Fig. 9. Myosin surface density determination and actin filament length dependence of unloaded shortening velocity. (A) Fluorescent quantitation of the binding of GFP-myosin to mAb-coated glass coverslips. The fluorescence from GFP-myosin bound to the surface was measured by placing the coverslip at a 45° angle to the light path in motility buffer in a standard 1 cm cuvette. The solid line is a fit of the myosin concentration dependence of iodinated adult skeletal muscle myosin binding to the mAb surfaces (Winkelmann et al., 1995). This provides a rapid method for determining the myosin surface density used in the in vitro motility assay. (B) Actin filament length dependence of the unloaded shortening velocity was used to estimate the duty ratio for WT and mutant GFP-myosin. The sliding filament velocity (v_exp) and actin filament length (l) data were fit to the equation

for each myosin surface density () (Harada et al., 1990; Uyeda et al., 1990). The data is weighted by the number of filaments (n) in each length bin. This is indicated for each data point by the vertical bars that are equal to 1/n^1/2. These data are for WT GFP-myosin at a myosin surface density of 830 mol/µm² and the G584R mutant at 414 mol/µm².