If I understand the original post correctly, the binding sites in question are not chemically identical. While it's possible that lattice may invert the order in which sites are occupied, it is not very likely given that affinity gap is sufficient to be observable by ITC.
Mutagenesis is a good option too. On Tue, 2013-11-19 at 17:12 +0100, Bärbel Blaum wrote: > Hello, > > we work with proteins that have typically several chemically identical > binding sites (viral capsid proteins fully assembled or as multimeric > assembly-intermediates). Depending on how long at which concentrations > they are soaked the chemically identical ligand pockets within one > asymmetric unit are typically occupied to different levels purely > because of individual crystal contacts and accessibility. I therefore > think that neither soaking with different concentrations nor B-factor > analysis are solid methods to determine some sort of relative > affinities. I'd suggest to design mutants for either binding site and > ITC measurements with the mutant proteins. This might also tell you if > some sort of co-op exists between both sites. > > Baerbel > > Quoting Ed Pozharski <epozh...@umaryland.edu>: > > > IMHO, while explaining binding affinity from a structure is fun, it does > > not prove anything. Assuming that I understand your situation > > correctly, you can (relatively) easily find out from experiment which > > pocket has higher affinity. Just do soaks with different ligand > > concentrations - the expectation is that the weaker binding site will > > become partially occupied first. > > > > On Tue, 2013-11-19 at 04:58 +0000, Xiaodi Yu wrote: > >> Hi Wei: > >> > >> Based on the structure, you can calculate the binding surface between > >> the protein and the ligand. Maybe the two binding pockets will give > >> you two different numbers. And the larger one usually can have the > >> higher binding affinity. You also can analyse how the ligand > >> interacts with the protein though hydrophobic or electrostatic > >> interaction , etc? the last, you may also compare the b factors of > >> the ligand or the protein binding pocket regions after you refining > >> the structure. These things may give you some hints about which > >> binding site is more strong. > >> > >> Dee > >> > >> > >> ______________________________________________________________________ > >> Date: Mon, 18 Nov 2013 22:45:58 -0500 > >> From: wei.shi...@gmail.com > >> Subject: Re: [ccp4bb] distinguish ligand binding sites within a > >> protein > >> To: CCP4BB@JISCMAIL.AC.UK > >> > >> Thank you so much for the suggestions, Tomas! Yes, my ligand is a > >> small molecule. I have the crystal structure of the ligands bound to > >> the protein, do I still need to computationally dock the ligand to the > >> two pockets, can I calculate the parameters of binding directly using > >> the crystal structure? > >> > >> Best, > >> Wei > >> > >> > >> > >> On Mon, Nov 18, 2013 at 9:03 PM, Tomas Malinauskas > >> <tomas.malinaus...@gmail.com> wrote: > >> Dear Wei Shi, > >> is your ligand a small molecule? If it is a small molecule, I > >> would > >> try to computationally dock the small molecule to two pockets > >> separately using AutoDock, and look at the estimated free > >> energies of > >> binding. > >> Best wishes, > >> Tomas > >> > >> On Mon, Nov 18, 2013 at 8:55 PM, 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 > >> > >> > >> > > > > -- > > Edwin Pozharski, PhD, Assistant Professor > > University of Maryland, Baltimore > > ---------------------------------------------- > > When the Way is forgotten duty and justice appear; > > Then knowledge and wisdom are born along with hypocrisy. > > When harmonious relationships dissolve then respect and devotion arise; > > When a nation falls to chaos then loyalty and patriotism are born. > > ------------------------------ / Lao Tse / > > > > > -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore ---------------------------------------------- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. ------------------------------ / Lao Tse /
