate by kinases is involved in many metabolic pathways and in control of enzyme activity through protein phosphorylation. To understand the processes catalyzed by these enzymes, it is important to measure the kinetics of individual elementary steps and conformation changes. Fluorescent nucleotides can directly report on the binding and release steps, and conformational changes associated with these processes. In single-molecule studies, fluorescent nucleotides can allow their role to be explored by following precisely the temporal and spatial changes in the bound nucleotide. Here, the selection of fluorophores and nucleotide modifications are discussed and methods are described to prepare ATP analogs with examples of two alternate fluorophores, diethylaminocoumarin and Cy3.

Key words

Fluorescent nucleotides ATP GTP Motor proteins TIRF microscopy 

Notes

Acknowledgments

We would like to thank the various coworkers, who have been involved in synthesis and use of fluorescent nucleotides and are coauthors of publications cited here. We thank the Medical Research Council, UK (C.P.T. and M.R.W.) and European Molecular Biology Organization (C.P.T) for financial support.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.MRC National Institute for Medical ResearchLondonUK
  2. 2.Institut für Zelluläre Physiologie, Physiologisches InstitutLudwig Maximilians UniversitätMunichGermany

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