Team:INSA-Lyon/Molecular
From 2014.igem.org
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<li> since its end gets near the polymerisation site, <b>the folded conformation may hinder</b> a little the <b>curli formation</b>, hence a comparison of curli production between tagged and wildtype-producing <i>E.coli</i> may give a hint to the right conformation; | <li> since its end gets near the polymerisation site, <b>the folded conformation may hinder</b> a little the <b>curli formation</b>, hence a comparison of curli production between tagged and wildtype-producing <i>E.coli</i> may give a hint to the right conformation; | ||
- | <li> according to litterature | + | <li> according to litterature, a His-tag chelates nickel by folding around it so that two Histidines may form a bond with one nickel. If the tag is folded against CsgA, not only would it have less "reach" than a floating one, but also it would be less able to fold over nickel ions. Hence, if the folded state is the prefered one, nickel chelation shouldn't be too high. |
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Revision as of 17:57, 17 October 2014
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.
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;