This part was used as template to construct BBa_K5396004.
This part was used as template to construct BBa_K5396004.
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===Protein Design===
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<p>Starting from the BaCBM2 structure model generated by the AlphaFold2 software, we performed docking assays with six types of plastic: polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET), nylon (NY), polyvinyl chloride (PVC) and polystirene (PS). We made the docking using Gnina software with relaxed parameters to screen many proteins and features for plastic affinity, which was calculated as shown below, where P stands for Protein and L for Ligand </p>
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<p>PL \xrightarrow{} P + L \quad \xrightarrow{} \quad K_D = \frac{[P][L]}{[PL]} </p>
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<p>
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Thereafter, the produced overlaps were removed by the docking assays using the ChimeraX software, as well as used for visualization and sequence manipulation. A reverse folding was then performed with the protein output from the docking using the LigandMPNN tool. The original protein set generated from the docking was filtered to maintain just unique positions, considering the associated score , without overlap between them. </p>
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<p>
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By doing that, 6.000 sequences were generated for each ligand, totalizing 6 plastics x 6.000 sequences = 36.000 sequences, as illustrated in Figure 1. The consensus sequence from the 36000 sequence originated our most optimized protein sequence sensitive for several plastics types was named as BARBIE1! </p>
<p style="font-size: 11px;"><b>Figure 1.</b> Protein-ligand docking representation of the BARBIE1 protein docked with PP, PE, PET, NY, PVC.</p>
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<p>The BARBIE1 protein was redocked with the same plastics as before, once more using Gnina software. The result comparing its affinity with BaCBM2 in silico assays performed are described in the barplot of Figure 5. Comparing the predicted affinity between the original and the modified protein, it is notable a substantial increase for all plastics, in particular for PE, PP, and PS, highlighting the effectiveness of the processing pipeline.
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</p>
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<p>Besides the monomers tests, we also wanted to test the affinity using different sizes of plastic in order to guarantee that this could be a valuable parameter to future analysis and experiments. Therefore, the tested ligands were PE and PET plastics with 50 and 25 repeating units, respectively. As a result, the previous behavior at maintaining a higher KD for BARBIE1 when compared to BaCBM2 was preserved.</p>
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=== Computational Modeling===
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<p>Proteins with carbohydrate-binding modules (CBM) can not only be found as single units but also in more units or as part of larger multi-domain proteins. In light of the importance of understanding the thermodynamics basis for structural composition, the newest version of Alpha Fold 3 (AF3) was used [doi.org/10.1038/s41586-024-07487-w]. The model was used to optimize protein-protein interaction and test high-order oligomers. To effectively model the water filter system closer to reality, the first step was to predict the state of the proteins.</p>
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<p>The proposed proteins to be evaluated in Alpha Pulldown are the B1-CBM (pink), BaCBM2 (blue), and 1A3N (green), which is a reference protein that forms the deoxy human hemoglobin.</p>
<p style="font-size: 11px;"><b>Figure X.</b> Protein comparative of the tested proteins. In (a), it is shown the BARBIE1 structure, in (b) the BaCBM2, and in (c) the 1A3N protein.
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</p>
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<p>With 4 subunits as shown in Figure X+1, the 1A3N multicomplex protein forms a pocket in the middle region to store oxygen molecules. Since this reference protein was resolved with more subunits, it can serve as a baseline for the prediction of the other proteins.
<p style="font-size: 11px;"><b>Figure X+2.</b> pTM and iPTM values calculated by AlphaFold3 for BARBIE1, BaCBM2, and 1A3N with different repeating units.
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</p>
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<p>As it can be seen in Figure X+2, both BARBIE1, BaCBM2, and 1A3N achieved high scores as monomers. On the other hand, only the 1A3N as a multimer achieved higher values, such as its two and three subunits iPTM, as expected. Therefore, it is possible to affirm that it is very unlikely for both B1-CBM and BaCBM2 to form a multimer, which can be assured by the deoxy human hemoglobin results. This result allows the modeling of B1-CBM as a monomer, which simplifies the system.
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</p>
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=== Interaction Properties===
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<p>In order to further our knowledge in the protein properties, we compared our resulting sequence BARBIE1 with the original BaCBM2 in different tests. Firstly, we compared each protein's tertiary structure, as shown in Figure X+3. As a result, it is notable the similarity between them, specially in the secondary structures, in which is notable the presence of the same amount of beta sheets. In the upper left part of the BARBIE1 structure, however, there is a notable difference between them.
<p style="font-size: 11px;"><b>Figure X+3.</b> Tertiary structure of BARBIE1 on the left (pink) and BaCBM2 on the right (blue).
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</p>
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<p>This subtable contrast between the original and modified protein is shown in Figure X+4, in which we aligned the structures using VMD and calculated the root mean square deviation (RMSD). As it is possible to analyze in the aligned structures, it is very similar visibly, with a resulting RMSD of 0.56 Å (RMSDs < 2 Å indicates high similarity).
<p style="font-size: 11px;"><b>Figure X+4.</b> Alignment of BARBIE1 (magenta) and BaCBM2 (blue) tertiary structures resulting in a RMSD of 0.56.</p>
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<p>After that, we assessed the electrostatic surface of each protein using the ChimeraX tool, which can be valuable for identifying binding sites for ligands, as well as stability and its behavior when solvated.</p>
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<p>In Figure X+5, both proteins are represented with the electrostatic surface. While on one hand the red parts stand for the negative electrostatic, the blue parts stand for the positive electrostatic. Therefore, it is notable a major presence of negative values for BARBIE1 when compared to BaCBM2, which may indicate a higher affinity with positive ions or positive charged ligands.</p>
<p style="font-size: 11px;"><b>Figure X+5.</b> Electrostatic surface represented in the left for BARBIE1 and in the right for BaCBM2. The red regions indicate positive electrostatic and the negative are represented as blue.</p>
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<p>Following on, we calculated the proteins hydrophobicities also using ChimeraX. In this way, we can at the same time understand the water interacting regions, corresponding to the more hydrophilic parts, and possible binding sites, generally indicated by hydrophobic parts. In Figure X+6, the hydrophilic regions are represented as blue and the hydrophobic regions as yellow.</p>
<p style="font-size: 11px;"><b>Figure X+6.</b> Hydrophobicity surface represented in the left for BARBIE1 and in the right for BaCBM2. The blue regions indicate hydrophilic and the hydrophobicity is represented as yellow.</p>
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<p>In general, while on one hand hydrophilic regions are exposed to aqueous solvents in the exterior of the structure, hydrophobic regions are buried in its interior. When confronting both structures, it is notable an inverse behavior on the BARBIE1 structure compared to BaCBM2: the hydrophobic parts are not buried, but exposed to the surface.
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Since more hydrophobic sites are exposed to the surface, it may point to a better understanding of our results. The better scores were achieved by choosing more hydrophobic amino acids in the protein primary structure, which enabled the creation of more pockets and the subsequent increase of KD. Therefore, the choice is notable for a higher plastic affinity, but also a lesser water solubility.
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</p>
===BARBIE1 molecular dynamics===
===BARBIE1 molecular dynamics===
Revision as of 17:30, 20 September 2024
Barbie1_RFP_3xMad10
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.
This part was used as template to construct BBa_K5396004.
BARBIE1 molecular dynamics
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.
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.
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.
Figure X+7. On the left, it is represented a single molecule of styrene. On the right, it is represented a molecule with 10 repeating units.
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.
Figure X+8. Representation of the solvated system of BARBIE1 with polystyrene.
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.
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.
Figure X+9. Minimum distances between each protein and the polystyrene molecule.
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.
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.
Figure X+10. Root mean square deviation for polystyrene trajectory.
