The motility assay was performed as described in detail previously (47, 48). Both WT and mutant protein(s) were purified simultaneously and studied on the same day to minimize variability. All the experiments were performed at 23°C. Glass coverslips (VWR micro cover glass) were coated with a mixture of 0.2% nitrocellulose (Ernest Fullam Inc.) and 0.2% collodion (Electron Microscopy Sciences) dissolved in amyl acetate (Sigma) and air-dried before use. A permanent double-sided tape (Scotch) was used to construct four channels in each slide, and four different experiments were performed on the same slide. Partially inactivated myosin heads in S1 preparations were removed by the “dead-heading” process before performing the motility assay. The process of dead-heading had the following steps: A 10-fold molar excess of F-actin was added to myosin in the presence of 2 mM ATP, the mixture was incubated for 15 min in an ice bucket, 50 mM MgCl2 was added to form F-actin Mg2+-paracrystals and incubated for 5 min, the dead heads bound to actin paracrystals were sedimented at 350,000g for 15 min, the supernatant was collected, and the sS1 concentration was measured using the Bradford reagent (Bio-Rad). Before any experiments, dead-headed sS1 was diluted in 10% ABBSA {assay buffer [AB; 25 mM imidazole (pH 7.5), 25 mM KCl, 4 mM MgCl2, 1 mM EGTA, and 1 mM DTT] with bovine serum albumin (BSA; 0.1 mg/ml) diluted in AB}, unless otherwise stated.

For motility experiments using pure actin, reagents were sequentially flowed through the channels in the following order: 10 μl of 4 μM SNAP-PDZ18 diluted in AB and incubated for 3 min, 20 μl of ABBSA to block the surface from nonspecific attachments and incubated for 2 min, 10 μl of a mixture of eight-residue (RGSIDTWV)–tagged human b-cardiac sS1 (~0.05 to 0.1 mg/ml) and incubated for 3 min, 20 μl of AB to wash any unattached proteins, and finally 10 μl of the GO solution [5 to 10 nM tetramethylrhodamine (TMR)–phalloidin (Invitrogen)–labeled bovine actin; 2 mM ATP (Calbiochem); an oxygen-scavenging system consisting of 0.2% glucose, glucose oxidase (0.11 mg/ml; Calbiochem), and catalase (0.018 mg/ml; Calbiochem); and an ATP regeneration system consisting of 1 mM phosphocreatine (Calbiochem) and creatine phosphokinase (0.1 mg/ml; Calbiochem) in ABBSA].

For regulated thin filament (RTF) motility experiments, reagents were sequentially flowed through the channels in the following order: 10 μl of 4 μM SNAP-PDZ18 diluted in AB and incubated for 3 min, 20 μl of ABBSA to block the surface from nonspecific attachments and incubated for 2 min, 10 μl of a mixture of eight-residue (RGSIDTWV)–tagged human ß-cardiac sS1 (~0.05 to 0.1 mg/ml) and incubated for 3 min, 10 μl of AB to wash any unattached proteins, 10 μl of 5 to 10 nM TMR-phalloidin (Invitrogen)–labeled bovine actin and incubated for 3 min, 10 μl of AB buffer to wash, 10 μl of 400 nM tropomyosin. troponin complex and incubated for 6 min, and 10 μl of the GO solution {2 mM ATP (Calbiochem), an oxygen-scavenging system [0.2% glucose, glucose oxidase (0.11 mg/ml; Calbiochem), and catalase (0.018 mg/ml; Calbiochem)], and an ATP regeneration system [1 mM phosphocreatine (Calbiochem) and creatine phosphokinase (0.1 mg/ml; Calbiochem)] in ABBSA}.

For all experiments, movies were obtained using a Nikon Ti-E inverted microscope with an AndoriXon+EMCCD camera (model DU885). All experiments were repeated with at least four different fresh protein preparations. At each condition, at least three different movies with duration of 30 s were recorded. Filament tracking and analysis of movies were performed using the previously published Fast Automated Spud Trekker method, as described earlier (48).

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