Difference between revisions of "Team:NU Kazakhstan/Model"

 
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<h1 style="color: #fff; border-bottom: none; font-weight: bold!important">Modelling</h1>
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<p>We have first started by looking at similar proteins in the Databank. We started with a Blast Search against all PDB proteins. We found two main homologs. The Sulfide: Quinone Reductase of <i><font color="black">Acidothiobacillus ferrooxidans</font></i> and that of the hyperthermophile <i><font color="black">Aquifex Aeolicus</font></i>. </p>
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<img src="https://static.igem.org/mediawiki/2018/a/ad/T--NU_Kazakhstan--modelling.jpg" class="img-fluid"><br>
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<center>Figure 1. Blast search</center>
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We have then used MODELLER Software to align the protein from <i><font color="black">A. Aeolicus</font></i> as the template to our target sequence from <i><font color="black">Leptolyngbya Hensonii</font></i>. (please see data in the supporting information). Upon comparing the DOPE (Discrete Optimized Protein Energy) profiles, we noticed that there are regions of clear discrepancies. We have chosen the best five models to compare, this is the comparison for the best model we have.
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<center><img src="https://static.igem.org/mediawiki/2018/d/d9/T--NU_Kazakhstan--modelling2.png" class="img-fluid"><br>Figure 2. <i><font color="black">A. Aeolicus</font></i> and <i><font color="black">L. Hensonii</font></i> comparison of DOPE profiles.</center><br>
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The energy of the model is a lot higher especially in the region between 200 and 250. This region is not far from the FAD-binding site (the site of electron transfer). Moreover, the sequence responsible for anchoring the <i><font color="black">A. Aeolicus</font></i> into the membrane is very different in our case. Based on the homology model, there should be a turn in this region of 376 to 412. However, the turn is incorrectly predicted as there is no PROLINE GLY sequence found in our target protein. Instead, a VAL GLY TRP sequence is found. However, sites for the binding of S<sub><font color="black">8</font></sub> and the binding of Quinone are the same. </p><br>
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<center><img src="https://static.igem.org/mediawiki/2018/f/fc/T--NU_Kazakhstan--modelling3.png" class="img-fluid"><br>Figure 3. A turn in a sequence responsible for anchoring the <i><font color="black">A. Aeoulicus</font></i> into the membrane.</center><br>
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<p>We have then decided to try the use of <i><font color="black">A. ferrooxidans</font></i> SQR as a template. While both <i><font color="black">A. Aeolicus</font></i> and <i><font color="black">A. ferrooxidans</font></i> share an identity of 42%. In the case of <i><font color="black">A. ferrooxidans</font></i>, the coverage of the query is 5 percent higher, leading us to believe that the template might be better. The DOPE profiles are compared below.
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<center><img src="https://static.igem.org/mediawiki/2018/2/20/T--NU_Kazakhstan--modelling4.png" class="img-fluid"><br>
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Figure 4. <i><font color="black">A. Aeolicus</font></i> and <i><font color="black">A. ferrooxidans</font></i> comparison of DOPE profiles.</center><br>
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<p>A first glance shows that the comparison is better. However, regions between 210 to 300 are still problematic. As in the case with the <i><font color="black">A. Aeolicus</font></i>, the CYS disulfide bridges appear not conserved between the models and the template. A very important note is that our sequence does not share the C-terminal sequences known for anchoring the SQR of <i><font color="black">A. Aeolicus</font></i> and <i><font color="black">A. ferrooxidans</font></i> into the membrane. It is possible that our target SQR associates with the membrane through another protein. In that case, we have decided to add an anchoring sequence to the protein for expression in Cyanobacteria. As with the <i><font color="black">A. Aeolicus</font></i> we were able to find the Cysteine residues responsible for electron transport in the active site. The three Cysteine residues responsible for electron transport are CYS 127, CYS 159, and CYS 345. </p>
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<center><img src="https://static.igem.org/mediawiki/2018/5/51/T--NU_Kazakhstan--modelling5.png" class="img-fluid"><br>Figure 5. Cysteine residues responsible for electron transport in the active site of <i><font color="black">A. Aeolicus</font></i>.</center><br>
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<p>In <i><font color="black">A. ferrooxidans</font></i>, the amino equivalent amino acids are CYS 128, CYS 160, CYS 356.</p>
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<center><img src="https://static.igem.org/mediawiki/2018/4/41/T--NU_Kazakhstan--modelling6.png" class="img-fluid"><br>
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Figure 6. Cysteine residues responsible for electron transport in the active site of <i><font color="black">A. ferrooxidans</font></i>
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<p><i><font color="black">A. ferrooxidans</font></i> SQR showing the Pentasulfur intermediate in the active site homologous to that of SQR from <i><font color="black">L. Hensonii</font></i> [1].
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Unlike <i><font color="black">A. Aeolicus</font></i>, there are no disulfide bridges in neither SQR of <i><font color="black">A. ferrooxidans</font></i> nor in SQR of <i><font color="black">L. Hensonii</font></i>. That is expected as disulfide bridges in <i><font color="black">A. Aeolicus</font></i> are associated with a hyperthermophilic activity of SQR in that species [2].</p><center>
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<div class="col-md-12"><img src="https://static.igem.org/mediawiki/2018/7/77/T--NU_Kazakhstan--togeth0er.png" class="img-fluid"><br><p>Figure 7. Protein structure of SQR from L. hensonii.
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<p>The image was generated using Visual Molecular Dynamics (VMD) software using the Internal Rendering Tachyon algorithm of the whole protein structure of SQR from L. hensonii based on the template from A. ferrooxidans.
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<h1> Modeling</h1>
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<p>Mathematical models and computer simulations provide a great way to describe the function and operation of BioBrick Parts and Devices. Synthetic Biology is an engineering discipline, and part of engineering is simulation and modeling to determine the behavior of your design before you build it. Designing and simulating can be iterated many times in a computer before moving to the lab. This award is for teams who build a model of their system and use it to inform system design or simulate expected behavior in conjunction with experiments in the wetlab.</p>
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<b>References</b><br>
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<p>1. &nbsp; Cherney, M. M., Zhang, Y., Solomonson, M., Weiner, J. H., & James, M. N. (2010). Crystal structure of sulfide: quinone oxidoreductase from Acidithiobacillus ferrooxidans: insights into sulfidotrophic respiration and detoxification. Journal of molecular biology, 398(2), 292-305.</p>
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<p>2. &nbsp; Marcia, M., Ermler, U., Peng, G., & Michel, H. (2009). The structure of Aquifex aeolicus sulfide: quinone oxidoreductase, a basis to understand sulfide detoxification and respiration. Proceedings of the national academy of sciences, 106(24), 9625-9630.</p>
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<h3> Gold Medal Criterion #3</h3>
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Convince the judges that your project's design and/or implementation is based on insight you have gained from modeling. This could be either a new model you develop or the implementation of a model from a previous team. You must thoroughly document your model's contribution to your project on your team's wiki, including assumptions, relevant data, model results, and a clear explanation of your model that anyone can understand.
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The model should impact your project design in a meaningful way. Modeling may include, but is not limited to, deterministic, exploratory, molecular dynamic, and stochastic models. Teams may also explore the physical modeling of a single component within a system or utilize mathematical modeling for predicting function of a more complex device.
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SCHOOL OF SCIENCE AND TECHNOLOGY <br> Nazarbayev University <br> Astana, Kazakhstan
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<h3> Inspiration </h3>
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<li><a href="https://2016.igem.org/Team:Manchester/Model">2016 Manchester</a></li>
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<li><a href="https://2016.igem.org/Team:TU_Delft/Model">2016 TU Delft</li>
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Latest revision as of 01:02, 18 October 2018

Bioremediation of Sour Crude Oil Waste using Cyanobacteria




We have first started by looking at similar proteins in the Databank. We started with a Blast Search against all PDB proteins. We found two main homologs. The Sulfide: Quinone Reductase of Acidothiobacillus ferrooxidans and that of the hyperthermophile Aquifex Aeolicus.



Figure 1. Blast search

We have then used MODELLER Software to align the protein from A. Aeolicus as the template to our target sequence from Leptolyngbya Hensonii. (please see data in the supporting information). Upon comparing the DOPE (Discrete Optimized Protein Energy) profiles, we noticed that there are regions of clear discrepancies. We have chosen the best five models to compare, this is the comparison for the best model we have.



Figure 2. A. Aeolicus and L. Hensonii comparison of DOPE profiles.

The energy of the model is a lot higher especially in the region between 200 and 250. This region is not far from the FAD-binding site (the site of electron transfer). Moreover, the sequence responsible for anchoring the A. Aeolicus into the membrane is very different in our case. Based on the homology model, there should be a turn in this region of 376 to 412. However, the turn is incorrectly predicted as there is no PROLINE GLY sequence found in our target protein. Instead, a VAL GLY TRP sequence is found. However, sites for the binding of S8 and the binding of Quinone are the same.



Figure 3. A turn in a sequence responsible for anchoring the A. Aeoulicus into the membrane.

We have then decided to try the use of A. ferrooxidans SQR as a template. While both A. Aeolicus and A. ferrooxidans share an identity of 42%. In the case of A. ferrooxidans, the coverage of the query is 5 percent higher, leading us to believe that the template might be better. The DOPE profiles are compared below.



Figure 4. A. Aeolicus and A. ferrooxidans comparison of DOPE profiles.

A first glance shows that the comparison is better. However, regions between 210 to 300 are still problematic. As in the case with the A. Aeolicus, the CYS disulfide bridges appear not conserved between the models and the template. A very important note is that our sequence does not share the C-terminal sequences known for anchoring the SQR of A. Aeolicus and A. ferrooxidans into the membrane. It is possible that our target SQR associates with the membrane through another protein. In that case, we have decided to add an anchoring sequence to the protein for expression in Cyanobacteria. As with the A. Aeolicus we were able to find the Cysteine residues responsible for electron transport in the active site. The three Cysteine residues responsible for electron transport are CYS 127, CYS 159, and CYS 345.



Figure 5. Cysteine residues responsible for electron transport in the active site of A. Aeolicus.

In A. ferrooxidans, the amino equivalent amino acids are CYS 128, CYS 160, CYS 356.


Figure 6. Cysteine residues responsible for electron transport in the active site of A. ferrooxidans

A. ferrooxidans SQR showing the Pentasulfur intermediate in the active site homologous to that of SQR from L. Hensonii [1]. Unlike A. Aeolicus, there are no disulfide bridges in neither SQR of A. ferrooxidans nor in SQR of L. Hensonii. That is expected as disulfide bridges in A. Aeolicus are associated with a hyperthermophilic activity of SQR in that species [2].



Figure 7. Protein structure of SQR from L. hensonii.


The image was generated using Visual Molecular Dynamics (VMD) software using the Internal Rendering Tachyon algorithm of the whole protein structure of SQR from L. hensonii based on the template from A. ferrooxidans.

References

1.   Cherney, M. M., Zhang, Y., Solomonson, M., Weiner, J. H., & James, M. N. (2010). Crystal structure of sulfide: quinone oxidoreductase from Acidithiobacillus ferrooxidans: insights into sulfidotrophic respiration and detoxification. Journal of molecular biology, 398(2), 292-305.

2.   Marcia, M., Ermler, U., Peng, G., & Michel, H. (2009). The structure of Aquifex aeolicus sulfide: quinone oxidoreductase, a basis to understand sulfide detoxification and respiration. Proceedings of the national academy of sciences, 106(24), 9625-9630.

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