Computational protocol: Relevance of the protein macrodipole in the membrane-binding process. Interactions of fatty-acid binding proteins with cationic lipid membranes

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Protocol publication

[…] A symmetric bilayer composed of 128 molecules of EDMPC was assembled, hydrated (~3500 water molecules), neutralized with Cl- ions, pre-equilibrated (200 ns) and placed in a 7 × 7 × 10 nm3 triclinic box containing a protein molecule, neutralizing Na+ and Cl- ions and ~104 water molecules. Four different initial configurations for ReP1-NCXSQ and two for L-BABP were generated by placing the proteins in different orientations and distances with respect to the interfacial plane. The distance between the protein center of mass (COM) and the cis hemilayer plane (determined by the phosphorous atoms average position) was between 2.5 and 4.5 nm, which is about the thickness of 7 to 13 layers of water.The GROMOS96 force field [] was used with the G53A6 set of parameters [] for the protein. The lipid parameters were from Kukol [] for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) modified by the addition of an ethyl group to the phosphate group. Kukol's atomic types, and also the charges for the glycerol group and the hydrocarbon chains were conserved, while an ab initio calculation for the headgroup remaining charges was performed using a PM3 optimized geometry. MOPAC partial charges were assigned using ATB webserver []. The charges and atomic types for the lipid molecule are specified in the Supporting Information (, ). The simple point charge (SPC) model of water was used []. The charges on the aminoacid side chains were according to the pKa estimated by the PROPKA web server []. The electrostatic interactions were handled with the SPME version of the Ewald sums [], with a real space cutoff of 0.9 nm. The van der Waals interactions were handled with the twin-range scheme with the short range cutoff at 0.9 nm and the long range cutoff at 1.4 nm. The simulations were carried out in the NPT ensemble using the v-rescale thermostat [] and Berendsen barostat []. The protein, the membrane, and the solvent were coupled separately to a temperature bath with a reference temperature of 320 K and a relaxation constant of 0.2 ps. The pressure was maintained constant by coupling to a reference pressure of 1 bar with a relaxation constant of 2.0 ps and a compressibility of 5×10−5 bar-1. Semi-isotropic coupling in the direction normal to the bilayer was applied. No dispersion corrections were applied neither in long range interactions nor in the pressure, to avoid artifacts in the mean area per lipid and in the bilayer thickness []. The bonds in the protein and the lipids were constrained using the LINCS algorithm []. For the bonds and angle of the water molecules, we used the SETTLE algorithm []. The time step for the integration of the equation of motion was 5 fs due to the use of virtual sites in the polar hydrogen atoms of the protein and the lipids []. The non-bonded list was updated every 4 time steps. Every run, whether of equilibration or production, was started with a different set of initial velocities in order to produce non-correlated trajectories. Production runs were performed without restrictions during 100 ns. The simulations and their analysis were performed with the GROMACS 4.5.4 software package [].Macrodipoles were computed using the GROMOS96 partial atomic charges localized according to the crystallographic structures. The electrostatic equipotential contour calculations were performed through the Finite Difference Poisson Boltzmann Equation method as implemented in APBS (Adaptive Poisson Boltzmann Solver) [] for VMD software package []. […]

Pipeline specifications

Application Protein structure analysis