Team:INSA-Lyon/Molecular

From 2014.igem.org

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<li><a href="#methods" onclick="$('#methods').slideToggle('slow')"><h1 align="left">Methods</h1></a><hr/></li>
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<li><a href="#methods" onclick="$('#methods').slideToggle('slow')"><h1 align="left"><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Methods</h1></a><hr/></li>
      
      
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<li><a href="#csgaEngineering" onclick="$('#csgaEngineering').slideToggle('slow')"><h1 align="left">CsgA Engineering</h1></a><hr/></li>
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<li><a href="#csgaEngineering" onclick="$('#csgaEngineering').slideToggle('slow')"><h1 align="left"><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />CsgA Engineering</h1></a><hr/></li>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/2/2b/CurlyonFoldedHis1.jpg" alt="floating His1" height="300px"/></div></td>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/b/be/CurlyonFloatingHis1.jpg" alt="folded His1" height="250px"/></div></td>
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<li><a href="#nichelation" onclick="$('#nichelation').slideToggle('slow')"><h1 align="left">Ni-Chelation</h1></a><hr/></li>
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<li><a href="#nichelation" onclick="$('#nichelation').slideToggle('slow')"><h1 align="left"><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Ni-Chelation</h1></a><hr/></li>
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After the structural changes brought by the tags we tried to determine the chelating activity of the tagged CsgA. So we simply added nickel ions in the neighborhood of the tags and let the minimisations and dynamics do their magic.</br></br>
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After the structural changes brought by the tags we tried to determine the chelating activity of the tagged CsgA. So we simply added nickel ions in the neighborhood of the tags and let the minimizations and dynamics do their magic.</br></br>
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The very first result we are going to talk about is that even without an His-tag, <b>the wildtype protein is able to chelate nickel</b>. That is something we didn't expect and were surprised to discover. We put some nickels next to CsgA (in a 10 Angstroms radius), and Sybyl computed the simulations (with AMBER FF002 library, cutoff at 12 Ang, during 10 to 20 000 fs). Nickel can be captured by either a <b>group of ketones</b> that are close to each other, but even more surprising, we observed histidines, aspartic acids and other amino acids already present in wildtype CsgA were "grabbing" the ions and litterally pulling them  between the beta strands <b>into the center of the protein in some cases</b>.</br></br></p>
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The very first result we are going to talk about is that even without an His-tag, <b>the wildtype protein is able to chelate nickel</b>. That is something we didn't expect and were surprised to discover. We put some nickels next to CsgA (in a 10 Angstroms radius), and Sybyl computed the simulations (with AMBER FF002 library, cutoff at 12 Ang, during 10 to 20 000 fs). Nickel can be captured by either a <b>group of ketones</b> that are close to each other, but even more surprising, we observed histidines, aspartic acids and other amino acids already present in wildtype CsgA were "grabbing" the ions and literally pulling them  between the beta strands <b>into the center of the protein in some cases</b>.</br></br></p>
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A far as the <b>folded conformation</b> is concerned, both His1-tag or His2-tag, cover already existing chelating sites of CsgA, but are still able to chelate nickel ions. The simulations showed that, though the tag could also fold a little to catch a nickel with two of its histidines, a bond between the nickel and both the tag AND CsgA often occured, thus stabilising the folded structure of the tag. However, with its reach decreased and the covering of the initial protein chelating sites, we concluded that such a conformation wouldn't bring much more chelating power to the protein.</br></br></p>
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A far as the <b>folded conformation</b> is concerned, both His1-tag or His2-tag, cover already existing chelating sites of CsgA, but are still able to chelate nickel ions. The simulations showed that, though the tag could also fold a little to catch a nickel with two of its histidines, a bond between the nickel and both the tag AND CsgA often occurred, thus stabilizing the folded structure of the tag. However, with its reach decreased and the covering of the initial protein chelating sites, we concluded that such a conformation wouldn't bring much more chelating power to the protein.</br></br></p>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/6/63/CurlyonHis1accNi.jpg" alt="folded His1 with Ni" width="300px"/></div></td>
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As for the <b>floating conformation</b> however, we managed to verify a property found in the <a href="http://www.tandfonline.com/doi/abs/10.1080/07391102.2003.10506903?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#.VEFESBZ-Tw0"> litterature</a>, which is that on a poly-Histidine tag, a nickel ion is chelated by the unprotoned nitrogen of the cycle of two histidines that are separated by a third  histidine. We also observed that through the folding of the tag around nickel ions so that histidines may chelate it, ketones groups were brought together, earning the ability to chelate an ion as well, and that the carboxyl group of its C-end was also able to chelate an ion. All in all, we determined that a floating tag should be able to chelate up to 5 extra nickel ions for the His1-tag, and 8 for the His2-tag.</br></br></p>
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As for the <b>floating conformation</b> however, we managed to verify a property found in the <a href="http://www.tandfonline.com/doi/abs/10.1080/07391102.2003.10506903?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#.VEFESBZ-Tw0"> literature</a>, which is that on a poly-Histidine tag, a nickel ion is chelated by the unprotoned nitrogen of the cycle of two histidines that are separated by a third  histidine. We also observed that through the folding of the tag around nickel ions so that histidines may chelate it, ketones groups were brought together, earning the ability to chelate an ion as well, and that the carboxyl group of its C-end was also able to chelate an ion. All in all, we determined that a floating tag should be able to chelate up to 5 extra nickel ions for the His1-tag, and 8 for the His2-tag.</br></br></p>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/9/98/CurlyonHis1floNi.jpg" alt="floating His1 with Ni" width="300px"/></div></td>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/9/9d/CurlyonHis2floNi.jpg" alt="floating His2 with Ni" height="250px"/></div></td>
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<p> For better understanding of the folding process, here's an outline of the chelation of a nickel ion (in the center) by a tag composed of three histidines.</br>
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<div align="center"><img src="https://static.igem.org/mediawiki/2014/a/a5/CurlyonHistag.png" alt="histag" height="300px"/></div></p>
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<i>"Then what is the point of tagging the original protein when it already chelates so much?"</i> are you going to ask me. The answer is quite simple : just for one protein, with only one His1-tag we can hope for a <b>25 % increase of its chelation power</b>. Now remember that CsgA is only the subunit of a fiber that is composed of thousands of them; that one bacterium bears hundreds, maybe thousands of these fibers; and that a culture of such bacteria allows for a countless number of bacteria as well. It's easy to understand how an increase of 25% of efficiency of the elementary unit of the fiber is already quite something! Well, that's provided the floating conformation is the preferred one, but as the <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu2">wet lab results</a> seem to prove that His2-tagged CsgA chelates better than His1-tagged CsgA, that already chelates more than wildtype CsgA, we can be confident in saying that <b>the floating conformation is actually the most present</b> in the fiber. However, as we are still unable to accurately quantify curli and CsgA, we unfortunately cannot provide any information regarding the difference between the theoretical chelating power of CsgA and the experimentally determined quantity of captured nickel.
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<i>"Then what is the point of tagging the original protein when it already chelates so much?"</i> are you going to ask me. The answer is quite simple : just for one protein, with only one His1-tag we can hope for a <b>25 % increase of its chelation power</b>. Now remember that CsgA is only the subunit of a fiber that is composed of thousands of them; that one bacterium bears hundreds, maybe thousands of these fibers; and that a culture of such bacteria allows for a countless number of bacteria as well. It's easy to understand how an increase of 25% of efficiency of the elementary unit of the fiber is already quite something! Well, that's provided the floating conformation is the preferred one. But since the <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu2">wet lab results</a> seem to prove that His2-tagged CsgA chelates better than His1-tagged CsgA, which chelates as much as wildtype CsgA, from this <b>we cannot really discriminate</b> between one conformation or the other from our experimental data, though these results seem to lead towards the folded conformation, where the second motif would be floating for His2-tag. Moreover, as we are still unable to accurately quantify curli and CsgA, we unfortunately cannot provide any information regarding the difference between the theoretical chelating power of CsgA and the experimentally determined quantity of captured nickel.
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Through our molecular study of a CsgA protein engineered with either <b>His1-tag</b> or <b>His2-tag</b>, we came to the conclusion that since it has a longer reach and its mobility makes it more available for chelation, using a tag positioned by the <b>C-terminus</b> of the protein <b>is more relevant than</b> placing it <b>in the middle of the sequence</b>, although doing so may provide a little more chelation power as long as the tag isn't too long.</br>
Through our molecular study of a CsgA protein engineered with either <b>His1-tag</b> or <b>His2-tag</b>, we came to the conclusion that since it has a longer reach and its mobility makes it more available for chelation, using a tag positioned by the <b>C-terminus</b> of the protein <b>is more relevant than</b> placing it <b>in the middle of the sequence</b>, although doing so may provide a little more chelation power as long as the tag isn't too long.</br>
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We also showed that there are  <b>two possible conformations</b> : one is <b>folded on the side of CsgA</b> and <i>a priori</i> does not increase the already-existing chelating power of CsgA; the other is a <b>"floating" conformation</b> where the tag is not attracted to the protein and is able to <b>improve its chelating power by up to 25%</b>!
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We also showed that there are  <b>two possible conformations</b> : one is <b>folded on the side of CsgA</b> and <i>a priori</i> does not increase the already-existing chelating power of CsgA; the other is a <b>"floating" conformation</b> where the tag is not attracted to the protein and is able to <b>improve its chelating power by up to 25%</b>! Unfortunately, the wetlab results are not sufficient to discriminate which conformation is more present <i>in vivo</i>.
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Latest revision as of 03:20, 18 October 2014

Curly'on - IGEM 2014 INSA-LYON

One of the main goals of our modeling work this year was to understand the structure of the curlin subunit protein, CsgA and its behavior when engineered with a tag constituted of either six histidines (that we will call His1-tag from now on) or twice that motif (His2-tag), since this peptide is known for its nickel chelation properties.
We then discussed over our results with the wet lab members to define a way to confirm the accuracy of our model, and so we were able to assess that, in accordance with literature, the best position for the tag was by the C-terminus end of the protein. We also determined that the His-tag was more likely to take a floating conformation instead of folding itself around CsgA.


  • Methods


  • CsgA Engineering


  • Ni-Chelation




Conclusion

Overall sum up

Through our molecular study of a CsgA protein engineered with either His1-tag or His2-tag, we came to the conclusion that since it has a longer reach and its mobility makes it more available for chelation, using a tag positioned by the C-terminus of the protein is more relevant than placing it in the middle of the sequence, although doing so may provide a little more chelation power as long as the tag isn't too long.
We also showed that there are two possible conformations : one is folded on the side of CsgA and a priori does not increase the already-existing chelating power of CsgA; the other is a "floating" conformation where the tag is not attracted to the protein and is able to improve its chelating power by up to 25%! Unfortunately, the wetlab results are not sufficient to discriminate which conformation is more present in vivo.

What we couldn't achieve

Unfortunately, having very little time and people, there are a few things we couldn't investigate as extensively as we wanted. Here are a few of those things:

  • more simulations with His2-tag. Since they took an awful lot of time, we only ran a handful of them;
  • modelisation of the docking between two CsgA proteins, and the influence of the His-tags, that our lack of experience prevented us from conducting;
  • find out just how many tags can be added without altering the protein properties of adherence and polymerization;