Computational protocol: The Dynamics of Ca2+ Ions within the Solvation Shellof Calbindin D9k

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[…] The number of simulations that were carried out, their lengths and compositions are given in .The force field selected for the simulation was Amber94 ported by Sorin and Pande . To optimize the interaction of the Ca2+ ion with carboxylate we modulated the Lennard–Jones parameters as described in , using the values: σ = 0.300616 nm; ε = 1.87 KJ·mol−1. The TIP3P water model was used in all simulations. The simulations were performed using the GROMACS 4 package of programs , , , . The WT Calbindin protein structure (PDB code 3ICB) given in the Protein Data Bank(PDB) was determined by X-ray crystallography at 0.23 nm . In order to simulate a Ca2+ depleted CaB (Apo-CaB), the two Ca2+ ions were removed from their binding sites and added at random locations. The water box extended to at least 1.2 nm from the molecule, for CaB simulations, and at least 2.0 nm from the Ca2+ ion for the free Ca2+ simulations. D, E and LYS residues were charged to reflect their state at physiological pH. Electroneutrality and physiological ionic strength of ∼0.150 M for the CaB simulations and 0.1 M for the free Ca2+ simulations were reached by adding appropriate amounts of Na+ and Cl− ions to the simulation boxes (as detailed in ). Prior to the dynamics simulation, internal constraints were relaxed by energy minimization. Following the minimization, an MD equilibration run was performed for 20 ps under position restraints. Finally, before all the production runs, further unconstrained equilibrations were performed for 200 ps. After the equilibration, an MD production run was performed for extended time frame, as described in . The time step for the simulation was 2 fs. The simulations were run under NPT conditions, using Berendsen's coupling algorithm for keeping the temperature and the pressure constant (P = 1 bar; τP = 0.5 ps; T = 300 K; τT = 0.1 ps) . VDW and short range electrostatic forces were treated using a cutoff of 1.2 nm. All simulations were carried out with periodic boundary conditions, using the particle mesh Ewald (PME) method for long range electrostatic forces. During the MD runs, the LINCS algorithm was used in order to constrain the lengths of all bonds; the water molecules were constrained using the SETTLE algorithm . Coordinates were saved every 1 ps. Protein images in were generated using VMD .The diffusion coefficients were calculated from the mean square displacement (MSD) curves between specific time points selected to give the most reliable results. For Ca2+ at specific shells from the protein, the MSD statistics used for calculation of the diffusion constant was such that it contained at least 8 different simulations which contributed to the MSD average and the MSD graph was near linear. The diffusion coefficient errors stated in the manuscript are estimates based on multiple calculations using different sets of simulations.Residence times were calculated by fitting single and double exponential functions to the residence time decay curves. The residence time decay curves denote the number of times a Ca2+ ion was found at the desired distance from the protein up to a given time span marked by the he abscissa. In these calculations an event where the Ca2+ ion changed its distance from the protein's surface for a period of 2 ps or less was not considered as termination of its residence period. All Apo-CaB simulations were used to generate these curves. For the exponential fitting procedure, only sections of the graph with a count higher than 10 were considered.Electrostatic potential calculation were performed using the APBS software 1.2.1 , with a grid spacing of 1.096×1.096×1.096 Å. The calculations were carried out for a solution having an ionic strength of 150 mM. The dielectric constant of the protein was set as 2 and solvent dielectric of 78.54. […]

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