S2Tag, a novel affinity tag for the capture and immobilization of coiled-coil proteins: Application to the study of human β-cardiac myosin

Figures

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  Figure 1. Mapping the epitope recognized by mAb 10F12.3 on skeletal myosin.A, cartoon of the structure of the striated muscle myosin and the HMM subfragment. B, rotary shadow electron micrographs of antibody-myosin complexes show the binding of the IgG1 mAb 10F12.3 to the S2 domain of the myosin rod. The arrows mark the bound mAb and >40% of the molecules had two antibodies simultaneously bound to the S2 domain of the rod. C, measurements map the epitope 44 nm from the head-rod junction. The rod is a continuous coiled-coil α-helix of 1096 residues/chain and measures 155 nm long. This yielded an estimate for the location of the epitope of 312 ± 28 residues from the start of the S2 domain. D, a series of truncations map the C-terminal limit of the epitope to 305 residues from the start of the S2 domain (the boxed SRE sequence). The N-terminal limit was determined by the loss of reactivity with the A295L point mutation. This defined the sequence A1140EKHRADLSRE1150 as the epitope. Residues highlighted in yellow are likely to be key to antibody recognition based on the mutational analysis. E, a model based on the structure of a Lethocerous myosin rod (PDB: 7KOG) flanking the S2Tag epitope…

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  Figure 2. Myosin concentration dependence for motility. An important factor for determining the actin filament gliding speed is the myosin surface density. Nitrocellulose-coated glass coverslips with bound mAb 10F12.3 were prepared under standard conditions and incubated with increasing concentrations of β-cHMM2.0. The actin gliding speed quantified for (A) WT cHMM2.0, and (B) S532P cHMM2.0 increases with motor density and is maximal at 5 to 10 μg/ml loading concentration. The maximal gliding speed is characteristic of the motor protein (Table 1). C, WT cHMM1.0 was bound by non-specific attachment directly to nitrocellulose-coated coverslips. Actin filament speed is slower than antibody captured β-cHMM2.0 (p < 0.0001, unpaired, two-tailed t test), and the non-specific binding requires an 8 - 10-fold higher concentration of myosin to saturate actin filament velocity. D, antibody capture with avian fast skeletal myosin produces maximum actin speed in the same range of loading protein concentration as the β-cHMM2.0 proteins. Motility assays of the β-cHMM proteins were done at 32 °C. The much faster skeletal myosin was measured at 27 °C to aid analysis. Plotted gliding speed is the mean ± SD…

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  Figure 3. Single-molecule actomyosin interactions of WT-cHMM bound via the S2Tag.A, cartoon of the 3-bead geometry (not to scale). B, sample trace of bead position and covariance for an optically trapped actin dumbbell interacting with WT-cHMM2.0 adhered to the pedestal bead through the 10F12.3 antibody-epitope interaction. C, cumulative distribution of attachment durations of actomyosin interactions. D, schematic of the two-substep power stroke of cHMM and associated product release. E, ensemble averages of actomyosin interactions aligned at start (Forward ensemble) or end (Reverse ensemble). F, histograms of substep sizes for single interactions. This data set included 516 interaction events for the WT-cHMM.

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  Figure 4. Mixed myosin motility assay.A, assay of WT cHMM2.0 (fast myosin) mixed with S532P cHMM2.0 (slow myosin) yields a linear relationship between actin filament velocity and the mole fraction of fast myosin consistent with a unitary force ratio, Fos/Fof = 1. Despite having a slower speed, S532P cHMM2.0 produces a force comparable to WT cHMM2.0. B, avian skeletal muscle myosin is a very much faster myosin than the β-CM, and the concave fit when assayed with the slower WT cHMM2.0 yields a force ratio, Fos/Fof = 3.3, suggesting the slower cardiac myosin generates higher force. The dotted line is the calculated fit if the force produced by each myosin isoform is equal. C, the S532P mutation imparts the same load on the skeletal myosin, Fos/Fof = 3.3, as the WT cHMM2.0. This result is consistent with balanced forces observed in the direct comparison of WT and S532P cHMM2.0 (Panel A). Two biological replicates and 2 or 3 technical replicates were measured for each data point. Error bars are the mean value ± 95% C.I.