Computational protocol: Calcium Induced Regulation of Skeletal Troponin — Computational Insights from Molecular Dynamics Simulations

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Protocol publication

[…] Molecular dynamic simulations were performed using the NAMD software package running on the XSEDE Lonestar supercomputer. The crystal structure files of fully Ca2+-saturated avian fast skeletal troponin molecule (1YTZ.pdb) and Ca2+-depleted TnC subunit (5TnC.pdb) were obtained from the Protein Data Bank .Our choice of PDB system was driven by the considerations that while both Ca2+- and apo-form crystal structures are available for the core domain of the skeletal troponin complex, 1YTZ.pdb is the only available structure in the Ca2+ form. The 5TnC.pdb crystal structure of Herzberg and James is the first crystal structure of EF-hand type Ca2+-binding proteins, is determined at 2 Å resolution, and is widely considered to be the gold standard for skeletal TnC structural description. The closeness of the two source species: Gallus gallus (1YTZ.pdb) and Meleagris gallopavo (5TnC.pdb) provided an additional level of complementarity. It should be noted that the selected PDB files have some missing residues. Specifically, in 1YTZ.pdb the 39 C-terminal amino acid residues (144–182) of TnI or the C-terminal residues of TnT2 (249–262), as well as the N-terminal tail domain of TnT, are not in the crystal set. We did not incorporate these residues in our simulations. These residues are considered to be flexible, at least in the absence of tropomyosin and it was demonstrated that the C-terminal mobile domain of TnI behaves independently from the core domain of the complex by NMR measurements , .In total we prepared and simulated 8 systems: 1) core troponin (TnI, TnC, TnT), Ca2+ present in sites I, II, III and IV (Tn4Ca2+); 2) core troponin (TnI, TnC, TnT), Ca2+ present in sites III and IV only (Tn2Ca2+); 3) TnC domain, Ca2+ present in sites I, II, III and IV (TnC4Ca2+); 4) TnC domain, Ca2+ present in sites III and IV (TnC2Ca2+); 5) TnC domain where site I was depleted, site II was saturated (TnCSite1noCa2+); 6) TnC domain, where site I was saturated, site II was depleted (TnCSite2noCa2+); 7) TnC domain, Ca2+ present in sites III and IV, with residue ALA24 mutated to ASP24 and LEU48 to GLU48 (TnCASP24GLU48), and 8) TnC domain, Ca2+ present in Site III and IV (5TnC). Protein initial coordinates for systems 1 through 7 were derived from 1YTZ.pdb and protein initial coordinates for system 8 were derived from 5TnC.pdb. Protein structure files (psf) were created using the molecular modeling package VMD and the plug-in program psfgen. Hydrogen atoms were added and the protein systems were solvated in explicit solvent environment. The CHARMM force field was used for the protein, water was considered as the TIP3P model . The total system size for the molecular structures ranged from 55582 to 87325 atoms. The molecular systems were energy minimized by conjugate gradient method and heated to 300 K. Simulations were performed with periodic boundary conditions in the NPT ensemble; temperature and pressure were kept constant by employing Langevin dynamics, at 1 atm and 300 K. Electrostatic interactions were computed by PME (Particle mesh Ewald) method. Non bonded interactions were treated with a cutoff using a switching function beginning at 10 Å and reaching zero at 14 Å. Data extraction, trajectory analysis, figures and visualizations were completed using VMD and MATLAB. In total, 16 simulations were run and 0.48 µs of simulation time were collected and analyzed. presents a list of systems, number and duration of simulations performed. […]

Pipeline specifications

Software tools NAMD, VMD, CHARMM
Application Protein structure analysis
Chemicals Calcium, Nitrogen, Oxygen