Hi Sue,
I fully agree with Thierry, you might have to cyrstallize the protein
under exclusion of dioxygen. There are many metallo proteins which have
to be crystallized that way. But a first attempt might be the TCEP as
already suggested by many others. If you need advice regarding
crystallization without O2, send me a note.
Good luck!
Guenter
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]
