Protein Labeling with Fluorescent Probes.

Thiol-reactive fluorescent dyes (ThermoFisher Scientific) were used for site-specific labeling of cysteines in MM variants. Using Alexa Fluor 488 as donor and Alexa Fluor 594 as acceptor provides a Föster distance of 5.4 nm (54, 55). The T165C variant was used to determine the dissociation constants of the artificial dimer and the R318C/N112C double mutant for smFRET measurement. To reduce cysteines, 250 μL of a protein solution at 30 to 40 mg/mL was incubated with 20 mM dithiothreitol (DTT) for 1 h on ice, following DTT removal by gel filtration on an S200 10/300 column equilibrated with buffer F (20 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 150 mM NaCl, 10 mM KCl). The concentrated proteins were mixed with the corresponding thiol-reactive dye in a 1:2 M ratio (protein:dye) and incubated for 1 h at room temperature (RT). In the case of double labeling by Alexa Fluor 488 and 594, the reaction was started with addition of Alexa Fluor 488 to the protein solution in a 1:0.66 (protein:dye) molar ratio. After 40 min of incubation at RT, Alexa Fluor 594 dye was added in a 1:2 (protein:dye) molar ratio and the reaction was continued for 1 h at RT. The labeling process was terminated by the addition of 10 mM DTT. After 5 min incubation at RT, insoluble material was removed by centrifugation and the sample was applied to a second round of SEC to remove unreacted dye and DTT. Labeling efficacy was calculated according to the manual of Thermo Fisher Scientific; a typical range was 90 to 100%. All fluorescence-based functional and GTP hydrolysis assays for the monomeric form of the MM constructs were performed in buffer 1 (20 mM HEPES-NaOH, pH 7.5, 150 mM KCl, 4 mM MgCl2). Experiments with the artificially dimerized form of the MM-HH variant were performed in buffer 2 (20 mM HEPES-NaOH, pH 7.5, 150 mM KCl, 4 mM MgCl2, 160 μM ZnSO4). All experiments were carried out at RT if not otherwise stated.