Team:Manchester/fattytest

Introduction
Since the model of the fatty acid biosynthesis pathways highlighted several key enzymes that could be altered to produce palm oil, including Delta9, Delta12 and FabA, which agrees with previous publications on these enzymes. Delta9 and Delta12 are involved in linear pathways, meaning that there are obvious reactants and products, so the overexpression of them can be measured directly via LC-MS and other techniques. However, FabA is a β-Hydroxydecanoyl Thiol Ester Dehydrase involved in a cyclic pathway [1] specifically the conversion of β-hydroxy acyl-ACP to Enol acyl-ACP as part of the fatty acid biosynthetic pathway [2]. Therefore characterising any specific change due to FabA overexpression will be challenging, as any products will automatically be involved in the proceeding stage of the cycle and it would therefore be difficult to determine the effect of overexpression. An alternative to measure the overexpression of FabA, would be through the addition of His-tags to either the N-terminal and or the C-terminal of FabA. However, depending on the structure of FabA, the addition of His-tags could potentially interfere with expression, protein folding, enzymatic functions and interactions [3, 4, 5, 6 and 7]. Several studies including work by [5] and [6], respectively showed that the addition of C-Terminus His-tags to proteins could interfere with enzyme activity and alter di-sulphide and therefore protein structure. These problems are particularly applicable to FabA, as it forms a homodimer, as shown by Leesong et al., 1996 [1]. Therefore, the addition of His-tag could potentially interfere with the interaction domain and thus the formation of a homodimer, which would be consistent with several reports [4, 5 and 6]. To address this issue, we decided to perform a molecular dynamics simulation using the GROMACS software package [8] on a structure of FabA, determined by X-ray crystallography by Leesong et al., 1996 [1]. This would allow for the trajectories of the N- and C-Termini of FabA over the course of the simulation to be studied, therefore allowing us to identify which terminal would be more suited for His-tag addition.

Installing GROMACS
The Groningen Machine for Chemical Simulation (GROMACS) Version 4.5.5 [8], was installed on a MacBook Pro 2011 model, operating OS X 10.8.3, with a 4 GB 1333 MHz DDR3 memory, 2.3 GHz Intel Core i5 processor and a 320 GB SATA disk drive. Prior to installing GROMACS, “command line tools” were installed within Xcode, Version 4.6.1. GROMACS was installed to single precision, with the source file downloaded from www.gromacs.org/Downloads. In addition to GROMACS, the FFTW library, Version 3.3.3 [9] was installed, with the source file downloaded from www.fftw.org/download.html.

Behind the scenes of the Molecular Dynamics Simulations
For the simulations of FabA, a dimeric structure of FabA (PDB ID: 1MKB) derived by X-ray Diffraction, with a resolution of 2 Å from an E.Coli expression system by [1] was used. The molecular dynamics simulation was performed within the GROMACS package using version 4.5.5 [8], with the AMBER99SB force field [10], the transferrable intermolecular potential 3P (TIP3P) water model [11] and the original crystal waters, from the X-Ray Crystallography with periodic boundary conditions. The methods for generating both topologies and parameters for G16bP are as described above. For all cases of the Protein in water, a protocol derived from the “Lysozyme in Water” GROMACS tutorial (by J. Lemkul, Department of Biochemistry, Virginia Tech) and optimized for FabA was used. Briefly, a cubic cell of 2 nm in diameter with the protein centered was used and filled with the generic single point charge 216 (SPC216) water configuration [12]. The system was neutralized to a salt concentration of 150 mM by adding Na+ and Cl-. Energy Minimization (EM) was performed using the Steepest Descent Algorithm [13] with a tolerance of 1000 KJ mol-1 nm-1, to remove any steric clashes or inappropriate geometries. In the simulation, the long-range electrostatic interactions were modeled using the Particle-Mesh- Eswald (PME) method [14 and 15] and the Linear Constraint Solver (LINCS) algorithm [16] to preserve chemical bond lengths. Temperature and pressure coupling were performed independently, using a modified Berendsen thermostat [17] at constant temperature of 300 K at a time constant of 0.1 ps and the Parinello-Rahman barostat algorithm [18] at a constant pressure of 1 bar, for a time constant of 2 ps and V-rescale, respectively. The Molecular Dynamics simulations trajectories were analysed with the GROMACS analysis tools [19], to output structural molecular dynamics trajectories and PyMOL (The PyMOL Molecular Graphics System, Education-Use-Only, Version 1.3 Schrödinger, LLC) used to visualize and create both still structures and videos. All calculations of progression plots from the simulations were produced using the GROMACS analysis tools with output files viewed in the Microsoft Excel 2011.

Getting a working GROMACS simulation for FabA
To study the motions of the N- and C-Termini of FabA with molecular dynamics, we first had to get an optimized system preparation protocol, which we based on the “Lysozyme in Water” GROMACS tutorial by [20]. Firstly, we built a simulation box of 2nm around FabA, which was sufficient to satisfy the minimum image convention and the simulation cut-off schemes without adding excess solvent. FabA had to then be solvated within this box by a solvent configuration compatible with the solvent model applied to the protein, in this case the SPC216 [12], which is compatible with the TIP3 water model [11]. The system is then neutralized through the addition of ions to a molarity of 150 mM, therefore allowing the system to reach a neutral state. Our results indicate that FabA is well solvated in SPC216 water and neutralized to a molarity of 150 mM in a cubic 2 nm cell.

Figure 3. Establishing, solvating and neutralising FabA, for simulation preparation. A. The establishment of a 2nm simulation cube around the dimerised structure of FabA. B. The solvation of the FabA structure by the addition of SPC216 water molecule as a solvent. C. Neutralisation of the solvated system by the addition of Na+ and Cl- ions. Na+ and Cl- ions are represented by Blue and Green spheres, respectively and SPC216 water is represented by Cyan coloured molecules.

Once we had prepared the system, it was relaxed by energy minimization. This ensures that there are no steric clashes or inappropriate geometries that exist, by applying the steepest descent algorithm [13]. With the minimized structure, the system of solvent and ions around the protein are equilibrated to become orientated about the protein solute at the same temperature, by an isothermal-isochoric ensemble [17]. Pressure is then applied using an isobaric-isochoric ensemble, thereby ensuring that the system reaches a proper density [18]. Our energy minimized structure shows a decrease from -6.75E+05, to a maximum energy plateau for the system of -1.05 E+06 after 1238 ps of minimization time. The temperature of the system quickly reaches the target value of 300 K remaining stable, with an average temperature of 299.82 K, the equivalent of 27 °C over the 100 ps equilibration. Over the course of the 100 ps equilibration stage, both the pressure and density of the system averages 0 bar and 1015.19 Kg m-3. This is close to the experimental value of 0 bar and 1000 Kg m-3 and the equivalent of Earth’s atmospheric pressure at sea level and the density of water. The pressure fluctuations are consistent with the applied isothermal-isobaric ensemble and is suggestive of compatible molecular dynamics conditions for simulations of FabA.

Figure 4. Graphs of the Energy Minimisation, Temperature, Pressure and Density equilibration of the FabA simulation system prior to simulation.