Team:Gothenburg-Sweden/Results

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  <p><strong>Structure Predictions in  2D</strong></p>
  <p><strong>Structure Predictions in  2D</strong></p>
  <p><img width="205" height="187" src="http://2010.igem.org/wiki/images/8/83/Results_clip_image002.jpg" alt="alpha.jpg"><img width="208" height="187" src="http://2010.igem.org/wiki/images/6/69/Results_clip_image004.jpg" alt="beta.jpg"><img width="204" height="187" src="http://2010.igem.org/wiki/images/f/f9/Results_clip_image006.jpg" alt="gamma.jpg"> <br>
  <p><img width="205" height="187" src="http://2010.igem.org/wiki/images/8/83/Results_clip_image002.jpg" alt="alpha.jpg"><img width="208" height="187" src="http://2010.igem.org/wiki/images/6/69/Results_clip_image004.jpg" alt="beta.jpg"><img width="204" height="187" src="http://2010.igem.org/wiki/images/f/f9/Results_clip_image006.jpg" alt="gamma.jpg"> <br>
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    Alpha                                Beta                         Gamma<br>
+
    Alpha                                    Beta                             Gamma<br>
   <img width="205" height="189" src="http://2010.igem.org/wiki/images/3/31/Results_clip_image008.jpg" alt="cfp_alpha.jpg"><img width="201" height="188" src="http://2010.igem.org/wiki/images/1/1f/Results_clip_image010.jpg" alt="yfp_gamma.jpg"><img width="205" height="189" src="http://2010.igem.org/wiki/images/7/73/Results_clip_image012.jpg" alt="cfp_gamma_yfp.jpg"> <br>
   <img width="205" height="189" src="http://2010.igem.org/wiki/images/3/31/Results_clip_image008.jpg" alt="cfp_alpha.jpg"><img width="201" height="188" src="http://2010.igem.org/wiki/images/1/1f/Results_clip_image010.jpg" alt="yfp_gamma.jpg"><img width="205" height="189" src="http://2010.igem.org/wiki/images/7/73/Results_clip_image012.jpg" alt="cfp_gamma_yfp.jpg"> <br>
      ECFP_Alpha                    EYFP_Gamma                  ECFP_Gamma_EYFP</p>
      ECFP_Alpha                    EYFP_Gamma                  ECFP_Gamma_EYFP</p>

Revision as of 08:52, 21 July 2010

Chalmers University of Technology

Results
 
 
primer design
 
 
primer design document
 
 
3D modelling
 
 

Since the structure of the subunits in our protein complex is not complete and the fluorescent proteins are to be studied along with the complex, we have modeled the entire complex along with the FPs computationally. There are two main steps involved in the process of modeling this complex computationally. First step is to predict the structure of each subunit and then to dock them to find how they bind to each other.


STRUCTURE PREDICTION
We obtained the entire primary structure (protein sequence) of all three subunits (snf1, sip2, snf4) and the fluorescent proteins (ECFP and EYFP) from the Protein Database (PDB). We need to find the structure of the 3 subunits and also the fusion proteins which are ECFP-snf1, EYFP-snf4 and ECFP-snf4-EYFP. So, here for the fusion protein sequence we combined the sequence of FPs and the subunits accordingly.
Now we just have the sequence of all 6 proteins including the fusion proteins and we need to find the structure of each one separately. The most prominent method used to find the tertiary structure of the proteins from its sequence is “homology modeling”.  Here they compare the target sequence to the database and they find the alignment and predict the structure from that alignment. We used this method and found the following predictions using soft-wares.

Structure Predictions in 2D

alpha.jpgbeta.jpggamma.jpg
Alpha                                 Beta                         Gamma
cfp_alpha.jpgyfp_gamma.jpgcfp_gamma_yfp.jpg
ECFP_Alpha                    EYFP_Gamma                  ECFP_Gamma_EYFP

DOCKING
We have got the structures so far and now we have to find how they can bind to each other. This can be obtained by means of “Protein Docking”. This method will obtain the surface of the molecules and try to bind them together by considering various factors like surface complementarily, electrostatics, bond angles etc. Here, we found the appropriate docking for two different approaches of tagging the FPs and just the subunits.

First, we docked all three subunits without the FPs. This is just to study how the subunits bind themselves normally. This is the predicted structure of the entire complex.

a-b-g.jpg
Predicted SNF1 Complex

 

Secondly, we docked the alpha subunit with ECFP on its N-terminal end, beta subunit and gamma subunit with EYFP on its N-terminal end. Here, from the picture below you can see that the distance between the FPs is 51.10Å. This would give some good results for the FRET analysis.

dock1

Predicted FPs orientation with SNF1 complex when they are tagged to alpha (snf1) and gamma (snf4) subunits to their N-terminal ends

 

The third docking is to find the binding of alpha, beta and gamma with FPs on its N- and C- terminal ends. Here, the distance between the two chromophores is shown as 92.70Å in the figure below. This is because of the reason that the FPs are oriented exactly in opposite directions. But in reality it might be closer than predicted which would help in FRET analysis.

dock2
Predicted FPs orientation in the SNF1 complex when they are tagged to the N- and C- terminal ends of the gamma (snf4) subunit

 

 
 
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