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. ***************************************************** 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
