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Dear Colleagues,
first of all thank you for the suggestions!
so plenty to try!
this is a collection of the answers so far,
I also found that a similar issue was raised in the 2000...
http://people.cryst.bbk.ac.uk/~ubcg09j/ccp4arc/bb2000/msg00949.html
You could try adding 50mM Glu and 50 mM Arg. These are supposed to help
with stabilising proteins in solution and improving their solubility -
never tried it though!
See: Golovanov, A. P., Hautbergue, G. M., Wilson, S. A. & Lian, L. Y.
(2004). A simple method for improving protein solubility and long-term
stability. J Am Chem Soc, 126, 8933-8939.
>
I do not know the answer to your question but yet have a suggestion: you
can try to crystallise your protein by (inverse) salting-in. Having a
reservoir with lower vapour pressure (e.g. by lower salt concentration)
should make your drop take up water, i.e. let the salt concentration go
down. According to your description the protein seems to precipitate
under these conditions, and if you find the right additive it might do
so in crystalline form. I remember such a case where we only needed
(initially) 4% PEG 6k, no salt and some buffer to stabilise the pH. The
protein was in 500mM NaCl and crystallised after about 5 days (and in
one out of a few thousand conditions such a crystal would be C2 instead
of P65 and diffract to 1.6A instead of 3.5A ;-) )
>
Rather than eliminate NaCl, you can address the problem by adding NaCl
to your reservoir solution.
If your protein is - by itself - not very soluble,
and if it has free cysteine residues, you can
try and modify them by carboxymethylation.
See:
http://www.ionsource.com/Card/cmc/method.htm
<https://webmail0.york.ac.uk/redirect/http*3a*2f*2fwww.ionsource.com*2fCard*2fcmc*2fmethod.htm>
This often increases solubility of proteins,
so that you can get away with lower salt
concentrations.
There are several structures in the literature,
where cysteine carboxymethylation was neccessary
for structure determination.
try to work above PI
try adding eg. sulphates or phosphates if they mimick the substrate
Check out the following reference:
Chemical screening methods to identify ligands that promote protein
stability, protein crystallization, and structure determination.
* *Proc Natl Acad Sci U S A. <javascript:AL_get(this, 'jour', 'Proc Natl
Acad Sci U S A.');> 2006 Oct 24;103(43):15835-40. Epub 2006 Oct 11.
Just a quick note: proteins happily crystallize upon salting-in - for
example Concanavalin A dissolves to 100+ mg/ml in concentrated NaCl, then
crystallizes upon dilution of salt (we did it via dialysis but there are
other ways too).
Another happy candidate is the nucleosome protein-DNA complex.
Salting-in is THE procedure to crystallize nucleosomes. We used
salting-in by vapor diffusion (both hanging drops and sitting drops).
Just to give an idea, in our case, if the concentration of the salt in
the stock solution was X, the final concentrations in the drop and
reservoir were X/2 and X/4, respectively.
We have a project in which we have a similar problem: need the salt to keep
the protein happy. My approach has been to use batch crystallization (no luck
yet) to avoid any issues with the salt. Since you make a droplet of known
concentrations, you can (if you wish) keep the salt in there at the
concentration you know you need to keep the protein happy and this will not
change over time.
>
Adding NaCl to 1 M in all reservoir should exactly compensate for
your higroscopy problem. While this will dilute all other components
in the crystallization cocktail, it does so in a predictable an
reproducible way and should not be a problem during screening.
Also an untested idea out of my head, your drops that pick water
actually reduce their NaCl concentration over time, which you say
reduces the solubiluty of your protein. Well, that's exactly what you
want to accomplish to crystallize it, a slow cross over the
solubility limit! Maybe 0.5M NaCl in the reservoir is a nice
intermediate solution?
**********************************
Stefano Benini Ph.D.
http://www.ysbl.york.ac.uk/~benini
<https://webmail0.york.ac.uk/redirect/http*3a*2f*2fwww.ysbl.york.ac.uk*2f*7ebenini>
York Structural Biology Laboratory
University of York
Heslington York YO10 5YW United Kingdom
Tel.:+44 1904 328276
Fax: +44 1904 328266
"verba volant scripta manent"
**********************************
