The Advanced Photon Source
a U.S. Department of Energy Office of Science User Facility

Relaxation at the Molecular Level

The molecular interactions between the proteins myosin and actin that generate force during muscle contraction are some of the most well-studied molecular interactions in biology. However, there are some congenital skeletal muscle disorders and types of heart failure where relaxation of the muscle, rather than the force generation part of the cycle, appears to be the problem, and there are currently no available treatments that affect relaxation specifically. A more detailed understanding of the dynamics of the relaxation process could help in the development of treatments that maintain or increase force generation while repairing defects in relaxation. Recent work conducted at the U.S. Department of Energy’s Advanced Photon Source (APS) used a unique transgenic mouse model, time-resolved small-angle x-ray diffraction, and molecular dynamics simulations to discover more about how myosin and actin interact during skeletal muscle relaxation. This research, published in the Journal of Physiology, demonstrates that this type of small-angle x-ray analysis may be of great value for uncovering the information needed to identify new treatments for neuromuscular disorders associated with impaired muscle relaxation kinetics.

The protein-protein interactions between myosin and actin to generate skeletal muscle contraction forces involve a cycle that starts with myosin thick-filament binding to actin thin filaments to form cross bridges. This is followed by conformational changes in the myosin that pull muscle filaments toward each other and then release the filament to enter relaxation. The process is dependent on many other proteins, calcium, and, like so many other cellular processes, the hydrolysis of adenosine triphosphate (ATP). In the laboratory, researchers have shown that other nucleotides can affect the dynamics of myosin cycling, including the naturally occurring 2-deoxy-ATP (dATP) that is a myosin activator that enhances the rate and magnitude of force development. However, the reason it attracted the interest of this research team is that the effects of dATP on contraction kinetics also offer an opportunity to study the relaxation process in more detail.

For the muscle experiments, the team of researchers from the Illinois Institute of Technology, the University of Washington, and Cornell University was able to take advantage of a transgenic mouse model in which the expression levels of an enzyme regulating the formation of dATP are elevated in skeletal muscle tissue, increasing the amount of dATP in the muscle 25-fold to a total of 1%-2% of total ATP. Comparison of time-resolved small-angle x-ray diffraction data from non-transgenic mice to the transgenic mouse model tissue obtained at the Biophysics Collaborative Access Team (Bio-CAT) 18-ID beamline at the APS allowed the team to make some discoveries about the effects of dATP on skeletal muscle relaxation (Fig. 1).

The main observations from comparison of small-angle x-ray diffraction data for the two muscles were that, although there were no differences in the kinetics of force development compared to the wildtype, the myosin in the dATP-enhanced muscles was closer to the actin, took more time for the force to drop, had a longer half-time for relaxation, and returned to the resting state more quickly. The high time resolution of the x-ray diffraction technique, enabled by the high-brightness x-rays from the APS, allowed the team to distinguish the nuanced effects of dATP by capturing the prolonged decay in force relative to the wildtype muscle as well as recording the faster restoration of the resting state, discrete steps that could easily have been missed with less sensitive techniques.

These findings led the team to ask what is different about myosin in the presence of dATP? For this they turned to molecular dynamics simulations. Modeling of myosin bound to dATP showed that it adopted more conformations with dATP than with ATP and that it particularly favored an extended conformation that may be more accessible to actin. In addition, solvent accessible surface area (SASA) analysis suggested that dATP increases the surface area of actin binding regions, increasing the exposure of positively charged residues near the negatively charged actin and potentially explaining the observation that myosin in the dATP muscles was closer to the actin filaments. This study demonstrate that the low-angle X-ray diffraction in intact mammalian muscle could be a powerful approach to study sarcomeric protein dynamics and  identify compounds for the treatment of neuromuscular disorders associated with muscle activation and/or relaxation.— Sandy Field

See: Weikang Ma1*, Matthew Childers2, Jason Murray2, Farid Moussavi-Harami2, Henry Gong1, Robert Weiss3, Valerie Daggett2, Thomas Irving1, and Michael Regnier2, “Myosin dynamics during relaxation in mouse soleus muscle and modulation by 2'-deoxy-ATP,” J. Physiol. 598(22), 5165 (2020). DOI: 10.1113/JP280402

Author affiliations: 1Illinois Institute of Technology, 2University of Washington, 3Cornell University

Correspondence: *wma6@iit.edu

The research was supported by National Institutes of Health (NIH) R01 HL128368, NIH R56 AG055594, NIH RM1 GM131981, and NIH U01 HL122199 to M.R., and K08 HL128826 to F.M.H. Use of the BioCAT facility was supported by grant 9 P41 GM103622 from the National Institute of General Medical Sciences of the National Institutes of Health. Use of the Pilatus 3 1M detector was provided by grant 1S10OD018090-01 from NIGMS. Research was also supported by the National Energy Research Scientific Computing Center, supported by the U.S. Department of Energy (DOE) Office of Biological Research, which is supported by the U.S. DOE under contract no. DE-AC02-05CH11231. The research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number P30AR074990. This research used resources of the Advanced Photon Source, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

The U.S. Department of Energy's APS is one of the world’s most productive x-ray light source facilities. Each year, the APS provides high-brightness x-ray beams to a diverse community of more than 5,000 researchers in materials science, chemistry, condensed matter physics, the life and environmental sciences, and applied research. Researchers using the APS produce over 2,000 publications each year detailing impactful discoveries, and solve more vital biological protein structures than users of any other x-ray light source research facility. APS x-rays are ideally suited for explorations of materials and biological structures; elemental distribution; chemical, magnetic, electronic states; and a wide range of technologically important engineering systems from batteries to fuel injector sprays, all of which are the foundations of our nation’s economic, technological, and physical well-being.

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Published Date
02.17.2021