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| − | ===BARBIE1 molecular dynamics===
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| − | <p>Following on, with a deeper understanding of BARBIE1 protein, it is now possible to study its interaction with plastic molecules. As a proof of concept of the system, only the polystyrene (PS) is analyzed in the experiments, as in other parts of the project. To better comprehend the binding between BARBIE1 with PS, we used Charmm-GUI to create the solution system for running on Gromacs.</p>
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| − | <p>Considering that building a microplastic particle is a very expensive task in computation terms, we used a plastic molecule instead. In most of the tests, the PS molecule was built with 10 repeating units, as shown in Figure X+7. For this kind of plastic in specific, there are three principal taticities formation, that is the extent to where the functional group is.
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| − | </p>
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| − | <p>For this particular case, polystyrene is composed of a hydrocarbon chain with phenyl groups, making it possible the creation of the particular taticites: atactic, syndiotactic, and isotactic. Since atactic is the only important for commercial purposes, as consequence the majority of PS microplastics must be atactic, which justified the creation of the ligand as atactic.
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| − | </p>
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| − | https://static.igem.wiki/teams/5396/registry/ps.png
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| − | <p style="font-size: 11px;"><b>Figure X+7.</b> On the left, it is represented a single molecule of styrene. On the right, it is represented a molecule with 10 repeating units.
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| − | </p>
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| − | <p>The created system simulated the one used in the wet lab experiment, which is in ambient temperature (300 K), neutral pH (pH=7), and 0.15 mM as salt concentration of NaCl. As represented in Figure X+8, the system is colored with its solvent as blue, BARBIE1 as magenta, the styrene molecule in gray and white, and the ions in green. The system configuration is setted with periodic boundary conditions (PBCs) with an edge distance of 15 Å.
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| − | </p>
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| − | https://static.igem.wiki/teams/5396/registry/barbie-ps.png
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| − | <p style="font-size: 11px;"><b>Figure X+8.</b> Representation of the solvated system of BARBIE1 with polystyrene.
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| − | </p>
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| − | <p>Aiming to keep on the comparison between BARBIE1 and BaCBM2, another similar system was generated for BaCBM2. It is fundamental to note that since calculating MDs for large molecules, such as proteins, it is not viable using Quantum Mechanics. For this reason, we are not able to check the actual bind between protein and ligand, but it is possible to check its energies and behavior through classical dynamics.</p>
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| − | <p>With the calculated systems, it was notable how rapidly both proteins get close to the plastic as a result of their affinity for them. For quantifying this proximity, we calculated the minimum distance between plastic and protein for each simulation, as shown in Figure X+9. It is relevant to realize that the peak generated in BaCBM2 simulation is given by a periodic boundary condition - i.e. the plastic is getting close to the borders and traveling to the other side of the box.
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| − | </p>
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| − | https://static.igem.wiki/teams/5396/registry/minimum-distances.png
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| − | <p style="font-size: 11px;"><b>Figure X+9.</b> Minimum distances between each protein and the polystyrene molecule.
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| − | </p>
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| − | <p>As it is possible to see in the lower part of the graph, the proximity between each component of the simulations are very stable, with a minimum distance average of 2.17 Å for BARBIE1. This is a very interesting result, since a hydrogen bond has an average distance between 2.6 and 3.3 Å, which is very relevant for understanding the interaction type we expect.</p>
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| − | <p>A possible complementary analysis to the minimum distance is the root mean square deviation, which calculates the deviation trajectory through the simulation. Essentially, it allows us to understand a molecule or atom's stability through time, which can be useful for protein-ligand interaction. In Figure X+10, we calculated the RMSD for polystyrene molecule.</p>
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| − | https://static.igem.wiki/teams/5396/registry/ps-trajectory.png
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| − | <p style="font-size: 11px;"><b>Figure X+10.</b> Root mean square deviation for polystyrene trajectory.
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| − | </p>
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| − | <p></p>
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| − | <p></p>
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| | <span class='h3bb'>Sequence and Features</span> | | <span class='h3bb'>Sequence and Features</span> |
BARBIE1 is a synthetic protein derived from BaCBM2 through a process of reverse engineering. It has the increased ability to bind to plastics when compared to BaCBM2.
The BARBIE1 protein is fused with the red fluorescent protein (RFP)[ ], which exhibits an excitation maximum at 558 nm and an emission maximum at 583 nm. This fusion enhances the visualization of BARBIE1 by fluorescence-based methods.
