Hi,
sorry, it should read "salt in" not "inverse salt in" ...
- J. -
Am 05.12.13 19:55, schrieb mesters:
Hi,
Showers of crystals often occur if the protein is not that
soluble/happy/stable in the solute. The solubility of a protein
depends on its concentration, its pI, pH of solute, temperature,
compounds in the solute (e.g. salts and small organic molecules),
construct used, to name a few. So, to increase the solubility, you
need to change one these parameters. Salts in general are said to
inverse "salt in" the protein thus making it more soluble. Hofmeister
published an interesting paper on this in 1888! NaCl for example is
placed in the middle of the Hofmeister series, neutral so to speak,
and practice shows it is often a good choice to start with at a
concentration of 150-200 mM. However, occasionally it is not a good
choice and will have negative effects.
The choice of salt basically depends on the pI of the protein and the
pH of the solute/crystallization condition. Have a serious look at the
chapter by Madeleine Riess-Kautt about the Hofmeister series in A.
Ducruix und R. Giegé book with title "Crystallization of Nucleic Acids
& Proteins. A practical Approach" (Oxford IRL-University Press (1992)).
For a "normal" hen egg white protein (pI/IEP below the pH, protein
overal negatively charged) he was working on, ammoniumsulfate will
precipitate (stabilize/crystallize) the protein while a perchlorate
salt (destabilize) will dissolve the protein completely at high
concentrations. As you generally need high protein concentrations for
starters in crystal growth, you thus need to add a salt that
"dissolves" the protein slightly without denaturing it (low
concentration 50 -200 mM in the solute). Then, you can add a second
salt (on the opposite site of the series) or precipitant like PEG to
crystallize it. However, for several proteins the pI is located above
the pH of the solute. In these cases the protein is positively charged
and the Hofmeister series turns around! We recently had a case like
this producing lost of tiny plate-like crystals. Changing the protein
concentration or the precipitant concentration, or hanging to sitting
etc. did not help at all. We finally found out we had to add some
ammoniumsulfate to "solubilize/dissolve" the protein first and got
very nice single and large crystals in the end.
In your case, try to replace the glycerol by the proper choice of
salt! You can also try to exchange MOPS (zwitterion) for another
buffer compound. All this will also change the crystallizaiton
behaviour. You will most probably need to redo the screening...
- J. -
