Engineering Dictyostelium discoideum myosin II for the introduction of site-specific fluorescence probes.
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.
Wakelin
Stuart
S
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
Conibear
Paul B
PB
Woolley
Robert J
RJ
Floyd
David N
DN
Bagshaw
Clive R
CR
Kovács
Mihály
M
Málnási-Csizmadia
András
A
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D024462:Myosin Type II / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D015800:Protozoan Proteins / Q000235:genetics; D014364:Tryptophan / Q000032:analysis
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D049452:Green Fluorescent Proteins; D000244:Adenosine Diphosphate; D014364:Tryptophan; D024462:Myosin Type II