Dear Mero,

will start with one question, you haven't mention the SDS-PAGE, in this case is your dimer dissociate ?
What i mean is that it seams the dimer is relatively strong (1M urea 20 mM Tricin), maybe the construct used for expression has a problem and you have a dimer "single chain" ?

Except this hypothesis, you can try more sever condition to denaturate this protein like 6M Guanidine-HCL. In my opinion, very rare are complex which are able to resist in this type of "buffer".

To try to give you an answer :
1 : In solution, you will have something like : monomer + monomer --> <-- dimer. It's an equilibrium, if the dissociation is very "slow" compare with the dimerization, the equilibirum is displaced to favorise the dimer form. That can explain why in SAXS and Native PAGE you observe dimer form. I think it's also concentration dependant. If you are in very low concentration, you increase the chance of monomer being.
(subsidiary question : did you perform the same purification method as the people from NMR ? just to see if you can obtain monomer in this condition)
One general observation, for the dimer in crystal structure, the cristal growing is allow because of the presence of "contacts" between partners well organized. That's seem that the contact that you describe are not necessarly the one in solution. Did you try to fit you Crital model in the SAXS envelope ? Is the SAXS model in accordance with the cristal structure ?

2 : i can't answer that or hypothesis something as large as that.
3 : if you achieve to obtain monomer, you can try to caracterise your complexe affinity by SPR or ITC.
4 : Which question would you solved with this refolding experiment ?, except if you think that the protein is misfolded.

Hope to help you.

Nicolas


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De : CCP4 bulletin board [[log in to unmask]] de la part de amro selem [[log in to unmask]]
Envoyé : samedi 26 septembre 2015 01:24
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Objet : [ccp4bb] Uncommon dimer confirmation

Dear cc4bb community,
i want to post a little bit confusing story, i am working on a protein  interesting antigen  of unknown function  , this protein contain N terminal poly q tract (11 residues) which is disordered  then starting domain with identity  percent up to 40 % other protein family members, in order to obtain good diffracting crystals , the poly q is removed. The structure is solved by Se-SAD up to 1.5 angstrom, the R f and R free are 15 and 18 respectively. The structure show strange confirmation in which two chains are interlocked like Olympic rings. The protein is dimer in solution; this is confirmed by SAXS, native PAGE and gel filtration. The problem is one of homologe protein is solved by NMR as monomer with open form. i tried to dissocaite this dimer using 1 m urea and 20 mm Tricin , low and high ph but this dimer is very stabe. The dimer interface consists of 4 salt brigds and 14 hydrogen bonds and 142 van der waals.
The a symmetric unite contain only one molecule, the other one is symmetric molecule
Now i am writing manuscript but many question jump in my mind.
1- If the homolgy is monomer and in open form and my protein in closed form. So where is my open protein form?
2- Is this common in nature
3- Is there are some techniques to validate this state of folding
4- The is the best intereprtation of this case
5- If refolding experiment should be considered or not.
I attached the homology and both monomer and dimer of my protein.
Thank you in advance
Mero