Team:INSA-Lyon/Molecular
From 2014.igem.org
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- | The results of the wetlab however, showed no particular decrease in curli formation between tagged and wildtype-producing <i>E.coli</i | + | The results of the wetlab however, showed <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu1">no particular decrease</a> in curli formation between tagged and wildtype-producing <i>E.coli</i>, and added to this, the nickel chelation seemed greater with the tags than without it, although the difference was hard to catch since <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu2">CsgA wildtype is already able to chelate nickel </a>. This means that <b>the floating conformation seems more likely to happen <i>in vivo</i> than the folded one</b>. |
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- | The simulations with a tag on a loop between two beta strands showed a tendency to <b>slighltly modify the structure of the beta sheets</b> around the tag | + | The simulations with a tag on a loop between two beta strands showed a tendency to <b>slighltly modify the structure of the beta sheets</b> around the tag, but we cannot say whether it would be enough to influence any of the curli properties. Moreover, it seems like modeling the behavior of beta-sheets is a really complex task that many molecular simulation programs do not guarantee to achieve perfectly. Thus the effects of the tag on the general structure of the protein are still questionable.</br></br></p> |
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Revision as of 17:55, 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;