Evaluation of structural alterations in actin by computational simulation and high-speed AFM to study the mechanism of myosin force generation
Biophysical Journal(2023)
Abstract
In this study, we used High-Speed Atomic Force Microscopy (HS-AFM) and computational simulation to evaluate the effect of actin oxidation on myosin force generation. For computational analysis, we implemented oxidation of certain tyrosine residues in the PDB structures of G-actin monomer and F-actin filaments. The selection of oxidized residues was based on our previous work, where oxidized residues in F-actin were identified by mass spectrometry. Computationally oxidized and non-oxidized PDB structures of the G-actin monomers were used to evaluate the electrostatic potential energy as well as for the simulation of the HS-AFM images. The comparison of the G-actin electrostatic maps revealed several spots affected by oxidation. A cross-section analysis around oxidized and non-oxidized residues in the simulated HS-AFM images of G-actin monomers revealed a shift in the height values (∼0.2 nm to 1 nm in magnitude). These differences were confirmed in the computationally oxidized F-actin structures and in experiments conducted with HS-AFM using F-actin oxidized in-vitro by an NO and O2 donor, SIN-1 (∼1.5 nm in magnitude). To better understand actin oxidation-induced impairment in contractility we determined the myosin head displacements while attached to non-oxidized or SIN-1-treated F-actin. We observed that adding myosin molecules to SIN-1-treated F-actin in the presence of ATP resulted in a change of the size of myosin head displacement. The myosin heads were able to produce short (below 1 nm), moderate (2-3 nm), and long (over 4 nm) displacements on the non-oxidized F-actin, but the frequency of long displacements was significantly decreased in the SIN-1-treated F-actin. These results suggest that oxidation decreases the pool of the weak bound myosin molecules and shortens the long displacements related to the Pi release step, thus reducing the force generation by myosin motors.
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Key words
actin,force,high-speed
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