Team:INSA-Lyon/Molecular

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<h4>MOLECULAR MODELISATION</h4>
<h4>MOLECULAR MODELISATION</h4>
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<a href="https://2014.igem.org/Team:INSA-Lyon/Modeling" class="hu-icon"><li class="iconmulti">MODELING</br>SUMMARY</li></a>
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<a href="https://2014.igem.org/Team:INSA-Lyon/Molecular" class="hu-icon"><li class="iconmulti">MOLECULAR</br>MODELISATION</li></a>
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<a href="https://2014.igem.org/Team:INSA-Lyon/CurliSynthesis" class="hu-icon"><li class="iconmulti">CURLI</br>SYNTHESIS</li></a>
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                         <p><div align = "justify">  
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<p>
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One of the main goals of our modeling work this year was to understand the <b>structure</b> of the curlin subunit protein, <b>CsgA</b> and its behavior when engineered with a tag constituted of either six histidines (that we will call <b>His1-tag</b> from now on) or twice that motif (<b>His2-tag</b>), since this peptide is known for its nickel chelation properties.</br>
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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 <b>best position</b> for the tag was by the <b>C-terminus end</b> of the protein. We also determined that the His-tag was more likely to take a <b>floating conformation</b> instead of folding itself around CsgA.
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One of the main goals of our modeling work this year was to understand the <b>structure</b> of the curlin subunit protein, <b>CsgA</b> and it's behavior when engineered with a tag constituted of either six histidines (that we will call <b>His1-tag</b> from now on) or twice that motif (<b>His2-tag</b>), since such a peptide  is known for its nickel chelation properties.</br>
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                        </p></div></br>
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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 <b>best position</b> for the tag was by the <b>C-terminus</b> of the protein. We also determined that the His-tag was more likely to take a <b>floating conformation</b> instead of folding itself around CsgA.
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                        </div></p>
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<ul style="list-style-type: none !important;">
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                        <h6 align="left">Methods</h6>
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<li><a href="#methods" onclick="$('#methods').slideToggle('slow')"><h1 align="left"><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Methods</h1></a><hr/></li>
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<ul id="methods" style="list-style-type: none !important;display:none;">
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<li>                            <div align = "justify"> <p>
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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 <b>Sybyl-X</b>, which is a program we started to use this year and that offers a number of useful tools as well as powerful calculation algorithms. </br>
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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: </p>
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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 <b>Sybyl-X</b>, which is a program we began to use this year that offers a number of useful tools as well as powerful calculation algorithms. </br>
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<p>
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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 <b>pdb file</b>, 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 publication that were able to fully characterize its shape, let alone define its 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 <b>M. Chapman</b> from the University of Michigan, who sent us his work and to whom we are really grateful.</br></br>
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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; </p>
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<p>
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provide the program with a <b>pdb file</b>, which is a file that gives the spatial coordinates of every atom in the molecule.</p>
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<p> 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 <b>M. Chapman</b> from the University of Michigan, who sent us his work and to whom we are really grateful.</br></br></p>
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   <td><img src="https://static.igem.org/mediawiki/2014/4/45/WtFace1.jpg" alt="CsgAWTSybyl" width="400px"/></div></td>
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<li> the <b>minimize</b> option, that searches for the minimal steric hindrance of the protein;
<li> the <b>minimize</b> option, that searches for the minimal steric hindrance of the protein;
<li> what we called a "<b>dynamic</b>", 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).
<li> what we called a "<b>dynamic</b>", 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).
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</ol><br/><br/>
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</ol><br/><br/><p>
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.<br/>
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.<br/>
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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 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 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 minimisations and dynamics take a lot of time, and since we could only run one simulation at a time, the whole study spread over several weeks.  
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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.  
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                            </div></p>
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                          </p></div></li>
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<h1 align="left">CsgA Engineering</h1>
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<li><a href="#csgaEngineering" onclick="$('#csgaEngineering').slideToggle('slow')"><h1 align="left"><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />CsgA Engineering</h1></a><hr/></li>
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          <ul id="csgaEngineering" style="list-style-type: none !important;display:none;">
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                             <li><div align = "justify"> <p>                         
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The first step of the study was to determine the <b>behavior of the CsgA protein once tagged with a poly-His peptid</b>. Hence we ran several simulations as described above with both His1-tag and His2-tag, in <b>C-terminus</b> position but also inserted in a loop <b>between two beta strands</b>, 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.
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The first step of the study was to determine the <b>behavior of the CsgA protein once tagged with a poly-His peptide</b>. Hence we ran several simulations as described above with both His1-tag and His2-tag, in <b>C-terminus</b> position but also inserted in a loop <b>between two beta strands</b>. 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.
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                            </div></p>
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                          </p> </div></li>
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<h5 align="left">Results</h5>
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<li><h5 align="left">Results</h5>
  <h6 align="left">In C-terminus</h6>
  <h6 align="left">In C-terminus</h6>
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The first observation we can make is that two main conformations of the tags came out of the simulations : they could either <b>fold along the side of Csga</b> ending with its extremity toward the N-terminus side of the protein or, on the contrary, remain "<b>floating</b>" 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 polymerisation according to litterature. However, in none of the simulation did it happen: the second motif always ended up folding in another direction.</br></br>
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The first observation we can make is that two main conformations of the tags came out of the simulations : they could either <b>fold along the side of CsgA</b> ending with its extremity towards the N-terminus side of the protein or, on the contrary, remain "<b>floating</b>" 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.</br></br></p>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/2/2b/CurlyonFoldedHis1.jpg" alt="floating His1" width="300px"/></div></td>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/2/2b/CurlyonFoldedHis1.jpg" alt="floating His1" height="250px"/></div></td>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/b/be/CurlyonFloatingHis1.jpg" alt="folded His1" width="300px"/></div></td>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/b/be/CurlyonFloatingHis1.jpg" alt="folded His1" height="250px"/></div></td>
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                             </div></p>
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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:
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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:
<ol>
<ol>
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<li> since its end gets near the polymerisation site, <b>the folded conformation may hinder</b> a little the <b>curli formation</b>, hence a comparison of curli production between tagged and wildtype-producing <i>E.coli</i> may give a hint to the right conformation;
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<li> since its end gets near the polymerization site, <b>the folded conformation may hinder</b> a little the <b>curli formation</b>, hence a comparison of curli production between tagged and wildtype-producing <i>E.coli</i> may give a hint to the right conformation;
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<li> according to litterature <font color="red">publi modmol</font>, 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.
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<li> 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.
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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><font color="green">lien vers les résultats biofilms</font>, 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 <font color="green">lien vers nichelation</font>. 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 results of the wet lab 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>. Though we do not know the extent to which the folded conformation may hinder polymerisation, we may conclude that <!--, 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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                             </div></p>
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                             </p></div>
  <h6 align="left">Inbetween beta-sheets</h6>
  <h6 align="left">Inbetween beta-sheets</h6>
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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<font color="red">image sybyl</font>, 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>
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The simulations with a tag on a loop between two beta strands showed a tendency to <b>slightly 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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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/d/d2/CurlyonHis1In1.jpg" alt="floating His1" width="500px"/></div></td>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/d/d2/CurlyonHis1In1.jpg" alt="floating His1" width="350px"/></div></td>
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What can be said however is that it doesn't seem to have any other conformation than a floating loop between the original amino acids of the protein on which it's 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 futures 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.</br></br>
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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.</br></br>
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                             </div></p>
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                             </p></div></li>
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          <h1 align="left">NiChelation</h1>
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<li><a href="#nichelation" onclick="$('#nichelation').slideToggle('slow')"><h1 align="left"><img src="https://static.igem.org/mediawiki/2014/d/d5/Insa_fleche_titre.png" width="20px" />Ni-Chelation</h1></a><hr/></li>
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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 magics.
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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 minimizations and dynamics do their magic.</br></br>
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                            </div></p>
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<a target="_blank" href="http://www.dailymotion.com/video/x27smzt_curly-on-chelation-of-nickel-by-his-tagged-csga_tech"><img src="https://static.igem.org/mediawiki/2014/f/f8/Modelo_chelation_of_nickel.png" alt="les filles au labo" width="500px"/></a><br /><a style="margin-left:120px" href="http://www.dailymotion.com/video/x27smzt_curly-on-chelation-of-nickel-by-his-tagged-csga_tech" target="_blank">CurLy&#039;on Nickel chelation - iGEM INSA-Lyon 2014</a> <i>
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<a style="color:black" href="http://www.dailymotion.com/iGEM_Lyon_2011" target="_blank"></a></i>
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</div></br>
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                            </p></div></li>
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<h5 align="left">Results</h5>
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<li><h5 align="left">Results</h5>
    <h6 align="left">Wildtype CsgA chelates nickel</h6>
    <h6 align="left">Wildtype CsgA chelates nickel</h6>
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The very first result we are going to talk about is that even without an His-tag, <b>the wildtype protein is able to chelate nickel</b>. That is something we didn't expect and were surprised to discover. Nickel can be captured by either a <b>group of ketones</b> that are close to each other, but even more surprising, we observed the couple of histidines already present in wildtype CsgA act like gates, "grabbing" the ions with their cycles, and litterally pulling them  between the beta strands <b>into the center of the protein</b>.</br></br>
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The very first result we are going to talk about is that even without an His-tag, <b>the wildtype protein is able to chelate nickel</b>. 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 <b>group of ketones</b> 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 literally pulling them  between the beta strands <b>into the center of the protein in some cases</b>.</br></br></p>
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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 <b>more than twenty ions</b>.
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<p>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 <b>more than twenty ions</b>.
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                             </div></p>
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                             </p></div>
    <h6 align="left">Chelation by the His-tags</h6>
    <h6 align="left">Chelation by the His-tags</h6>
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For the <b>folded conformation</b> the tag, be it His1-tag or His2-tag, covers already existing chelating sites of CsgA, but is still able to chelate nickel ions. The simulations showed that, though it could also fold a little to catch a nickel with two of it's histidines, a bond between the nickel and both the tag AND CsgA often occured, thus being chelated but also 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.</br></br>
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A far as the <b>folded conformation</b> 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 occurred, thus stabilizing 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.</br></br></p>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/6/63/CurlyonHis1accNi.jpg" alt="folded His1 with Ni" width="300px"/></div></td>
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   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/6/63/CurlyonHis1accNi.jpg" alt="folded His1 with Ni" height="250px"/></div></td>
-
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/7/72/CurlyonHis2accNi.jpg" alt="folded His2 with Ni" width="300px"/></div></td>
+
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/7/72/CurlyonHis2accNi.jpg" alt="folded His2 with Ni" height="250px"/></div></td>
</tr>
</tr>
<tr>
<tr>
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</table>
</table>
</div></br>
</div></br>
-
 
+
<p>
-
As for the <b>floating conformation</b> however, we managed to verify a property found in the litterature <font color="red">publi modmol</font>, 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 nickel ions for the His1-tag, and 8 for the His2-tag.</br></br>
+
As for the <b>floating conformation</b> however, we managed to verify a property found in the <a href="http://www.tandfonline.com/doi/abs/10.1080/07391102.2003.10506903?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed#.VEFESBZ-Tw0"> literature</a>, 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.</br></br></p>
<div align="center">
<div align="center">
<table>
<table>
<tr>
<tr>
-
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/9/98/CurlyonHis1floNi.jpg" alt="floating His1 with Ni" width="300px"/></div></td>
+
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/9/98/CurlyonHis1floNi.jpg" alt="floating His1 with Ni" height="250px"/></div></td>
-
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/9/9d/CurlyonHis2floNi.jpg" alt="floating His2 with Ni" width="300px"/></div></td>
+
   <td><div align="center"><img src="https://static.igem.org/mediawiki/2014/9/9d/CurlyonHis2floNi.jpg" alt="floating His2 with Ni" height="250px"/></div></td>
</tr>
</tr>
<tr>
<tr>
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</div>
</div>
 +
<p> For better understanding of the folding process, here's an outline of the chelation of a nickel ion (in the center) by a tag composed of three histidines.</br>
 +
<div align="center"><img src="https://static.igem.org/mediawiki/2014/a/a5/CurlyonHistag.png" alt="histag" height="300px"/></div></p>
                             </div></p>
                             </div></p>
-
                             <p><div align = "justify">
+
                             <div align = "justify"><p>
-
<i>"Then what is the point of tagging the original protein when it chelates so much more than the tag itself?"</i> are you going to ask me. The answer is quite simple : just for one protein, with only one His1-tag we can <b>increase its chelation power by 25%</b>. Now remember that CsgA is only the subunit of a fiber that is composed of thousands of them; that one bacterium bear 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 prefered one, but as the wetlab results <font color="green">liens vers résultats ICP</font> 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 <b>the floating conformation is actually the most present</b> 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.
+
<i>"Then what is the point of tagging the original protein when it already chelates so much?"</i> 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 <b>25 % increase of its chelation power</b>. 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 since the <a href="https://2014.igem.org/Team:INSA-Lyon/Results#contenu2">wet lab results</a> seem to prove that His2-tagged CsgA chelates better than His1-tagged CsgA, which chelates as much as wildtype CsgA, from this <b>we cannot really discriminate</b> between one conformation or the other from our experimental data, though these results seem to lead towards the folded conformation, where the second motif would be floating for His2-tag. Moreover, 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.
-
                             </div></p>
+
                             </p></div></li>
 +
</ul>
 +
</ul>
 +
</br></br>
-
  <h1 align="left">Conclusion</h1>
+
  <h1 align="left">Conclusion</h1></hr>
    <h6 align="left">Overall sum up</h6>
    <h6 align="left">Overall sum up</h6>
-
                             <p><div align = "justify">
+
                             <div align = "justify"><p>
Through our molecular study of a CsgA protein engineered with either <b>His1-tag</b> or <b>His2-tag</b>, 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 <b>C-terminus</b> of the protein <b>is more relevant than</b> placing it <b>in the middle of the sequence</b>, although doing so may provide a little more chelation power as long as the tag isn't too long.</br>
Through our molecular study of a CsgA protein engineered with either <b>His1-tag</b> or <b>His2-tag</b>, 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 <b>C-terminus</b> of the protein <b>is more relevant than</b> placing it <b>in the middle of the sequence</b>, although doing so may provide a little more chelation power as long as the tag isn't too long.</br>
-
We also showed that for the tags there exist <b>two possible conformations</b> : one is <b>folded on the side of CsgA</b> and <i>a priori</i> does not increase the already-existing chelating power of CsgA; the other is a <b>"floating" conformation</b> where the tag is not attracted to the protein and is able to <b>improve its chelating power by up to 25%</b>!
+
We also showed that there are  <b>two possible conformations</b> : one is <b>folded on the side of CsgA</b> and <i>a priori</i> does not increase the already-existing chelating power of CsgA; the other is a <b>"floating" conformation</b> where the tag is not attracted to the protein and is able to <b>improve its chelating power by up to 25%</b>! Unfortunately, the wetlab results are not sufficient to discriminate which conformation is more present <i>in vivo</i>.
-
                             </div></p>
+
                             </p></div>
    <h6 align="left">What we couldn't achieve</h6>
    <h6 align="left">What we couldn't achieve</h6>
-
                             <p><div align = "justify">
+
                             <div align = "justify"><p>
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:
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:
<ul>
<ul>
<li> more simulations with His2-tag. Since they took an awful lot of time, we only ran a handful of them;
<li> more simulations with His2-tag. Since they took an awful lot of time, we only ran a handful of them;
-
<li> modelisation of the docking between two CsgA proteins, and the influence of the His-tags, that our lack of experience prevented us to conduct;
+
<li> modelisation of the docking between two CsgA proteins, and the influence of the His-tags, that our lack of experience prevented us from  conducting;
-
<li> find out just how many tags can be added without altering the protein properties of adherence and polymerisation;
+
<li> find out just how many tags can be added without altering the protein properties of adherence and polymerization;
</ul>
</ul>
-
                             </div></p>
+
                             </p></div>
</div>
</div>

Latest revision as of 03:20, 18 October 2014

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




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 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;