Hi,
You may want to have a look at the two papers below.
Experimental determination of van der waals energies in a biological system.
Wear MA, Kan D, Rabu A, Walkinshaw MD. Angew Chem Int Ed Engl.
2007;46(34):6453-6.
The First Direct Determination of a Ligand Binding Constant in Protein
Crystals.
Wu Sy S, Dornan J, Kontopidis G, Taylor P, Walkinshaw MD.
Angew Chem Int Ed Engl. 2001 Feb 2;40(3):582-586.
Regards
Iain
Quoting Jacob Keller <[log in to unmask]> on Mon, 27 Jun
2011 12:04:35 -0500:
> Yes, I think you are right--the somewhat counterintuitive case I was
> thinking of was, for example, when:
>
> Kd = 20nM
> [L] = 20uM
> [Po in crystal] = 20mM
>
> In this case, even though [L] = 20uM, since [L] is 1000 x Kd, the
> occupancy should be ~100%, and [PL] at equilibrium should be about
> 20mM, so in the crystal, the total [L] should be ~20mM. This explains,
> among other things, why bromophenol blue makes crystals bluer than the
> surrounding solution--the Kd is probably significantly lower than the
> BB concentration in the drop.
>
> Thanks for your clarifications!
>
> Jacob
>
> The question would remain, then, whether there is any utility in
> titrating ligands into crystals, and monitoring occupancies as a
> readout for binding. Although crystallization conditions are horribly
> non-physiological, perhaps there would be utility in the case where
> there are multiple known binding sites of various affinities, and
> other methods would have trouble resolving the binding events. One
> could start with:
>
> 1. totally saturated conditions, set occ=1 for all sites, refine B's, then
> 2. fix B's at this value, and refine the occ's in a subsequent series
> of dilutions.
>
> All of this is not totally theoretical--I am considering a set of
> experiments along these lines, where there really are multiple sites
> of varying affinity.
>
> *******************************************
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> cel: 773.608.9185
> email: [log in to unmask]
> *******************************************
>
>
Dr. Iain McNae
School of Biological Sciences
Institute of Structural and Molecular Biology
The University of Edinburgh
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