Computational protocol: Stability and Membrane Orientation of the Fukutin Transmembrane Domain: A Combined Multiscale Molecular Dynamics and Circular Dichroism Study

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Protocol publication

[…] We created a model of the 36-residue transmembrane domain of fukutin (FK1TMD) by threading the FK1TMD sequence to an idealized α-helix using Modeller 9v7 (). The resulting helix was evaluated for stereochemical integrity using Procheck (). This model was used for all of the atomistic simulations and also for creating the coarse-grained version of FK1TMD. [...] Atomistic simulations were performed using GROMACS 4.0.7 (www.gromacs.org) (−). The simulations used an extended united atom version of the GROMOS96 force field (). Berger parameters were used for phospholipids, as described in ref (). Water molecules were treated explicitly using the SPC water model (). Sodium and chloride ions were used to neutralize the charge of the simulation systems. All lipid and protein bonds were constrained using the LINCS algorithm (), and water molecules were constrained using the SETTLE algorithm (), allowing a time step of 2 fs to be used. The velocity rescaling thermostat, with a time constant for coupling of 0.1 ps, was used to maintain the system temperature (). The Berendsen barostat, with a time constant of 1.0 ps, was used to maintain the system pressure at 1.0 bar (). Electrostatic interactions used a cutoff of 1.0 nm, with interactions beyond this cutoff treated using the smooth particle mesh Ewald (PME) method (). The van der Waals interactions also used a cutoff of 1.0 nm with a long-range dispersion correction applied for the energy and pressure. The neighbor list was updated every 10 steps. The system components of each simulation are summarized in Table I of the . All simulations were run for 50 ns. The conformational properties of FK1TMD were analyzed using GROMACS tools and DSSP (). Visualization was conducted with VMD (). [...] All CG simulations were performed using GROMACS 4.0.7 (www.gromacs.org) (−) with the MARTINI CG force fields. All simulations involved self-assembly of a lipid bilayer in the presence of one FK1TMD molecule from a random configuration of protein, lipids, ions, and water as described in refs (−). The CG parameters for DPPC, DLPC, POPC, ions, and water molecules are given in ref (). The parameters for the 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol 4,5-bisphosphate (PIP2) lipid headgroup were derived through matching the bond and angle distributions from a CG inositol 1,4,5-trisphosphate (IP3) () simulation to atomistic simulations of IP3 performed using the GROMOS carbohydrate parameters (). The remainder of the PIP2 lipid parameters were the same as those used for the CG DPPC lipid. Parameters for amino acids are given in refs (), (), and (). The integrity of the FK1TMD helix was retained by implementing an elastic network model as described in ref (). For simulations of FK1TMD in the more complex Golgi Apparatus membrane, we used the MARTINI2.0 force field because of the greater range of lipid models available (). The complex GA membrane was composed of 50% PC, 20% phosphatidylethanolamine (PE), 12% PIP2, 8% phosphatidylserine (PS), and 10% sphingomyelin (SM). All phospholipids had palmitoyl fatty acid tails in both the sn-1 and sn-2 positions of the glycerol backbone. The ratio of phospholipid to cholesterol was 16:1 (0.16). This composition is similar to that of the GA (). As with the atomistic simulations, analyses of the CG simulations were performed using GROMACS tools and locally written code, and visualization was conducted with VMD (). […]

Pipeline specifications

Software tools MODELLER, PROCHECK, GROMACS, P-LINCS, VMD, MARTINI
Application Protein structure analysis
Chemicals Phosphatidylcholines