Hello
I wish to thank everyone for all the helpful replies. A summary
follows:
While some expressed surprise at the zinc lability, others related
tales of difficulty keeping zinc in a protein.
Suggested ways of overcoming the problem included:
1) Use of TCEP as a reducing agent
2) Allowing Zn to leave and filling the site with Au or Cd
3) Limiting oxygen during crystallization - crystallization in a glove
bag or degassed solutions.
One person noted that TCEP decomposition products include phosphate
and as a result ZnPO4 crystals can form.
I'm off to try suggestion (3). Hope it works and I don't have to
resort to suggestion (2).
Thanks again for the help.
Sue
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
[EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>
Dr. Sue A. Roberts
Biochemistry & Molecular Biophysics
University of Arizona
520 621 8171
[EMAIL PROTECTED]
