We had a zinc-finger containing protein that we were soaking with different
heavy atom compounds. It turns out KAu(CN)2 provided the best diffraction
of several soaks. We found out it was because the gold had replaced the
zinc ion and was coordinated by the Cys and His's. The lab nickednamed the
protein Goldfinger! Although we didn't have the problem with disulfides,
perhaps a similar gold soak might help if you tried to crystallize the
protein in the presence of TCEP as well.
HTH
----- Original Message -----
From: "Sue Roberts" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Thursday, September 25, 2008 4:35 PM
Subject: losing zinc during crystallization
> Hello Everyone
>
> I've been trying to crystallize a zinc-containing enzyme for what
> seems to me to be an eternity. The protein contains stoichiometric
> zinc (1 zinc/ protein monomer) when isolated and the zinc is required
> for activity. Each crystal we've obtained has lost the zinc and
> contains a disulfide bond between two cysteine residues that should be
> zinc ligands (based on structures of similar proteins).
>
> We've tried crystalizing in the presence of reducing agents,
> crystallizing with substrate analogs, and supplementing the
> crystallization drops with zinc with no success (and combinations of
> these approaches). We've obtained a variety of crystals and
> determined structures, but none contain any zinc.
>
> Attempts to insert zinc into the crystal (zinc + reducting agent or
> zinc alone) have not been successful.
>
> Does anyone have any tricks to suggest that might help?
>
> Thanks in advance.
>
> Sue
>
> Dr. Sue A. Roberts
> Biochemistry & Molecular Biophysics
> University of Arizona
> 520 621 8171
> [log in to unmask]
>
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