New paradigms in actomyosin energy transduction: Critical evaluation of non‐traditional models for orthophosphate release

Månsson, Alf; Ušaj, Marko; Moretto, Luisa; Matusovsky, Oleg S.; Velayuthan, Lok Priya; Friedman, Ran; Rassier, Dilson E.

doi:10.1002/bies.202300040

Cited by 6 publications

(3 citation statements)

References 105 publications

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“…Our conclusion that force develops before Pi release and that the same force is maintained after Pi release on myofibrils is consistent with previous observations on fast-twitch single fibers [31,7,61,60,36], slow-twitch single fibers [33,64], and single molecule experiments [70]. This conclusion is at variance with that of X-ray crystallographic studies [38] (see also [45,41]), which concluded that force develops after Pi release. There may be several reasons for this disparity.…”

Section: Force Associated With Each Cb Statesupporting

confidence: 91%

Oscillatory work and the step that generates force in single myofibrils from rabbit psoas

Kawai,

Iorga

2024

Pflugers Arch - Eur J Physiol

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The elementary molecular step that generates force by cross-bridges (CBs) in an active muscle has been under intense investigation in the field of muscle biophysics. The question asked here is whether this step is before phosphate (Pi) release or after its release. It is known that an increase in the concentration of Pi ([Pi]) diminishes isometric force in Ca 2+ activated fibers, indicating a tight coupling between the force-generating step and the Pi release step. We investigated the effect of Pi on oscillatory work production in single myofibrils and found that Pi-attached state(s) to CBs is essential for its production. Oscillatory work is the mechanism that allows an insect to fly by beating its wings, and it also has been observed in skeletal and cardiac muscle fibers, implying that it is an essential feature of all skeletal muscle types. With our studies, oscillatory work disappears in the absence of Pi in experiments using myofibrils. This suggests that force is generated during a transition between steps of oscillatory work production and that the states involved in its production must have Pi attached. With sinusoidal analysis, we obtained the kinetic constants around the Pi release steps, established a CB scheme, and evaluated force generated (and supported) by each CB state. Our results demonstrate that force is generated before Pi is released, and the same force is maintained after Pi is released. Stretch activation and/or delayed tension can also be explained with this CB scheme and forms the basis of force generation and oscillatory work production.

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Section: Force Associated With Each Cb Statesupporting

confidence: 91%

Oscillatory work and the step that generates force in single myofibrils from rabbit psoas

Kawai,

Iorga

2024

Pflugers Arch - Eur J Physiol

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“…Moreover, blocking orthophosphate from reaching the exit tunnel with the mutation S217A or exposure to high concentrations of orthophosphate affected neither the size nor the rate of the power stroke, implying that the power stroke occurs before orthophosphate enters the exit tunnel [ 64 ]. However kinetic models showed that the timing of orthophosphate release from the active state after the power stroke was not consistent with experimental observations of the [Pi]-independence of maximal shortening velocity and monotonous decrease in isometric force with increased [Pi] [ 62 , 65 ]. This led to the multi-step orthophosphate release kinetic model, which predicts that orthophosphate leaves the active site but is trapped at an intermediate secondary binding site before completion of the power-stroke [ 65 ].…”

Section: Discussionmentioning

confidence: 77%

“…In our adaptation of the Davydov soliton model, straightening of the relay helix is proposed to be an early step in the power stroke that is initiated within picoseconds of ATP hydrolysis by energy transfer from the vibrationally excited orthophosphate molecule to the relay helix. Whether orthophosphate release from the active site occurs before or after the power stroke is a highly debated issue [ 61 , 62 ]. It has been determined experimentally by Förster resonance energy transfer (FRET) studies that the timescale for orthophosphate release ranges from 3 to 50 ms and that the timescale for the power stroke is 2–3 ms [ 61 ].…”

Section: Discussionmentioning

confidence: 99%

Study of the Myosin Relay Helix Peptide by Molecular Dynamics Simulations, Pump-Probe and 2D Infrared Spectroscopy

Freedman,

Tuszynski

2024

IJMS

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The Davydov model was conjectured to describe how an amide I excitation created during ATP hydrolysis in myosin might be significant in providing energy to drive myosin’s chemomechanical cycle. The free energy surfaces of the myosin relay helix peptide dissolved in 2,2,2-trifluoroethanol (TFE), determined by metadynamics simulations, demonstrate local minima differing in free energy by only ~2 kT, corresponding to broken and stabilized hydrogen bonds, respectively. Experimental pump-probe and 2D infrared spectroscopy were performed on the peptide dissolved in TFE. The relative heights of two peaks seen in the pump-probe data and the corresponding relative volumes of diagonal peaks seen in the 2D-IR spectra at time delays between 0.5 ps and 1 ps differ noticeably from what is seen at earlier or later time delays or in the linear spectrum, indicating that a vibrational excitation may influence the conformational state of this helix. Thus, it is possible that the presence of an amide I excitation may be a direct factor in the conformational state taken on by the myosin relay helix following ATP hydrolysis in myosin.

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Probing actin‐activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence

Berg,

Velayuthan,

Tågerud

et al. 2024

Cytoskeleton

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Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit from functional studies of a wide range of point‐mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus‐free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin‐activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero‐length cross‐linking of human cardiac myosin II motor fragments (sub‐fragment 1 long [S1L]) to surface‐immobilized actin filaments in a configuration with maintained actin‐activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin‐activated rate constant (kcat). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin‐activated ATPase, but also a third exponential process consistent with non‐specific ATP‐binding outside the active site.

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New paradigms in actomyosin energy transduction: Critical evaluation of non‐traditional models for orthophosphate release

Cited by 6 publications

References 105 publications

Oscillatory work and the step that generates force in single myofibrils from rabbit psoas

Oscillatory work and the step that generates force in single myofibrils from rabbit psoas

Study of the Myosin Relay Helix Peptide by Molecular Dynamics Simulations, Pump-Probe and 2D Infrared Spectroscopy

Probing actin‐activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence

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