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Actin nitrosylation and its effect on myosin driven motility

Heather M. Bansbach William H. Guilford

*Corresponding author: William H. Guilford guilford@virginia.edu

Molecular2016,3,426doi:10.3934/molsci.2016.3.426

The cytoskeletal protein actin can be nitrosylated, and others have shown that nitrosylation of actin can affect actin filament polymerization. However, the effects of nitrosylation on its interactions with the motor protein myosin are unknown. We therefore measured the effect of S-nitrosylation on the interactions of several actin isoforms with myosin. We used a modified coumarin switch assay to determine the number of nitrosylated cysteines in α-skeletal muscle, α-smooth muscle, and non-muscle (β and γ) actin in response to in vitro treatment with nitroso-L-cysteine—an endogenous nitric oxide (NO) donor. We also measured actin filament velocity over heavy meromyosin (HMM) using an in vitro motility assay, the isometric force generated by HMM using a laser trap, and the actin activated ATPase rates of HMM. We found that all three isoforms of actin were nitrosylated equally at ~2 sites per monomer. Nitrosylation of skeletal muscle a-actin reduced the velocity of actin filaments over HMM in a dose dependent fashion. The sliding velocities of all actin isoforms over HMM were reduced equally by ~24% when nitrosylated with 50 µM donor. Our data are consistent with actin nitrosylation causing an increase in the time myosin remains bound to actin during its hydrolytic cycle.

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