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    • br Methodology In this paper

    2024-01-09


    Methodology In this paper, mechanical properties of G-actin are examined. To do so, three different external tensile loads are exerted to the protein. As illustrated in Fig. 1, Arp2/3 complex binds to the one sides of mother LY2940680 and leads to the growth of a new daughter filament at a distinctive 70° angle. The latter figure shows a tensile load in the daughter filament with the tensile force decomposed into orthogonal components and . Therefore, three different cases of external loading are considered in this paper. Fig. 2 represents the three considered cases of exerting external force to the G-actin. In Fig. 2(a) simple tension in the filament is modeled however in Fig. 2(b) and (c) the tensile load on the daughter filament is considered by decomposition into the two orthogonal component. In axial tensile modeling (Fig. 2(a)) bottom of the G-actin is fixed, however in the lateral loading (Fig. 2(b) and (c)) bottom and up parts of the G-actin are fixed and the side of the G-actin is subjected to the lateral loads (normal and tangential). Moreover, in each case of loading, both states of nucleotide state of G-actin are simulated to study the effects of the nucleotide on the mechanical properties of the G-actin. Therefore, as a whole, six different cases of study are considered and simulated in this paper.
    Materials and methods In this section, a series of steered MD simulations are carried out in order to investigate the mechanical properties of the G-actin molecule. Atomic coordinates used for ATP G-actin and ADP G-actin are taken from the crystal structure reported by Kabsch et al. (1990) (PDB code: 1ATN) and Oda et al. (2009) (PDB code: 2ZWH), respectively. MD and SMD simulations are performed based on classical force field CHARMM (MacKerell et al., 1998), implemented in the MD program of NAMD (Phillips et al., 2005). Missing hydrogen atoms were added to the proteins with the program PSFGen plugin in the NAMD distribution and then each G-actin was solvated in a 9 × 9 × 12 nm3 orthorhombic box using TIP3 water model and to neutralize the net charge, potassium chloride (KCl) ions were added with the concentration of 150 mM using Visual Molecular Dynamics (VMD) (Humphrey et al., 1996) autoionize plug-in. The size of the water box was considered in a way to ensure that the entire protein was embedded in water during the entire simulation. van der Waals interactions were calculated by using simple spherical cutoff at a distance of 14 Å a switching function is employed which is starting at 10 Å and is zeroed at a cutoff of 12 Å. In all simulations, chemical bonds involving hydrogen atoms were restrained using the SHAKE algorithm. Periodic boundary conditions (PBC) were applied in the simulations for more realistic simulations of aqueous solutions. The pressure and temperature are kept constant at 1 atm and 310 K using the Nose–Hoover Langevin piston method with a decay period of 100 fs and a damping time constant of 50 fs. Electrostatics interactions were calculated using particle mesh Ewald (PME) to account for long-range effects. An integration time step of 1 fs was employed. Each system was minimized by the conjugate gradient algorithm for 10,000 steps, heated for 0.3 ns to the required temperature and equilibrated at the constant temperature for 1 ns. To exert external forces to G-actin that induce axial and lateral deformation, steered molecular dynamics (SMD) has evolved into a useful tool which imitates directly the basic idea of an AFM experiment (Lu et al., 1998). In this method, the following potential is added to the Hamiltonian of the system: where and represent the position of center of masses of SMD atoms at time t and initial time, respectively. In Eq. (1), v denotes pulling velocity, is pulling direction and kSMD is SMD virtual harmonic spring constant. The net force applied on the pulled atoms can be expressed as: As indicated in Eq. (2), FSMD is a function of kSMD and depends on it. Previous studies have considered a constant value for kSMD, however in this paper, we carried out a set of simulations with nine different virtual spring constants k values varying from 1.5 to 9.5 kCal/mol/Å2. For each kSMD value, three simulations were performed for statistical purposes. In all of the simulations, the pulling velocity is set to 0.1 Å/ps.