Team:INSA-Lyon/molecular

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.

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