Team:Freiburg/Modelling

From 2011.igem.org

(Difference between revisions)
(The Idea)
(Modelling: Rational protein design)
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We received this pdb file with a C-score= 0.53. Obviously this was not a realistic model, since the folding would surely be impaired by so many Histidines everywhere. But is was useful to find several good locations, were the Nickel could fit in in respect to the requirements it has for binding ligands. We manually fit in a Nickel ion, as it was crystallized in the mentioned PDB file, and measured the distances and evaluated the 3dimensional orientation of the Histidines towards the Nickel. Histidines can coordinate ligands with their free electron pair pointing planar away from the imidazole ring. What we further realized from the structure file was, that the end of the LRR segments were “open”, that means, the hydrophobic core of the protein was exposed and as it is visible in the prediction, curled in on one end into a sort of helix. This means the protein folding is not reliable and the structure needs some caps on both ends to stabilize the LRR core motif.
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We received this pdb file with a C-score of 0.53. Obviously this was not a realistic model, since the folding would obviously be impaired by so many Histidines everywhere. But is was useful for finding several good locations where the Nickel could fit in in respect to the requirements it has for binding ligands. We manually fit in a Nickel ion, as it was crystallized in the mentioned PDB file, and measured the distances and evaluated the 3dimensional orientation of the Histidines towards the Nickel. Histidines can coordinate ligands with their free electron pair pointing planar away from the imidazole ring. What we further realized from the structure file was, that the ends of the LRR segments were “open”, meaning the hydrophobic core of the protein was exposed and as it is visible in the prediction, curled in on one end into a sort of helix. This means the protein folding is not reliable and the structure needs some sor of cap on either end in order to stabilize the LRR core motif.
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A solution to this was shown by Schmidt et al 2010, who crystallized the TLR-4 receptor.  
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A solution to this was shown by Schmidt et. al. 2010, who crystallized the TLR-4 receptor.  
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In this very nice piece of work he dissected the TLR4(PDB: 3FXI) into 3 parts, since it was to large and unhandy to be crystallized at once
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In this very useful work he dissected the TLR4(PDB: 3FXI) EDIT into 3 parts, since it was too large and unhandy to be crystallized at once..
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To overcome this problem of an exposed hydrophobic core, he used the N- and C-terminal protein fragments of a LRR protein derived from hagfish. They tried a variety of different versions of how to glue together his fragments and these N- and C-terminal caps until he found a working one.
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To overcome this problem of an exposed hydrophobic core, he used the N- and C-terminal protein fragments of a LRR protein derived from hagfish. They tried a variety of different versions of how to glue together his fragments and these N- and C-terminal caps until they found a working one.
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We used this knowledge for our purpose and took the same sequences and attached them to our protein sequence. These caps partially still show the typical LRR consensus sequences (which luckily is highly conserved in all kingdoms of life!), which made it possible to fit them onto our stack of LRR loops in the right position.  
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We used this knowledge for our purpose, applied the same sequences and attached them to our protein sequence. These caps do still in parts show the typical LRR consensus sequences (which luckily is highly conserved in all kingdoms of life!), which made it possible to fit them onto our stack of LRR loops in the right position.  
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To the outer end the protein has a helix on the N-terminal side shielding off the core and the C-terminus the LRR disappears slowly turn by turn with more and more hydrophilic amino acids replacing the LRR consensus sequence.
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To the outer end the protein has a helix on the N-terminal side, shielding off the core and the C-terminus while the LRR disappears slowly turn by turn with more and more hydrophilic amino acids replacing the LRR consensus sequence.
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After evaluating the C-Scores of I-TASSER we saw that our favorite versions 1,2 and 4 gave acceptable scores. We then reverse translated them with a codon usage optimized for ''E. coli'', using http://www.bioinformatics.org/sms2/rev_trans.html
After evaluating the C-Scores of I-TASSER we saw that our favorite versions 1,2 and 4 gave acceptable scores. We then reverse translated them with a codon usage optimized for ''E. coli'', using http://www.bioinformatics.org/sms2/rev_trans.html
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The sequence was handed over to our sponsor ATG:biosynthetics, who ran their own analysis on it, to optimize the sequence for RNA trafficking and secondary structures. In pictures below the alignment of our reverse translated sequence and the returned sequence is shown: ours above, ATG:biosynthetics sequence below. Underlined in red are the mutations the company introduced to optimize the expression. They also were so kind to synthesize the three genes for us as a gift.
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The sequence was handed over to our sponsor ATG:biosynthetics, who ran their own analysis on it, to optimize the sequence for RNA trafficking and secondary structures. In pictures below the alignment of our reverse translated sequence and the returned sequence is shown: ours above, ATG:biosynthetics sequence below. Underlined in red are the mutations the company introduced to optimize the expression. They also were so kind as to synthesize the three genes for us as a gift.

Revision as of 01:29, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!