Fig. 3.

Characterization of functional MHC-B. a Single ATP turnover experiment monitoring Trp-fluorescence decay (K_obs = 0.073 s⁻¹). b F-actin co-sedimentation assay. In the presence of F-actin, MHC-B is detected in the pellet fraction (P) but is released upon addition of ATP and found in the supernatant (SN). c F-actin-stimulated ATPase activity of MHC-B, yielding V_max = 0.257 s⁻¹ and K_M = 23.1 µM. Error bars represent the s.e.m., of two biological replicates (MHC-B from different purifications), for which three technical replicates have been carried out each. d Stopped-flow analysis measuring dissociation of the actin:MHC-B complex. The inset illustrates example traces of the increase in pyrene fluorescence when 20 µM ATP was mixed with 50 nM pyrene-labelled actin and varying concentrations of MHC-B (5–1000 nM). The increase in fluorescence can be described by an exponential function in which the rate constant is proportional to the ATP concentration and the amplitude of the fluorescence change is proportional to the fractional saturation of actin with MHC-B. The actin affinity derived by this experiment was 10.3 ± 3.7 nM (average of three independent measurements). e Left panel: Structural alignment of ADP(red)-bound MHC-B (50 kDa domain in green, N-terminal domain in lilac, converter in orange) and human beta-cardiac myosin (PDB code: 4db1, gray). Right panel: Close-up view of the nucleotide-binding pocket of MHC-B in the ADP-bound state, overlaid with corresponding active site residues of the cardiac myosin. The ADP molecule (red) is shown together with the 2Fo-Fc omit electron density, contoured at 1.5σ