Hello everybody.
I'm working with a proteins with oxidase activity that are active when I have it in the cell but lose the activity when I purify it with detergents or polymers.
My question if you know any recent structure prediction program that allows you to know which cofactors, metal ions etc may be necessary for the activity of the protein, because I think that when purifying it, these cofactors are lost... and thus the loss of activity.
Thanks in advance :)..
Hola Enrique,
I recently used i-Tasser to predict my protein structure and after you get your results, the software proposes also metal cofactors that could bind within the predicted structure. For me it predicted correctly as my protein has no known crystal structure but cofactors are known.
Regarding the loss of activity after protein purification, please, make sure you have no EDTA in your buffers. Also, we have found out in our lab, that IMAC columns could remove metal clusters from our protein. Our proteins of interest (nitrogenases) have Fe-S clusters that are quite exposed to the solvent which makes the oxygen labile. If you provide more information about your purification protocol, I might able to give you more insight.
:)
If you have the sequence of your protein, you can go in a bioinformatic way. BLAST your sequence against UniprotKB and check the protein families with major similarities within your sequence. UniprotKB gives annotations about bindings sites of cofactors in families of proteins. Also, you can BLAST directly against PDB to check structural similarities of solved proteins and verify cofactor binding sites (and then create a homology model using I-TASSER, Modeller, etc.).
If you don't have sequence information, the question is trickier. Maybe changing some experimental variables as suggested above could give an insight into the chelation of metals of importance in your protein.
Dear your problem lies in purification led loss of activity. To preserve the active domain to retain activity you have to provide a solution capping around the protein to keep it intact.
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