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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


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;