Team:INSA-Lyon/Molecular

From 2014.igem.org

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We then discussed over our results with the wetlab members to define a way to confirm the accuracy of our model, and so we were able to assess that, in accordance with litterature, the <b>best position</b> for the tag was by the <b>C-terminus</b> of the protein. We also determined that the His-tag was more likely to take a <b>floating conformation</b> instead of folding itself around CsgA.  
We then discussed over our results with the wetlab members to define a way to confirm the accuracy of our model, and so we were able to assess that, in accordance with litterature, the <b>best position</b> for the tag was by the <b>C-terminus</b> of the protein. We also determined that the His-tag was more likely to take a <b>floating conformation</b> instead of folding itself around CsgA.  
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As for the <b>floating conformation</b> however, we managed to verify a property found in the litterature <font color="red">publi modmol</font>, 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 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"> 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 nickel ions for the His1-tag, and 8 for the His2-tag.</br></br></p>
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<i>"Then what is the point of tagging the original protein when it chelates so much more than the tag itself?"</i> are you going to ask me. The answer is quite simple : just for one protein, with only one His1-tag we can <b>increase its chelation power by 25%</b>. Now remember that CsgA is only the subunit of a fiber that is composed of thousands of them; that one bacterium bear 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 prefered one, but as the wetlab results <font color="green">liens vers résultats ICP</font> 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 chelates so much more than the tag itself?"</i> are you going to ask me. The answer is quite simple : just for one protein, with only one His1-tag we can <b>increase its chelation power by 25%</b>. Now remember that CsgA is only the subunit of a fiber that is composed of thousands of them; that one bacterium bear 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 prefered one, but as the <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu2">wetlab 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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Revision as of 17:51, 17 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 it's 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 wetlab members to define a way to confirm the accuracy of our model, and so we were able to assess that, in accordance with litterature, the best position for the tag was by the C-terminus 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 for the tags there exist 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%!

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 to conduct;
  • find out just how many tags can be added without altering the protein properties of adherence and polymerisation;