Based on my own experience with zinc-metalloenzymes with thiolate
ligands, it's usually more a problem to get the zinc OUT than get it IN.
Zinc is pretty thiophilic, so removing it once ligated in a multiple Cys
environment is often difficult. Have you tried TCEP as a reducing agent
for protecting properly zinc-populated protein?
Cheers,
--
------------------------------------------------------------------------
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [log in to unmask]
Sue Roberts wrote:
> 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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