Computational protocol: Ion–ion interactions in the denatured state contribute to the stabilization of CutA1 proteins

Similar protocols

Protocol publication

[…] MD simulations were performed using each subunit of three EcCutA1 mutants: an SH-free mutant with Td = 85.6 °C (Ec0SH = EcCutA1_C16A/C39A/C79A), a hydrophobic mutant also lacking SH groups with Td = 113.3 °C (Ec0VV = Ec0SH_S11V/E61V), and an ionic mutant with Td = 136.8 °C (Ec0VV_6 = Ec0VV_A39D/S48K/H72K/S82K/Q87K/T88R).MD simulations were performed using GROMACS software (ver. 4.5.5),. The missing atoms in the coordinate file of Ec0SH (PDB ID, 4Y65), which are three N-terminal residues of the B subunit and eight N-terminal residues of the C subunit, were modeled in QUANTA2000 (Accelrys) using the coordinates of N-terminal residues of the A subunit as a reference. The structures of Ec0VV and Ec0VV_6 were modeled using FoldX, based on the structure of Ec0SH. Hydrogen atoms were added to each protein. The models were solvated in water boxes with a minimum distance of 1.2 nm between the protein and the box. Counter-ions were added to the model to neutralize any net charge. The periodic boundary condition was adopted and the long-range electrostatic interactions were computed using the Particle-Mesh-Eward (PME) method. The GROMOS 43A1 force field and SPC/E water model were employed. The system was weakly coupled to a heat bath by velocity rescaling with a relaxation time of 0.1 ps. A Parrinello–Rahman barostat was used to maintain a pressure constant at 1.0 bar for 300 K and 6.0 bar for 400 K or 450 K with a relaxation time of 0.5 ps. Hydrogen atoms were constrained using LINCS, and MD simulations at 300 K, 400 K, and 450 K were conducted with an integration time step of 1 femtosecond (fs). Energy minimizations were done to remove bad van der Waals contacts. Next, the temperature was raised from 50 K to 300 K in increments of 50 K, with 10,000 integration steps at each temperature and a harmonic constraint of C-alpha atoms. Thereafter, the ensemble was equilibrated through four 100-picosecond (ps) cycles with gradually released harmonic constraints: 1000, 100, 10, and 1 kJ mol−1 nm−2. The subsequent MD stages for the EcCutA1 mutants were carried out without any restraint at 300 K. When the system temperature was increased to 400 K or 450 K from 300 K, pressure coupling was not set during 1000 ps at 400 K or 450 K. The obtained MD trajectories were analyzed using GROMACS software. The calculations for RMSD of Cα atoms and the radius of gyration were performed using the commands ‘gmx rms’ and ‘gmx gyrate,’ respectively. For the salt bridges, ‘gmx saltbr’ was used with the option t = 0.4 nm, which means that groups that were never closer than this distance were not plotted. For the calculation of ASA values of each atom, the results of ‘gmx sasa’ (atomarea.xvg: average area per atom) were used. For the trajectory of secondary structures, the command ‘do_dssp’ was used. For the average α-helix at each residue, the command ‘g_helix’ was used. […]

Pipeline specifications

Software tools GROMACS, FoldX, P-LINCS
Application Protein structure analysis
Organisms Escherichia coli
Diseases Genetic Diseases, Inborn