β-cardiac myosin construct motility assay surface occupancy estimation. (a) β-cardiac myosin constructs were attached to the motility assay glass surface via their C-terminal EGFP to preadhered anti-GFP nanobodies. Increasing the applied myosin concentration increased surface EGFP fluorescence intensity. (b) Surface EGFP fluorescence intensity increased as a function of β-cardiac myosin construct concentration infused into motility assay flow-cell. Fluorescence intensity followed saturable binding behavior with an apparent dissociation constant, K_app (nM): K_appS1 WT = 93 ± 27, K_app2HEP WT = 116 ± 42, K_app15HEP WT = 143 ± 32, K_app25HEP WT = 181 ± 36, K_app2HEP E525K = 152 ± 38, K_app15HEP E525K = 191 ± 45, and K_app25HEP E525K = 153 ± 34. No difference in the surface EGPF intensity across WT and E525K mutants (P = 0.98). (c) Data in b normalized to fluorescence at 700 nM β-cardiac myosin concentration and converted to surface occupancy. Data are mean ± SD of at least three experiments from separate protein preparations.