Re: [ccp4bb] Promoting oligomer dissociation

[Michael Kenneth Fenwick]

Many thanks Jacob and Mark for your questions/suggestions. In response:

 So what happened with the non-reducing gel? (If the DTT was fresh, there 
 should be no problem, but if not...)
The gel is very clear, it shows the same exact pattern as the reducing gels. 
Both high and low MW fractions run at the same MW. I used -80C stocks of the 
native protein for which the DTT was buffer exchanged out (3, ~15-fold 
exchanges via concentration/dilution). Technically, there is a tiny amount in 
there. But I don't think it's disulfides based on this experiment. Concerning 
the freshness of added DTT, it's pretty much as fresh as our solid DTT at -20C 
is fresh. I make all buffers containing DTT just before use and I do it by 
first adding all the components to ddH2O except the DTT; then I put the buffer 
at -20C until it gets cold ~4C. Then I pH it and add the DTT and move it to the 
column.

you're right, I read too quickly over the 2 orders, and understood 2-fold. 
Is it possible the oligomer peak contains higher aggregates that inhibit 
crystallisation and do not separate well on your gel filtration column? In 
that case, perhaps very high-g centrifugation could remove them, or a gel 
filtration column that separates better in that MW-range. Or perhaps the tail 
fractions of the oligomer peak on your current column might be already 
conformationally pure enough.
Concerning this point made by Mark, there might be something to this. On gels 
these fractions do tend to appear slightly dirty in that there are a couple 
very faint bands at high MW. Also, When I rerun individual peak fractions back 
through gel filtration, the high MW samples sometime result in additional 
light-absorbing matter at even higher MW closer to the void fractions. 
Crystallization drops for the high MW fractions appear less soluble even at 
lower concentrations than the lower MW fractions. However, the unclipped, lower 
MW samples also are less soluble than their clipped counterparts (which raises 
the issue of the importance of the tag). I'm still playing with optimization of 
these samples as well because who knows really. 

[ccp4bb] Promoting oligomer dissociation

[Michael Kenneth Fenwick]

Hi,

I have a protein that shows high and low MW peaks on gel filtration (which run 
at the same MW on SDS-PAGE). There is a slow equilibrium because rerunning the 
individual peaks on gel filtration a couple days later shows both peaks. The 
higher MW peak is ~2 orders of magnitude more dominant...the lower MW peak is 
not yielding much. However, as nature would have it I've only gotten the lower 
MW peak fractions to crystallize, and only when the affinity tag is clipped 
(the higher MW species is resistant to clipping). I would like to do something 
to shift the equilibrium towards the lower MW species. So far, I've tried 
(without success or clues for success) changing pH, increasing NaCl from 200  
to 600mM, adding glycerol to 12%. Things that I've seen in papers but have not 
yet tried are temperature jumps, other salts, limited Gu/urea, Arg/glu, 
dioxane, limited SDS/triton.

Any suggestions are greatly appreciated, thanks very much,
Mike

Re: [ccp4bb] Promoting oligomer dissociation

[Michael Kenneth Fenwick]

Thanks for all your suggestions so far...as a quick reply to some:

You say the fractions are in equilibrium - how about keeping the oligomer 
fraction each time and adding it to the subsequent preparation?
I did this once. The equilibrium is sort of a gift that keeps on giving, but 
the problem is the amount it's giving. In the end, this might be the only way 
to go, but it's very tempting to find a chemical solution.

Do you have a reducing agent in your solutions? I.e., maybe you are seeing 
disulfides?
For native preps I used 1mM DTT throughout...for the SeMet prep I used 3mM DTT. 
To confirm it's not S-Ss, I'm about to run SDS-PAGE lacking reducing agent.

Changing buffer from Tris/Hepes into phosphate or citrate or acetate, or if 
higher pH borate?
When I tried lower pH I used citrate. I might give the others a try.

Stay away from SDS/Triton because they will almost certainly kill your 
crystallization and it will be hard, very hard if not impossible to get rid
of them.
Thanks for the tip!

Another person off the bulletin board suggested differing how cell lysis is 
done 
I used sonication. 

Re: [ccp4bb] generating separate pdb files

[Michael Kenneth Fenwick]

Hi Charlie,

This may not be what you want, but this perl script seemed to work just now on 
1g9e.pdb


$base='1g9e';open(IN,$base.pdb);@indata = IN;$i=0;

foreach $line(@indata) {

if($line =~ /^MODEL/) {++$i;$file=${base}_$i.pdb;open(OUT,$file);next}

if($line =~ /^ENDMDL/) {next}

if($line =~ /^ATOM/ || $line =~ /^HETATM/) {print OUT $line}

}



Hope it helps you,

Mike