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?
Is it a hypothetical question, or experimental result?
From a simplistic viewpoint, concentration of the protein does not
affect the Kd, i.e. the concentration of free ligand at which the
sites are half occupied.
All the usual equations work, but you have to remember
that [Ligand] refers to concentration of _free_ ligand,
and since the protein concentration is so high (100 uM to 1 mM say)
in the drop, that free ligand is likely to be a small fraction of total.
Thus if you add 1 pM ligand to the drop, you will not get significant
binding even with infinitely high affinity. If you dialyze your crystal
against liters and liters of 1 pM ligand solution, you might get
high occupancy, and that would indeed mean the Kd is < 1 pM, but
i don't think that is very practical. Basically you need to add equimolar
ligand plus an additional concentration say 10 x the Kd to satisfy the
Maybe if you soak a single tiny crystal in a reasonalble volume of
buffer at 1 pM, it would be enough? say (0.1 mm)^3 crystal containing
1 mM biding sites (which corresponds to a rather large ASU, most
crystals would be higher) you would need 10^6 times the volume
or 1 ml of 1 pM solution to have one ligand for every site.
But then the free ligand concentration would be zero.
Jacob Keller
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Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
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