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Crosslinking of telokin to chicken gizzard smooth muscle myosin
1Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo.
Abstract:
Telokin, which is a candidate for one of the factors stabilizing dephosphorylated myosin filaments in smooth muscle cells, was subjected to a chemical crosslinking study to determine its binding location on the myosin molecule. Telokin labeled with 5-iodoacetamidofluorescein (Fl-telokin) retained the nature of unlabeled telokin: it bound to dephosphorylated gizzard myosin with a stoichiometry of 1 mol/mol myosin and induced filament assembly. The carboxyl groups of Fl-telokin were activated with 1-ethyl-3-(3-dimethyl-amino-propyl) carbodiimide and N-hydroxysuccinimide and then crosslinked to myosin. The production of three fluorescent peptides was observed on an SDS-gel, accompanying a decrease in the amount of regulatory light chain (LC20). The molecular weights of these products estimated to be > 200, 62, and 41 kDa. When unlabeled telokin was crosslinked to myosin of which LC20 was exchanged with 5-[[2-[(iodoacetyl)amino]ethyl]amino]-naphthalene-1-sulfonic acid-labeled LC20, the 62- and 41-kDa bands were also fluorescent. These results suggest that the > 200, 62, and 41-kDa species are telokin crosslinked with a heavy chain, with 2 LC20, and with 1 LC20, respectively. Myosin crosslinked with unlabeled telokin showed an extra structure with a small projection at the head-rod junction on electron microscopy and this structure was proved to be telokin by decorating it with anti-telokin antibodies. In addition, dephosphorylated myosin crosslinked with Fl-telokin was incapable of folding into the 10S conformation at 0.2 NaCl in the presence of MgATP, and assembled into filaments at 0.15 M NaCl in the presence of MgATP. Thus, telokin may bind to LC20 and a heavy chain region at the head-rod junction and suppress folding into the 10S conformation, leading to the assembly of myosin into filaments.
Insights
Telokin stabilizes dephosphorylated myosin filaments by binding to the heavy chain and regulatory light chain (LC20). This interaction suppresses myosin
Area of Science:
- Muscle physiology
- Protein biochemistry
- Molecular cell biology
Background:
- Telokin is a myosin-binding protein implicated in smooth muscle contraction.
- Dephosphorylated myosin filaments require stabilization for proper function.
- Understanding telokin's precise binding site on myosin is crucial for elucidating its regulatory role.
Purpose of the Study:
- To determine the specific binding location of telokin on the myosin molecule using chemical crosslinking.
- To investigate how telokin binding affects myosin filament assembly and conformation.
Main Methods:
- Chemical crosslinking of fluorescently labeled telokin (Fl-telokin) to dephosphorylated gizzard myosin.
- SDS-PAGE analysis to identify crosslinked products and their molecular weights.
- Electron microscopy and antibody decoration to visualize and confirm telokin's location on myosin.
Main Results:
- Fl-telokin crosslinked to myosin, forming fluorescent peptides of >200 kDa (heavy chain), 62 kDa (2x LC20), and 41 kDa (1x LC20).
- Electron microscopy revealed an extra structure at the myosin head-rod junction upon telokin crosslinking.
- Telokin binding prevented myosin from adopting the 10S conformation and promoted filament assembly.
Conclusions:
- Telokin binds to both the myosin heavy chain and the regulatory light chain (LC20).
- Telokin's binding site is located at the head-rod junction of myosin.
- Telokin stabilizes myosin filaments by inhibiting the 10S conformation, thereby promoting filament assembly.