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• PROJECT

  • 
    PROJECT
  • PROJECT SUMMARY
  • ACHIEVEMENTS

• WETLAB

  • 
    WETLAB
  • WETLAB SUMMARY
  • RESULTS
  • NOTEBOOK
  • PROTOCOLS
  • DATA PAGE

• MODELING

  • 
    MODELING
  • MODELING SUMMARY
  • MOLECULAR MODELISATION
  • CURLI SYNTHESIS
•   HUMAN PRACTICE
  • 
    HUMAN
    PRACTICE
  • HUMAN PRACTICE SUMMARY
  • SYNBIO PERCEPTION
  • PROJECT'S INTEREST

• TEAM

  • 
    TEAM
  • MEMBERS & ATTRIBUTIONS
  • SPONSORS
  • PARTNERSHIPS
  • HAVE FUN WITH IGEM
  • GALLERY

MOLECULAR MODELISATION

• Methods

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• For a numerical molecular model, what is required in the first place is the program that will be used. As far as we were concerned, we chose to use Sybyl-X, which is a program we started to use this year and that offers a number of useful tools as well as powerful calculation algorithms.
  Then we had to find a file containing the structure of CsgA. Indeed, reproducing it from scratch (we only had its amino acid sequence) would be close to impossible since what the program allows us to do is only theoretical, so it cannot know the right conformation of a complex protein just like that. Thus you have two possibilities:

  - 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;

  - 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 so there aren't a lot of publications that were able to fully characterize their shape, let alone define their spatial features with certainty. Thus 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 only managed to get a pdb file of the CsgA protein 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.
  
  

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

  1. the minimize option, that searches for the minimal steric hindrance of the protein;
  2. what we 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 acids, their position is totally arbitrary. They can be put wherever and however we want around 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 the atoms slightly differently. Thus we had to run a lot of simulations, analyze and compare the results before we could conclude. And since both minimizations and dynamics take a lot of time, and we could only run one simulation at a time, the whole study spread over several weeks.

• CsgA Engineering

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• The first step of the study was to determine the behavior of the CsgA protein once tagged with a poly-His peptide. 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. With this, we could see if inserting the tag in the middle of the sequence had any kind of impact on the molecular structure causing a problem. 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.

• Results

  In C-terminus

  The first observation we can make is that two main conformations of the tags came out of the simulations : they could either fold along the side of CsgA ending with its extremity towards the N-terminus side of the protein or, on the contrary, remain "floating" away from the protein like a flag. 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 CsgA polymerization described in the literature. However, in none of the simulations did it happen: the second motif always ended up folding in another direction.
  
  

  Folded conformation	Floating conformation

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

  1. since its end gets near the polymerization 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 literature, 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 preferred one, nickel chelation shouldn't be too high.

  The results of the wet lab however, showed no particular decrease in curli formation between tagged and wildtype-producing E.coli. Though we do not know the extent to which the folded conformation may hinder polymerisation, we may conclude that the floating conformation seems more likely to happen in vivo than the folded one.

  Inbetween beta-sheets

  The simulations with a tag on a loop between two beta strands showed a tendency to slightly modify the structure of the beta sheets 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.
  
  

  In-loop insertion seems to slightly alter the protein's structure

  
  

  What can be said however is that the tag doesn't seem to have any conformation other than a floating loop between the original amino acids of the protein on which its been hooked. We can also question the usefulness of such a construct for our project since it has neither the reach nor the rotation freedom of the floating C-terminus tag. It might be interesting for future teams that would want to maximize the number of tags to add to their CsgA though, providing the structure alteration isn't too important, which is an information we didn't have enough time to investigate this year.
  
  

• Ni-Chelation

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• After the structural changes brought by the tags we tried to determine the chelating activity of the tagged CsgA. So we simply added nickel ions in the neighborhood of the tags and let the minimisations and dynamics do their magic.
  
  

  
  CurLy'on Nickel chelation - iGEM INSA-Lyon 2014

  
  

• Results

  Wildtype CsgA chelates nickel

  The very first result we are going to talk about is that even without an His-tag, the wildtype protein is able to chelate nickel. That is something we didn't expect and were surprised to discover. We put some nickels next to CsgA (in a 10 Angstroms radius), and Sybyl computed the simulations (with AMBER FF002 library, cutoff at 12 Ang, during 10 to 20 000 fs). Nickel can be captured by either a group of ketones that are close to each other, but even more surprising, we observed histidines, aspartic acids and other amino acids already present in wildtype CsgA were "grabbing" the ions and litterally pulling them between the beta strands into the center of the protein in some cases.
  
  

  The natural protein can chelate nickel by itself.

  
  

  After witnessing this we ran simulations with wildtype CsgA alone and a lot of nickel ions, and realized that its chelating power was quite high as it could retain a bit more than twenty ions.

  Chelation by the His-tags

  A far as the folded conformation is concerned, both His1-tag or His2-tag, cover already existing chelating sites of CsgA, but are still able to chelate nickel ions. The simulations showed that, though the tag could also fold a little to catch a nickel with two of its histidines, a bond between the nickel and both the tag AND CsgA often occured, thus stabilising the folded structure of the tag. However, with its reach decreased and the covering of the initial protein chelating sites, we concluded that such a conformation wouldn't bring much more chelating power to the protein.
  
  

  Nickel chelation of folded His1-tag	Nickel chelation of folded His2-tag

  
  

  As for the floating conformation however, we managed to verify a property found in the litterature, which is that on a poly-Histidine tag, a nickel ion is chelated by the unprotoned nitrogen of the cycle of two histidines that are separated by a third histidine. We also observed that through the folding of the tag around nickel ions so that histidines may chelate it, ketones groups were brought together, earning the ability to chelate an ion as well, and that the carboxyl group of its C-end was also able to chelate an ion. All in all, we determined that a floating tag should be able to chelate up to 5 extra nickel ions for the His1-tag, and 8 for the His2-tag.
  
  

  Nickel chelation of floating His1-tag	Nickel chelation of floating His2-tag

  "Then what is the point of tagging the original protein when it already chelates so much?" are you going to ask me. The answer is quite simple : just for one protein, with only one His1-tag we can hope for a 25 % increase of its chelation power. Now remember that CsgA is only the subunit of a fiber that is composed of thousands of them; that one bacterium bears hundreds, maybe thousands of these fibers; and that a culture of such bacteria allows for a countless number of bacteria as well. It's easy to understand how an increase of 25% of efficiency of the elementary unit of the fiber is already quite something! Well, that's provided the floating conformation is the preferred one, but as the wet lab results seem to prove that His2-tagged CsgA chelates better than His1-tagged CsgA, that already chelates more than wildtype CsgA, we can be confident in saying that the floating conformation is actually the most present in the fiber. However, as we are still unable to accurately quantify curli and CsgA, we unfortunately cannot provide any information regarding the difference between the theoretical chelating power of CsgA and the experimentally determined quantity of captured nickel.

Conclusion

Overall sum up

What we couldn't achieve