Protein crosslinking before crystallization

From:

Opher Gileadi

Hi all Does anyone have experience or insights on intramolecular crosslinking of proteins before crystallization? The idea is to lock a felxible (multidomain) protein into a restricted conformation. Thanks, Opher

Hi Opher, I seem to recall some old papers in which glutarldehyde was used (in the RESERVOIR only) in order to promote crystallization of proteins with suspected inter-domain flexibility. Unfortunately, I don't have specific references in mind. Cheers, Boaz I found this paper to be very helpful for a similar experiment. "Elucidation of the mechanism and end products of glutaraldehyde cross-linking reaction by x-ray structure analysis". Wine et al. Biotechnology & Engineering, Vol. 98, No. 3, October 15, 2007. Hi Opher, I have a project where a protein was made 'cysless' and selective cysteines were introduced to allow for directed crosslinking using MTS and aldrithiol activated reagents (see Toronto Research Company and Pierce for a range of reagents of different lengths). A side product of these crosslinks was a dramatic improvement in crystal diffraction. Katherine Rank Rayment Lab Thanks all for the comments. I was thinking of crosslinking a protein that hasn't crystallized. Cystein engineering seems a good idea but depends on the availability of a good model. We'll be trying mild crosslinking using bifunctional reagents of various lengths (I suspect glutaraldehyde will not be the best option). Opher