Polypropylene microplate

Catalyst mix A (an aqueous solution of copper sulfate (50 mM) and sodium ascorbate (250 mM) was prepared according to the literature procedure [10 (link)]. A 25 mM solution of each alkyne in dimethyl sulfoxide and a 50 mM solution of each azide in dimethyl sulfoxide were also prepared. To a 96-well polypropylene microplate (Greiner (Kremsmünster, Austria), 655201) were added a solution of an alkyne (10 µL/well), a solution of an azide (7 µL/well), dichloromethane (33 µL/well) and catalyst mix A (50 mL/well) and the plate was left to stand at ambient temperature, allowing dichloromethane to evaporate from the system. After 48 h, the reactions were monitored by TLC, and the consumption of all the starting alkynes was confirmed. Then, dimethyl sulfoxide (8 µL/well) was added. At this point, the final concentration of triazoles should be 10 mM.

Catalyst mix A (an aqueous solution of copper sulfate (50 mM) and sodium ascorbate (250 mM) was prepared according to the literature procedure [10 (link)]. A 25 mM solution of each alkyne in dimethyl sulfoxide and a 50 mM solution of each azide in dimethyl sulfoxide were also prepared. To a 96-well polypropylene microplate (Greiner (Kremsmünster, Austria), 655201) were added a solution of an alkyne (10 µL/well), a solution of an azide (7 µL/well), dichloromethane (33 µL/well) and catalyst mix A (50 mL/well) and the plate was left to stand at ambient temperature, allowing dichloromethane to evaporate from the system. After 48 h, the reactions were monitored by TLC, and the consumption of all the starting alkynes was confirmed. Then, dimethyl sulfoxide (8 µL/well) was added. At this point, the final concentration of triazoles should be 10 mM.

Single turnover experiments were performed in a fluorescence plate reader (Tecan model – Infinite M200 PRO). Experiments were performed with the WT and mutant versions of human β-cardiac 25-hep HMM and 2-hep HMM in a 96-well plate (Greiner polypropylene microplate) by mixing 100 nM myosin in a buffer containing 10 mM Tris pH 7.5, 4 mM MgCl₂, 1 mM EDTA, 1 mM DTT, and 5 mM potassium acetate with 2′-(or-3′)-O-(N-Methylanthraniloyl) adenosine 5′-triphosphate (mant-ATP, Thermo Fischer Scientific) at a final concentration of 100 nM^{24 (link)}. After 10 s, 2 mM ATP was added, followed by measuring the fluorescence signal at 470 nm after excitation at 405 nm. Fluorescence was recorded every ~2 s for 16 min total and the traces were normalized and plotted^{24 (link)}. The kinetic traces were fitted to a bi-exponential decay function which yielded the amplitudes and rates of the fast (DRX rate) and slow (SRX rate) phases.