I am working with a protein/ligand complex that is very resistant. to getting a stable complex. The first thing to try is to go to saturating ligand concentrations, even if it seems that it is excessive. It is possible that binding affinity is significantly different.
My biggest problem is precipitant interfering with binding, combined with relatively low substrate solubility. I tried transferring to a different condition, but it seems that most precipitating agents have a solubility affect on the ligand as well. I ended up screening conditions just to understand ligand solubilities. I even tried things like DMSO additives. Eventually, I found conditions where the ligand is fairly soluble, but also stabilizes the protein. I found that ethylene glycol helped binding even though glycerol inhibited it. I also changed the pH, and did the soak at room temperature instead of in the cold room. Even then, I used saturating ligand concentrations. Joe Krahn Nian Huang wrote: > Dear All, > > I have been trying to get a protein-ligand complex crystal for a long > time. Here, ligand is either substrate or product for this kinase. I > have tried many methods: soaking with apo-crystal, co-crystal with > different concentration of ligand and screen for new conditions. I got > a couple of co-crystals with the new conditions, but still no ligand > was found in the active site. > > Anybody has some new ideas that I can try? I have been using glycerol > as my cryoprotectant. Could cryoprotectant have some negative effect > on co-crystal? I will try to collect some data at room temperature in > the future. > > Thank you in advance for your suggestion. > > Nian > > Dept of Biochemistry > UT Southwestern Medical Center
