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Modeling of a protein with disulfide bond - (Mar/03/2014 )

Hello,

I have designed a fusion protein containing a motif with two cys that I want to make a disulfide bond and create a circular motif . The protein sequence also contains a cys that should not make a bond with motif's cys.  After protein modeling using I-TASSER server, i could not recognize any disulfide bond with SPDBV viewer. Could anyone help me with this problem. How can I predict that the disulfide bond is created correctly?

 

Thanks 

-Mariam-

Hi Mariam.  The sulfhydryl groups on your cysteines will react and cross-link with each other as long as they are in an oxidative environment.  Predicting which 2 of the 3 will form the intrachain disulfide bond is not possible with the information you have provided, but if you were to express this protein you may end up with more than one combination of disulfides, e.g. Cys1 linked to Cys3 in one molecule and Cys2 linked to Cys3 in another.  Unless one of the sulfhydryl groups is completely inaccessible, you can not really predict which disulfides will form.

-dimensionsbio-

Hi Mariam.  The sulfhydryl groups on your cysteines will react and cross-link with each other as long as they are in an oxidative environment.  Predicting which 2 of the 3 will form the intrachain disulfide bond is not possible with the information you have provided, but if you were to express this protein you may end up with more than one combination of disulfides, e.g. Cys1 linked to Cys3 in one molecule and Cys2 linked to Cys3 in another.  Unless one of the sulfhydryl groups is completely inaccessible, you can not really predict which disulfides will form.

Thanks for your reply.

In this situation what can I do before testing the protein in vitro? It is really important to predict if the two cys of motif form disulfide bond and make the motif circular. If not the motif won't have its native function! I tried to in silico predict the possibility of each disulfide bond formation using different bioinformatic tools like DiANNA,.. But the result was not satisfactory since the probability of the cys1 linkind to cys3 was the highest!!

-Mariam-

Isn't it possible to treat with suitable  protease site if present in protein chain - note size on PAGE!!! reduce disulfe bonds and again run on PAGE.

-Inbox-

Isn't it possible to treat with suitable  protease site if present in protein chain - note size on PAGE!!! reduce disulfe bonds and again run on PAGE.

But I don't know how to recognize the site of disulfide bonds after treating with protease? Is there any size difference on PAGE if the disulfide bond is formed between cys1-cys3 or cys2-cys3??

-Mariam-

Isn't you protein sequence known? you can predict site of cleavage by any available soft package. I am just speculating about separation on PAGE. You need to read more into it.

 Following link might be helpful to you ( I don't have access to whole article so read only abstract).

http://www.sciencedirect.com/science/article/pii/0003269788900280?via=ihub

-Inbox-

http://www.ncbi.nlm.nih.gov/pubmed/22899260

-Inbox-

Thank you very much for introducing such useful articles. 

Good luck

-Mariam-