differentiating differently folded structures of the same protein - what methods? reply asap plz (Aug/13/2006 )

Hi all,

I am working on this highly hydrophobic protein, with a single disulphide linkage, no free cysteine - its a recombinant protein. But certain native peptide map results show that the folding of the protein is not the desired - active one.

1. Could this only be due to different hydrophobic interactions - which lead to a different refolding?

2. Plz tell me the other methods that are capable of differentiating differently folded structures for the same protein? like a CD.. what else?

3. how can i know which particular aminoacids are involved in this misfolding?



I need quick replies please:)



Thanks a ton!

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If your protein is full of hydrophobic patches, it might well be membrane-bound or -associated. Ifyou are playing around with it in solution, that might be the source of your problems - the protein is either refolding/misfolding to bury more of the hydrophobic regions, or it could be aggregating, to do the same thing. Either way, you lose activity of whatever it does.

You might be able to differentiate your isoforms by gel filtration. Try a variety of conditions, including a chaotropic salt (to dissociate the multimers, if that is the problem) as well as some hydrophobic buffers (I think most GF columns can handle acetonitrile and they can all handle alcohols). You should see different forms eluting at different times.

You would have to have pure isoforms in order to really see what is happening by CD; if you ran the mix, it would not be possible to determine what the CD spectrum *should* look like (i.e., the folded, "active" form).

As for finding out the misfolding/refolding "culprits", you might have to go from the peptide maps to see what reisdues become buried, then try to mutate them to something more benign, see if it affects the activity (it wouldn't do to destroy the active site, now would it ;-) )

Good luck with whatever you end up doing...

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I wonder if short chain phospholipids can help in this case.

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Hi all and Thanks lot swanny:)

Exactly, a peptide map. Actually, what hinted me of the differences in the folding is a peptide map, where my profiles for the known folded standard and this protein i m trying to purify did not match under native conditions. Yet they did match under reduced-denatured conditions. So, i suppose that means my aminoacid sequences r same but they r differently folded.

Both the forms do have the activity, i dont have efficient / accurate methods to determine how active they are.



For Cd, as u said i need some pure folded form, which i do have.

Its not a membrance protein and i dont have major differences between the two forms on a gel filtration.



Anything else that i cud tell u abt this so that u can help me out with yr suggestions?

Plz do tell me.. thanks!

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If you have access to a reverse-phase HPLC system, you could try a gentle buffer system, say, EtOH, on a short chain column, like a C4. Your most hydrophobic proteins should stick best, so you could potentially separate. Alternately, have you investigated hydrophobic interaction chromatography?

You said you have a standard form; what does its CD spectrum look like? If you bought it somewhere, how did they purify it? Is there anything else you could tell us about the protein, or would you have to shoot us if you did?

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