Figure 1.
Summary of actomyosin kinetics and experimental system.(A) Kinetic scheme for myosin (M) attachment to actin (A). D, ADP; T, ATP; Pi, phosphate. Kw, equilibrium constant for weak binding of myosin to actin; kws, forward rate constant for the weak to strong transition. We assume that k-ws is insignificant. See text for other rate constants. (B) Top: Mf/A assay showing SMM-Spy Snoop-LMM (RLC-LMM) cofilament (orange) labeled with QD-SpyC (blue dot) attached to the RLC in the lever arm domain (pink) and QD-SnoopC (red dot) moving over biotinylated actin (green) attached to PEG brush-coated coverslip (not shown). Filaments are shown widely separated for clarity with S2 (black) detached completely from the filament backbone, which may not reflect the actual process (see text). Pre-working step, green box 1; post-working step, gold box 2; and after full extension of S2, gray box 3, by other working heads (not shown) giving L = 40–50 nm. The myosin head then must return to its starting position relative to the filament backbone, purple box 4. Bottom: Graph of mixed-kinetic model predictions of single head behavior in a filament. Black arrows indicate approximate predicted dwell time and J value (see text) predictions at 10 µM [ATP]. Red line, filament backbone trajectory. Blue line, predicted single myosin head behavior. Lines are shown nearly coincident for clarity but could be separated depending upon the relative positions of the two QDs. Colors in the bar above the graph correspond to the state of the myosin head. The entire cycle time is not shown for clarity. (C) Expressed constructs and proteins. Pink, RLC (UniProtKB, P02612). Black, SpyTag002 (VPTIVMVDAYKRYK; Keeble et al., 2017). Gray, Xrcc4-Spc42 (Andreas et al., 2017; Drennan et al., 2019). Green, SnoopTag (KLGDIEFIKVNK; Hatlem et al., 2019). Thin black line, GSGESG linker. Lengths reflect approximate relative sequence lengths. RLC-Spy, SpyTag on C terminus of RLC. Snoop-LMM, SnoopTag on the N terminus of the Xrcc4-Spc42 domain, then LMM 1728–1979 (National Center for Biotechnology Information accession no. NP_990605.2). In RLC-LMM cofilament, colors match constructs above. Backbone formed by assembled LMM domains (orange), two heads (orange) and S2 domain (black); Xrcc4-Spc42 domain is not visible. 705 nm QD (red dot) chemically coupled to a SnoopC (green diamond) interacting with SnoopTag (green box) of LMM construct (orange). Enlarged region shows 605 nm QD (blue dot) chemically coupled to SpyC (blue diamond) interacting with SpyTag of RLC construct in myosin head (orange). See Materials and methods for details.

Summary of actomyosin kinetics and experimental system. (A) Kinetic scheme for myosin (M) attachment to actin (A). D, ADP; T, ATP; Pi, phosphate. Kw, equilibrium constant for weak binding of myosin to actin; kws, forward rate constant for the weak to strong transition. We assume that k-ws is insignificant. See text for other rate constants. (B) Top: Mf/A assay showing SMM-Spy Snoop-LMM (RLC-LMM) cofilament (orange) labeled with QD-SpyC (blue dot) attached to the RLC in the lever arm domain (pink) and QD-SnoopC (red dot) moving over biotinylated actin (green) attached to PEG brush-coated coverslip (not shown). Filaments are shown widely separated for clarity with S2 (black) detached completely from the filament backbone, which may not reflect the actual process (see text). Pre-working step, green box 1; post-working step, gold box 2; and after full extension of S2, gray box 3, by other working heads (not shown) giving L = 40–50 nm. The myosin head then must return to its starting position relative to the filament backbone, purple box 4. Bottom: Graph of mixed-kinetic model predictions of single head behavior in a filament. Black arrows indicate approximate predicted dwell time and J value (see text) predictions at 10 µM [ATP]. Red line, filament backbone trajectory. Blue line, predicted single myosin head behavior. Lines are shown nearly coincident for clarity but could be separated depending upon the relative positions of the two QDs. Colors in the bar above the graph correspond to the state of the myosin head. The entire cycle time is not shown for clarity. (C) Expressed constructs and proteins. Pink, RLC (UniProtKB, P02612). Black, SpyTag002 (VPTIVMVDAYKRYK; Keeble et al., 2017). Gray, Xrcc4-Spc42 (Andreas et al., 2017; Drennan et al., 2019). Green, SnoopTag (KLGDIEFIKVNK; Hatlem et al., 2019). Thin black line, GSGESG linker. Lengths reflect approximate relative sequence lengths. RLC-Spy, SpyTag on C terminus of RLC. Snoop-LMM, SnoopTag on the N terminus of the Xrcc4-Spc42 domain, then LMM 1728–1979 (National Center for Biotechnology Information accession no. NP_990605.2). In RLC-LMM cofilament, colors match constructs above. Backbone formed by assembled LMM domains (orange), two heads (orange) and S2 domain (black); Xrcc4-Spc42 domain is not visible. 705 nm QD (red dot) chemically coupled to a SnoopC (green diamond) interacting with SnoopTag (green box) of LMM construct (orange). Enlarged region shows 605 nm QD (blue dot) chemically coupled to SpyC (blue diamond) interacting with SpyTag of RLC construct in myosin head (orange). See Materials and methods for details.

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