The W501Y mutation in Dictyostelium myosin II decreased actin affinity 6-fold and abolished the 10% ATP-induced tryptophan fluorescence increase observed in the wild-type construct.
The study confirms that Trp-501 is the primary ATP-sensitive tryptophanyl residue in Dictyostelium myosin II.
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 et al. (1999) studied this question. W501Y mutation in Dictyostelium myosin II vs. Wild-type construct was evaluated on Tryptophan fluorescence emission upon addition of ATP. The W501Y mutation in Dictyostelium myosin II decreased actin affinity 6-fold and abolished the 10% ATP-induced tryptophan fluorescence increase observed in the wild-type construct.