Hi Monica, Yes, I am thinking of doing mutagensis to disrupt binding and do ITC again. Any suggestions on which and how many residues I should mutate to disrupt the binding of the ligand? As I mentioned before that one of the two binding sites, which I am thinking of mutating first, has the following interactions with the ligand: 5 residues are involved in forming 7 pairs of the hydrogen bonds with the ligands, 1 residue is involved in electrostatic interaction with the ligand, 14 residues (including 3 that forms the hydrogen bonds with the ligands) are involved in hydrophobic contacts with the ligand. I am not sure how many residues I should mutate in order to completely abolish the ligand binding to this pocket assayed by ITC. I also checked the same proteins from other species which could potentially bind the same ligand in the pocket, and found out that: 3 out of 5 that forms hydrogen bonds with the ligands are conserved, 1 residue that forms electrostatic interaction is conserved, 1 residue that is involved in the hydrophobic contacts is conserved.
Any suggestions or ideas about which and at least how many residues I should mutate to abolish the binding? Any rules or criteria to predict which residues might be more important and should be mutated first? Thank you so much! Also, I used DSXONLINE to rank the two binding sites according to binding free energy and get the prediction there, and will go on to use some other programs to see whether I get the same prediction. Best, Wei On Wed, Dec 11, 2013 at 11:22 PM, Monica Mittal <monica.mitta...@gmail.com>wrote: > Hi > I also have same the case for one of the protein i am working on and I can > suggest that you can try simple mutagenesis approach for one of the binding > site andcompare the affinity with the wildtype and repeating the same for > the second binding site. This will give you a better idea of which site is > high affinity one. > > Hope it works. > Monica > > > On Tue, Nov 19, 2013 at 7:25 AM, Wei Shi <wei.shi...@gmail.com> wrote: > >> Hi all, >> I got the crystal structure of a transcription factor, and every monomer >> binds two molecules of the same ligand in different binding pockets. And I >> also did the ITC experiment, titrating the ligand into the protein, and got >> a U-shaped curve. The binding affinity for the first binding site is higher >> than the second binding site. >> I am wondering whether I could computationally determine from the >> protein-ligand complex structure that which binding site has higher >> affinity for the ligand and correlate the binding sites with the parameters >> I got from ITC experiment. >> Thank you so much! >> >> Best, >> Wei >> > >
