I had success using crystallography to measure the Ca2+ affinity (in the
mM range) for a Ca2+ dependent enzyme.
See:
Characterization and implications of Ca2+ binding to pectate lyase C.
Herron SR, Scavetta RD, Garrett M, Legner M, Jurnak F.
J Biol Chem. 2003 Apr 4;278(14):12271-7.
We measured the occupancy of the Ca2+ ion using three different pH's and
3-4 different Ca2+ concentrations. The presence of the Ca2+ ion altered
the conformation of two residues in the binding pocket. In several of the
Ca2+ soak experiment the occupancy was between 35% and 70%, where both
orientations of the side chains could be modeled separately and their
occupancy values refined (see attached picture). We confirmed our
crystallographic Kd approach using tryptophan fluorescence. Since it was
difficult to measure mM binding affinities using dialysis or titration
calorimetry, we turned to crystallography (since we had lots of crystals
and beam time).
Steve
Jacob Keller wrote:
>Dear Crystallographers,
>
>what is the dogma with regard to affinities in crystals? For example,
>if I soak three crystals in 1pM, 1nM, and 1uM compound X, and they all
>show equivalent density, does that mean that the affinity is really
>better than 1pM, or is the crystal of such a high local concentration
>(~600mg/mL) that it will be fully occupied at nearly any
>concentration, provided external ligand concentration does not change
>due to binding in the crystal? I guess there is also the problem that
>the crystallization solutions are very non-physiological, but
>neglecting that, is there any straightforward way to think of this, or
>is there a good reference?
>
>Jacob Keller
>
>
>
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