Fab:Peptide complex crystallizationHi Christine, I never worked with Fab, but I do have a little experience with DMSO.
I had a case in which I could grow my crystal in 10-15% DMSO - to use it as cryoprotectant. But in another case, 5% DMSO inhibited my crystal's growth. When I had DMSO as cryo, I found lots of pyramid shaped electron densities on my protein surface, which should be the DMSO molecules. These DMSO molecules sit in little indents on protein surface where the surrounding environments are relatively hydrophobic. My ligand (sugar) binding was not affected. I think DMSO is generally thought to weaken interactions, but not too much. For example, in PCR, DMSO is used for increasing the specificity of the primers, i.e., weakening the base pairing a little. If you are going to add 1/10V of the 10% DMSO peptide solution to 1V protein, then the final DMSO concentration is only 1%. I do not think that will be too detrimental for the protein to crystallize, or for the peptide to bind. But beyond 5%, I would be cautious and will try gel filtering or dialyzing the mixture if the initial screen fails (but it may work!). Zhijie From: Harman, Christine Sent: Monday, July 19, 2010 1:37 PM To: [email protected] Subject: [ccp4bb] Fab:Peptide complex crystallization Hi all, I was wondering if anyone has had any experience with setting up Fab:peptide complexes for crystallization. I have Fab protein and peptide and I am not sure how to add the peptide to the Fab protein. My peptide is hydrophobic and thus not very soluble in standard aqueous buffers and barely soluble in some organics. The Fab protein I have is currently in 100mM Sodium Acetate pH5, 150mM NaCl. My current plan is to dissolve lyophilized peptide first in 10% DMSO and use this stock to add to Fab protein in a 20:1 or 10:1 molar ratio. I am not sure of the effects of residual DMSO on peptide binding to Fab or on crystallization. I have read one paper in which they rave about DMSO as an additive in their crystallization, but that protein was not Fab like at all. Any insight you all might have on this would be greatly appreciated. Thanks in advance, Christine
