Dear Wei, I would put something big in the binding site like a tryptophan, to physically block access of the ligand. Check beforehand in coot if and at which position such a residue would fit best. Best, Herman
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Wei Shi Gesendet: Donnerstag, 12. Dezember 2013 06:04 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] distinguish ligand binding sites within a protein 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<mailto: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<mailto: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
