blue native PAGE andmolecular mass analisys

ChenHA hzhen at freeuk.com
Mon May 15 19:46:16 EST 2000



Valeria Maida wrote:
> 
> I agree (I mean there is a fast equilibrium between the two species) but as
> I want to prepare a sample for NMR, I can NOT play too much with pH and
> also with salt concentration. 

You can play about with the pH and salt concentration for
NMR, as long as your protein doesn't precipitate at
different pH.  High pH and salt concentration is not good
for NMR, but still doable.  Everything depends on your
protein, although for a tetramer, I suppose you must
optimise your condition, but I think there could be a
reasonable pH range within which you could try (say 2-7?)

> By the way, what I observed is that
> increasing the amount of NaCl the gel filtration peak move towards the
> dimeric form but NMR told us that the dimeric specie seems a molten globule
> one, even if the tetramer is well folded. 

This tells me that the protein-protein interaction is
mediated by charged species. I would therefore suspect
histidines, glutamates or aspartates.  At a simplistic
level, if it is glutamates or aspartates, at low pH when
these residues are protonated, you should get only dimer. 
If it is histidines, you can try higher pH.  I don't know
how you can tell if the dimeric specie is a molten globule
(this is just my ignorance, but is there such a thing as
dimeric molten globule?).  Part of the structure (probably
the protein-protein interaction site) may simply be more
flexible as a dimer, but fold into a stable structure when
forming tetramer.  Are you just looking at chemical shift
changes, or by other kinds of analysis?

> So my problem is to have one
> specie (dimer or tetramer) AND well folded AND, at the same time,
> omogeneous. Gel filtration seems not good to test omogeneity, maybe either
> BN-page is not, I don't know.
> Any idea how to move the equilibrium towards a well folded tetramer or
> dimer (not using chaotrops)?

If I'm correct in my thinking, there is no point in
separating the dimer and tetramer by gel filtration, simply
because if they are in equilibrium, given enough time, you
will get both dimer and tetramers reappearing. Therefore I
think playing with pH and salt concentration will be useful.
If it is charge-charge interaction, low salt and buffer
concentration will enhance such interaction.  You can
therefore use minimal buffer concentration (say 0 - 5 mM ?)
and see if you get more tetramer.  Also, as I said before,
changing the pH is likely to shift the equilibrium if it is
mediated by charged specie, therefore the easiest thing to
do (do a pH titration, increasing/decreasing by ~0.5 pH unit
for each titration point).

You can also try mutagenesis of specific residue which can
enhance or destroy the protein-protein interaction. 
However, that may be a lot of work if you are not familiar
with it.

Crosslinking of the subunits is possible but might be
difficult or the result useless.  Other people may have
better suggestions. 

> thank you for the suggestions
> Vale
> 
> ---




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