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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 such a 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 likely to take floating conformation instead of folding itself around CsgA.

For a numerical molecular model, what is needed before anything else is the program that will be used. As far as we were concerned, we chose to use Sybyl-X, which we began to use this year, and offers a number of tools as well as powerful calculation algorithm. Then we had to find a file containing the structure of CsgA. Indeed, reproducing it from scratch only from its amino acid sequence would be close to impossible since what the program allows us to do is only theoretical, it cannot know the right conformation of a complex protein just like that. Thus you have to whether build the protein yourself, by specifying the sequence, angles and distances between amino acids, which will almost certainly result in failure for a protein as complex as CsgA; or you can provide the program with a pdb file, which is a file that gives the spatial coordinates of every atom in the molecule. However, curli are extremely stable and hard to characterize and we weren't able to find any such file in the banks we searched, be it the protein data bank, uniprot, and many others. We managed to get a pdb file of the CsgA protein only thanks to the generosity of professor M. Chapman from the University of Michigan who sent us his work and to whom we are really grateful.image CsgA

To work on the protein, we mainly used two functions of Sybyl-X :

1. the minimize option, that searches for the minimum steric hindrance of the protein;
2. what called a "dynamic", that is a simulation of the behavior of the protein under a set of conditions such as a magnetic force field (that can represent the effect of water for instance).

These tools were used once the His-tag and/or nickel ions were added, showing the behavior of the protein with these new elements around it.
However, keep in mind that when adding new atoms or amino acid, their position is totally arbitrary, they can be put wherever and however we want aroud the protein. It means that what is observed once is far from being enough to conclude, since it is very possible that the result would be different by placing them even a slight bit differently. Thus we had to run a lot of simulations and analyse and compare their results before we could conclude. And since both minimisation and dynamics take a lot of time and we could only run one simulation at a time, the whole study took a really long time.

The first step of the study was to determine the behavior of the CsgA protein once tagged with a poly-His peptid. Hence we ran several simulations as described above with both His1-tag and His2-tag, in C-terminus position but also inserted in a loop between two beta strands, to see if it had any kind of impact on the molecular structure or if inserting the tag in the middle of the sequence may cause a problem on the structural level. Although the parts had already been conceived with the tags in C-terminus, it was interesting to be able to make the comparison for future considerations.

The first observation we can make is that two main conformations of the tags came out of the simulations : it could either fold along the side of Csga image accolée ending with its extremity toward the N-terminus side of the protein or, on the contrary, remain "floating" away from the protein like a flag image flottante. For His2-tag, we were worried that when the first motif folded against CsgA the second motif might end up blocking the side of the protein supposedly involved in the CsgA polymerisation according to litterature. However, in none of the simulation did it happen, the second motif always ended up folding in another direction.

However aside from this there was no way to discriminate which conformation was more likely than the other. To validate one model or the other, we could only rely on two hypotheses:

1. since its end get near the polymerisation site, the folded conformation may hinder a little the curli formation, hence a comparison of curli production between tagged and wildtype-producing E.coli may give a hint to the right conformation;
2. according to litterature publi modmol, 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.

The results of the wetlab however, showed no particular decrease in curli formation between tagged and wildtype-producing E.colilien vers les résultats biofilms, and added to this, the nickel chelation seemed greater with the tags than without it, although the difference was hard to catch since CsgA wildtype is already able to chelate nickel lien vers nichelation.

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