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Journal of Muscle Research & Cell Motility

, Volume 23, Issue 7–8, pp 673–683 | Cite as

Engineering Dictyostelium discoideum myosin II for the introduction of site-specific fluorescence probes

  • Stuart Wakelin
  • Paul B. Conibear
  • Robert J. Woolley
  • David N. Floyd
  • Clive R. Bagshaw
  • Mihály Kovács
  • András Málnási-Csizmadia
Article

Abstract

Dictyostelium discoideum is a useful host for the production of constructs for the analysis of structure–function relationships of myosin. Here we describe the use of myosin II constructs containing a single tryptophan residue, at different locations, for probing events at the nucleotide binding site, the relay loop and the communication path between them. GFP fusions have also been expressed at the N- and C-termini of the myosin motor to provide sensitive probes of the actomyosin dissociation reaction in microscope-based kinetic assays. We report on the fluorescence anisotropy of these constructs in the context of their use as resonance energy transfer probes.

Keywords

Tryptophan Tryptophan Residue Resonance Energy Transfer Nucleotide Binding Nucleotide Binding Site 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Stuart Wakelin
    • 1
  • Paul B. Conibear
    • 1
  • Robert J. Woolley
    • 1
  • David N. Floyd
    • 1
  • Clive R. Bagshaw
    • 1
  • Mihály Kovács
    • 2
  • András Málnási-Csizmadia
    • 2
  1. 1.Department of BiochemistryUniversity of LeicesterLeicesterUnited Kingdom
  2. 2.Department of BiochemistryEötvös Loránd UniversityBudapest, Pázmány Péter sétány 1/CHungary

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