Muscle contraction and a range of critical cellular functions rely on force-producing interactions between myosin motors and actin filaments, powered by turnover of adenosine triphosphate (ATP). The relationship between release of the ATP hydrolysis product ortophosphate (Pi) from the myosin active site and the force-generating structural change, the power-stroke, remains enigmatic despite its central role in energy transduction. Here, we present a model with multistep Pi-release that unifies current conflicting views while also revealing additional complexities of potential functional importance. The model is based on our evidence from kinetics, molecular modelling and single molecule fluorescence studies of Pi binding outside the active site. It is also consistent with high-speed atomic force microscopy movies of single myosin II molecules without Pi at the active site, showing consecutive snapshots of pre- and post-power stroke conformations. In addition to revealing critical features of energy transduction by actomyosin, the results suggest enzymatic mechanisms of potentially general relevance.

Rationale: Airway smooth muscle (ASM) plays a key role in airway hyperresponsiveness (AHR) but it is unclear whether its contractility is intrinsically changed in asthma.Objectives: To investigate whether key parameters of ASM contractility are altered in subjects with asthma.Methods: Human trachea and main bronchi were dissected free of epithelium and connective tissues and suspended in a force-length measurement set-up. After equilibration each tissue underwent a series of protocols to assess its methacholine dose-response relationship, shortening velocity, and response to length oscillations equivalent to tidal breathing and deep inspirations.Measurements and Main Results: Main bronchi and tracheal ASM were significantly hyposensitive in subjects with asthma compared with control subjects. Trachea and main bronchi did not show significant differences in reactivity to methacholine and unloaded tissue shortening velocity (Vmax) compared with control subjects. There were no significant differences in responses to deep inspiration, with or without superimposed tidal breathing oscillations. No significant correlations were found between age, body mass index, or sex and sensitivity, reactivity, or Vmax.Conclusions: Our data show that, in contrast to some animal models of AHR, human tracheal and main bronchial smooth muscle contractility is not increased in asthma. Specifically, our results indicate that it is highly unlikely that ASM half-maximum effective concentration (EC 50 ) or Vmax contribute to AHR in asthma, but, because of high variability, we cannot conclude whether or not asthmatic ASM is hyperreactive.

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High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin–troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

Myosin-based molecular motors are responsible for a variety of functions in the cells. Myosin II is ultimately responsible for muscle contraction and can be affected by multiple mutations, that may lead to myopathies. Therefore, it is essential to understand the nanomechanical properties of myosin II. Due to the lack of technical capabilities to visualize rapid changes in nonprocessive molecular motors, there are several mechanistic details in the forcegenerating steps produced by myosin II that are poorly understood. In this study, high-speed atomic force microscopy was used to visualize the actin−myosin complex at high temporal and spatial resolutions, providing further details about the myosin mechanism of force generation. A two-step motion of the double-headed heavy meromyosin (HMM) lever arm, coupled to an 8.4 nm working stroke was observed in the presence of ATP. HMM heads attached to an actin filament worked independently, exhibiting different lever arm configurations in given time during experiments. A lever arm rotation was associated with several nonstereospecific long-lived and stereospecific short-lived (∼1 ms) HMM conformations. The presence of free P i increased the short-lived stereospecific binding events in which the power stroke occurred, followed by release of P i after the power stroke.