A detailed method of MST for determination of the dissociation constant for the binding of two proteins was described previously (7). For all interactions, the unlabeled protein partner was titrated into a fixed concentration of the fluorescently labeled partner (42 nM). Binding experiments between sS1 and proximal S2 were performed as described before (7). We carried out binding experiments between 25-hep HMM and C0-C7 by two methods. In one case, C0-C7 was titrated against 42 nM 25-hep myosin, and in the other, 25-hep HMM was titrated against 42 nM fluorescently labeled C0-C7. Sixteen such serially diluted concentrations of the unlabeled protein partner were prepared to generate one full binding isotherm. 25-hep and C0-C7 binding reactions were carried out in a buffer containing 10 mM imidazole (pH 7.5), 4 mM MgCl2, 1 mM EDTA, 1 mM DTT, 50 mM KAc, 1 mM ATP, and 0.05% Tween 20.

Samples were loaded into NT.115 premium treated capillaries (NanoTemper Technologies) after the reaction was incubated in the dark at 23°C for 30 min. The samples were then mounted in the Monolith NT.115 apparatus (NanoTemper Technologies) for binding measurements. All the data were recorded at 23°C. When myosin was held constant and C0-C7 was titrated, the myosin’s GFP fluorescence was measured by a blue light-emitting diode (LED) at 30% excitation power (blue filter; excitation, 460 to 480 nm; emission, 515 to 530 nm) and IR-Laser power at 60% was used. C0-C7 was fluorescently labeled with a commercial His-tag labeling kit according to the manufacturer’s instructions (NanoTemper Technologies). His-labeled C0-C7 fluorescence was measured by a red LED at 60 to 90% excitation power (red filter; excitation, 605 to 645 nm; emission, 680 to 685 nm) and IR-Laser power at 60 or 80% was used. Data analysis was performed with software NTAffinity Analysis (NanoTemper Technologies), where the binding isotherms were derived from the raw fluorescence data. At least two independent measurements each from at least two different preparations of protein were carried out in each case. Representative binding curves are shown in Figs. 2 and 4.

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