Hi,
We had a paper where we looked at Kd of arginine in the arginine
repressor-DNA complex (p. 248-249).
JMB,2010, *399*, pp.240-254.
Maia
Jacob Keller wrote:
> 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]
> *******************************************
>
>
>
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