Probing nucleotide dissociation from myosin in vitro using microgram quantities of myosin
The detailed kinetic analysis of novel myosin motors is often limited by the quantity of stable protein available for study. We show here that the use of coumarin based fluorescent ADP analogues allows the assay of ADP affinities and dissociation rate constants in a flash photolysis apparatus using μg quantities of the rabbit muscle myosin S1. We go on to use the analogues to characterise two other rat muscle myosin S1 and the motor domain of Dictyostelium cytoplasmic myosin II. The results show that the fluorescence change for the binding of a coumarin based ADP analogue to a myosin motor domain is variable in sign as well as amplitude for the different proteins. The analysis also provided estimates of the affinities of caged-ATP for S1 which were ≤10 μM for muscle S1s and >200 μM for the non-muscle myosin.
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