Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescence labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdot’s were imaged in time with total internal reflection fluorescence microscopy then spatially localized to 1-3 nanometers using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full length β-cardiac myosin (MYH7). Average Qdot-actin velocity matches measurements with rhodamine-phalloidin labeled actin. The sHMM Qdot-actin velocity histogram contains low velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm, and, larger compliance than sHMM depending on MYH7 surface concentration. Low duty cycle skeletal and cardiac myosin present challenges for a single molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space then characterized using super-resolution. The approach provides quick, quantitative, and inexpensive step-size measurement for low duty cycle muscle myosin.

Muscle myosin cyclically hydrolyzes ATP to translate actin. Ventricular cardiac myosin (βmys) moves actin with three distinct unitary step-sizes resulting from its lever-arm rotation and with step-frequencies that are modulated in a myosin regulation mechanism. The lever-arm associated essential light chain (vELC) binds actin by its 43 residue N-terminal extension. Unitary steps were proposed to involve the vELC N-terminal extension with the 8 nm step engaging the vELC/actin bond facilitating an extra ~19 degrees of lever-arm rotation while the predominant 5 nm step forgoes vELC/actin binding. A minor 3 nm step is the unlikely conversion of the completed 5 to the 8 nm step. This hypothesis was tested using a 17 residue N-terminal truncated vELC in porcine βmys (Δ17βmys) and a 43 residue N-terminal truncated human vELC expressed in transgenic mouse heart (Δ43αmys). Step-size and step-frequency were measured using the Qdot motility assay. Both Δ17βmys and Δ43αmys had significantly increased 5 nm step-frequency and coincident loss in the 8 nm step-frequency compared to native proteins suggesting the vELC/actin interaction drives step-size preference. Step-size and step-frequency probability densities depend on the relative fraction of truncated vELC and relate linearly to pure myosin species concentrations in a mixture containing native vELC homodimer, two truncated vELCs in the modified homodimer, and one native and one truncated vELC in the heterodimer. Step-size and step-frequency, measured for native homodimer and at two or more known relative fractions of truncated vELC, are surmised for each pure species by using a new analytical method.

Cardiac and skeletal muscle myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin. Myosin has a motor domain containing ATP and actin binding sites and a lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into the linear displacement known as step-size. The myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to myosin filament based contraction regulation and to the response of the muscle to disease. Myosin step-size was measured using a novel quantum dot (Qdot) assay that previously confirmed a 5 nm step-size for fast skeletal myosin and multiple unitary steps, most frequently 5 and 8 nm, and a rare 3 nm displacement for β cardiac myosin (βMys). S15 phosphorylation in βMys is now shown to change step-size distribution by advancing the 8 nm step frequency. After phosphorylation, the 8 nm step is the dominant myosin step-size resulting in significant gain in the average step-size. An increase in myosin step-size will increase the amount of work produced per ATPase cycle. The results indicate that RLC phosphorylation modulates work production per ATPase cycle suggesting the mechanism for contraction regulation by the myosin filament.

The labeling of muscle fiber proteins with iodoacetamido)tetramethylrhodamine (IATR) was reinvestigated with the purified 5' or 6' isomers of IATR. Both isomers modify the myosin heavy chain within the 20-kDa fragment of myosin subfragment 1 (S1) but with different rates, and only the 5'-IATR alters K(+)-EDTA- and Ca(2+)-activated ATPases. Absorption spectroscopic and ATPase studies of probe stoichiometry indicate that for 5'-IATR there are two probes per myosin sulfhydryl 1 (SH1). Quantitative fluorograms of the SDS-PAGE gels confirm that there are one covalent and one noncovalent probe per SH1 when S1 is labeled with 5'-IATR (5'-IATR-S1) and that there are one covalent and two noncovalent probes per S1 when S1 is labeled with 6'-IATR (6'-IATR-S1). The 5'- and 6'-IATR probes have similar fluorescent lifetimes when bound to S1, but quenching studies with potassium iodide show that 5'-IATR-S1 has a single class of strongly bound chromophores while 6'-IATR-S1 has two or more classes of chromophores. It is possible that 5'-IATR labels SH1 as a dimer. The polarization anisotropies of 5'- and 6'-IATR-S1 indicate that 5'-IATR is immobilized, while 6'-IATR is moving independently, on the surface of S1. The emission spectrum from 5'-IATR-S1 is unaffected by the addition of MgATP, while 6'-IATR-S1 shows a spectral shift and total intensity change. When labeling muscle fibers, 5'-IATR labels myosin SH1 and differentiates between the fiber physiological states by indicating cross-bridge rotation in quantitative agreement with previous results [Burghardt et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7515]. 6'-IATR reacts preferentially with actin in muscle fibers and does not differentiate between fiber physiological states as expected for an actin probe. The stereospecificity of the rhodamine isomers for SH1 indicates features of the local protein structure. The experimental results are used with theoretical methods for determining molecular structure to suggest a qualitative scheme for the specific interaction of 5'-IATR with its binding pocket on the surface of S1.

Ventricular myosin (βMys) is the motor protein in cardiac muscle generating force using ATP hydrolysis free energy to translate actin. In the cardiac muscle sarcomere, myosin and actin filaments interact cyclically and undergo rapid relative translation facilitated by the low duty cycle motor. It contrasts with high duty cycle processive myosins for which persistent actin association is the priority. The only pharmaceutical βMys activator, omecamtive mecarbil (OM), upregulates cardiac contractility in vivo and is undergoing testing for heart failure therapy. In vitro βMys step-size, motility velocity, and actin-activated myosin ATPase were measured to determine duty cycle in the absence and presence of OM. A new parameter, the relative step-frequency, was introduced and measured to characterize βMys motility due to the involvement of its three unitary step-sizes. Step-size and relative step-frequency were measured using the Qdot assay. OM decreases motility velocity 10-fold without affecting step-size, indicating a large increase in duty cycle converting βMys to a near processive myosin. The OM conversion dramatically increases force and modestly increases power over the native βMys. Contrasting motility modification due to OM with that from the natural myosin activator, specific βMys phosphorylation, provides insight into their respective activation mechanisms and indicates the boilerplate screening characteristics desired for pharmaceutical βMys activators. New analytics introduced here for the fast and efficient Qdot motility assay create a promising method for high-throughput screening of motor proteins and their modulators.