Dear all,

I'm sending a summary of useful advices which I received on
my email concerning the crystallization of a very acidic
protein. I would like to thank all the people who
responded!

Have a nice day!

Kornelius


There are an number (WT & mutants) of X-ray structures
published on xylose isomerase from A. missouriensis (see
e.g. 1XIM).
This is a highly negatively-charged protein with a pI of
3.2-3.5.

RNase P protein is quite basic (20-25% Arg/Lys).
Crystallization conditions (Stams et al., Science v 280 p
752, 1998) are not particularly informative for your
problem, although notably it could only be crystallized at
3 or 24 mg/ml. DLS revealed that the protein was a monomer
or dimer, respectively, in solution under these conditions.
 
Before cocrystallization with another protein, I would be
inclined to try crystallization from high [salt] or in the
presence of polyamines.
 
*****************************************************

This is a difficult problem. It reminds me of the opposite:
when you have a protein with many positive charges and it
is meant to interact with a negatively charged polymer
known as DNA. When you omit the DNA, frequently you cannot
crystallize the protein presumably because the repulsive
positive charges keep the protein from assuming the correct
conformation. Along that thought, you might try to find
(more) positively charged particles to counteract your
protein charges. I cannot think of positively charged
polymers very quickly, but they must exist and/or it must
be possible to make those. Maybe (arbitrary thought) you
could try positively charged detergent molecules?
 
You write that you have apparently decent CD data
confirming you protein folding. Do you have information on
the protein aggragation? (I would somehow not encourage
dynamic light scattering, it is a pain in the neck.) Size
exclusion chromatography or analytical ultracentrifugation
could help in assessing this. 
 
I am asking about these things because, if you had
confirmation of the aggragation state (notably the
knowledge that the protein does NOT aggragate), then you
could try to use SAXS to determine the global shape and
perhaps the positions of the individual domains. It would
also tell you if the protein is fully folded, or partially
folded (which would be of great importance for
crystallization). It would also tell you how these
parameters change as function of environmental parameters
(pH, ions present, additives), so you might experimentally
determine which conditions/additives help your protein to
be 'best behaved' for crystallization.
 
*****************************************************

Have you run your protein sequence through the FoldIndex
server (http://bip.weizmann.ac.il/fldbin/findex) to see if
it is even predicted to be completely folded? When you have
a protein with many charges, those charged areas are likely
not to be folded, but just hanging out into solvent (since
their interactions will be very favorable).
  
*****************************************************

The problem with these highly negatively charged proteins
is that they are extremely soluble. It is hard to get them
out of solution. You mentioned you tried concentrations up
to 50 mg/ml. This does not surprise me.
Ten years ago we managed to crystallize a halophilic 2Fe-2S
ferredoxin and determined its structure. The protein was
crystallized from 4 M phosphate, pH 7.
It was the only salt that brought the protein out of
solution.
The reference is 
F. Frolow, M. Harel, J.L. Sussman, M. Mevarech, and M.
Shoham. (1996) Insights into protein adaptation to a
saturated salt environment from the crystal structure of a
halophilic 2Fe-2S ferredoxin. Nature Structural Biology
3:451-457. 

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We have worked with a highly basic protein that refused to
even precipitate at concentrations lower than 100 mg/ml.
What finally worked was to co-crystallize it with 
monoclonal Fab that were available from collaborators.

You might consider trying favorite additives for DNA
crystallization,
e.g., cobalt hexamine, spermine, spermidine, etc.

*****************************************************

We managed to crystallize a halophilic protein (very
acidic) in its presumably natural medium (3M NaCl) + around
2M ammonium sulfate. On the other hand, we completely
failed (so far) with other halophilic protein around these
conditions and many others. Have you checked the
proteolytic digestion pattern of your protein ? Could there
be some flexible regions the prevent crystallization ?
 


 ----------------------------------------------
 Kornelius Zeth
 Max Planck Institute for Developmental Biology
 Dept. Protein Evolution
 Spemannstr. 35
 72076 Tuebingen, Germany
 [EMAIL PROTECTED]
 Tel -49 7071 601 323
 Fax -49 7071 601 349

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