Related Experiment Videos
Cold-sensitive mutants G680V and G691C of Dictyostelium myosin II confer dramatically different biochemical defects
B Patterson1, K M Ruppel, Y Wu
1Department of Molecular and Cell Biology, University of Arizona, Tucson, Tucson, Arizona 85721, USA. Bruce_Patterson@tikal.biosci.arizona.edu
Abstract:
Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.
Insights
Two myosin mutants, G680V and G691C, reveal distinct defects in muscle motor function. G680V shows impaired ATP hydrolysis and strong actin binding, while G691C releases phosphate prematurely, affecting the mechanochemical cycle.
Area of Science:
- Biochemistry
- Molecular Biology
- Muscle Physiology
Background:
- Cold-sensitive myosin mutants are valuable for studying myosin motor function.
- Understanding myosin's mechanochemical cycle is crucial for muscle contraction.
Purpose of the Study:
- To biochemically characterize two cold-sensitive myosin mutants, G680V and G691C.
- To elucidate the specific functional deficiencies caused by each mutation in the myosin motor.
Main Methods:
- Biochemical characterization of G680V and G691C myosin mutants.
- Analysis of nucleotide binding affinity, ATPase activity, and actin-binding states.
Main Results:
- G680V mutant displays increased nucleotide affinity, reduced ATPase activity, and a novel strong actin-binding state, suggesting a paused mechanical stroke.
- G691C mutant shows elevated basal ATPase activity, indicating premature phosphate release and bypassing the mechanical stroke.
Conclusions:
- G680V and G691C mutations, despite proximity, differentially impact the myosin mechanochemical cycle.
- These mutants provide insights into distinct steps of myosin function, particularly actin interaction and ATP hydrolysis.