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 <[email protected]>:
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: [email protected]
Subject: Re: [ccp4bb] distinguish ligand binding sites within a
protein
To: [email protected]
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
<[email protected]> 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
<[email protected]> 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
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Bärbel Blaum, Ph.D.
Interfakultäres Institut für Biochemie (IFIB)
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Germany
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