Dear Sue,
This is a very interesting case! Normally the Zn-S bond is quite strong - so
it's an unusual situation to have. Did you already attempt to purify the
protein in the presence of tiny quantities of Zn? Also, what buffers and
other components are you using during purification, and what is the chemical
composition of the crystallization solution (if you don't mind telling)?
Finally, does the purified protein have high activity before
crystallization? In other terms - does it have full Zn occupancy before you
crystallize it - or does the disulphide form during expression or
purification? This would change some of the other solutions that you might
attempt.
Cheers,
Artem
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Sue
Roberts
Sent: Thursday, September 25, 2008 5:36 PM
To: [log in to unmask]
Subject: [ccp4bb] 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
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