Cryo-atomic force microscopy of smooth muscle myosin
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Published on: May 7, 2020
1Department of Molecular Physiology, University of Virginia Health Sciences Center, Charlottesville 22906-0011, USA.
Abstract:
The motor and regulatory domains of the head and the 14-nm pitch of the alpha-helical coiled-coil of the tail of extended (6S) smooth-muscle myosin molecules were imaged with cryo atomic force microscopy at 80-85 K, and the effects of thiophosphorylation of the regulatory light chain were examined. The tail was 4 nm shorter in thiophosphorylated than in nonphosphorylated myosin. The first major bend was invariant, at approximately 51 nm from the head-tail junction (H-T), coincident with low probability in the paircoil score. The second major bend was 100 nm from the H-T junction in nonphosphorylated and closer to a skip residue than the bend (at 95 nm) in thiophosphorylated molecules. The shorter tail and distance between the two major bends induced by thiophosphorylation are interpreted to result from melting of the coiled-coil. An additional bend not previously reported occurred, with a lower frequency, approximately 24 nm from the H-T. The range of separation between the two heads was greater in thiophosphorylated molecules. Occasional high-resolution images showed slight unwinding of the coiled-coil of the base of the heads. We suggest that phosphorylation of MLC20 can affect the structure of extended, 6S myosin.
Insights
Thiophosphorylation shortens smooth muscle myosin tails by melting the coiled-coil structure. This structural change, affecting myosin
Area of Science:
- Biophysics
- Structural Biology
- Muscle Physiology
Background:
- Smooth muscle myosin is a critical motor protein involved in muscle contraction.
- The structure of myosin, particularly its tail domain, influences its motor activity.
- Post-translational modifications, like phosphorylation, are known to regulate protein function.
Purpose of the Study:
- To investigate the structural effects of thiophosphorylation on extended 6S smooth muscle myosin.
- To visualize the molecular architecture of myosin using high-resolution imaging techniques.
- To understand how regulatory light chain phosphorylation impacts myosin tail conformation.
Main Methods:
- Cryo atomic force microscopy (cryo-AFM) was employed to image myosin molecules at low temperatures (80-85 K).
- The study focused on the motor and regulatory domains of the myosin head and the alpha-helical coiled-coil of the tail.
- Analysis included measuring distances between structural features and assessing coiled-coil integrity.
Main Results:
- Thiophosphorylation resulted in a 4 nm shorter myosin tail compared to nonphosphorylated myosin.
- A previously unreported bend was observed approximately 24 nm from the head-tail junction.
- The distance between the two myosin heads increased upon thiophosphorylation, suggesting structural alterations.
Conclusions:
- Thiophosphorylation of the regulatory light chain (MLC20) induces structural changes in the myosin tail, likely through coiled-coil melting.
- These structural modifications, including tail shortening and altered bend positions, may affect myosin's interaction with actin and its motor function.
- The findings highlight the role of phosphorylation in modulating the conformation and potentially the activity of smooth muscle myosin.
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