Functional characterisation of Dictyostelium myosin II with conserved tryptophanyl residue 501 mutated to tyrosine.
We created a Dictyostelium discoideum myosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in a Dictyostelium strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin in Dictyostelium. Additionally, we expressed the W501 Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased, but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission.
Batra
R
R
Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Manstein
D J
DJ
D019943:Amino Acid Substitution; D000818:Animals; D004023:Dictyostelium / Q000378:metabolism*; D008958:Models, Molecular; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D014443:Tyrosine; D014364:Tryptophan; D009218:Myosins