Here is my two cents...
How strong zinc is captured by the protein is very protein dependent: I
always thought that a great case for this variability was the protection
of RING ubiquitin ligases against NEM. Cysteine-catalyzing HECT
ubiquitin ligase are killed by NEM; RING finger ligases, where zinc is
assumed to be only structural, are thought to be NEM resistant... Well,
until you actually do the experiment and see that some RING ligases are
also inactivated by NEM! My theory was that I had a case of weak zinc
coordination, where oxidation of the zinc-coordinating cysteines was a
big issue. DTT made it worse, TCEP kept the protein unaggregated the
longest.
That's just one protein, though.
Engin
Artem Evdokimov wrote:
Please note that TCEP decomposes and one of the decomposition products
is phosphate. Enough TCEP and you might have Zinc Phosphate crystals
which can sometimes look very odd and ‘protein looking’.
Artem
------------------------------------------------------------------------
*From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf
Of *Jennifer Han-Chun Tsai
*Sent:* Thursday, September 25, 2008 6:18 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] losing zinc during crystallization
I don't know if anyone had experience of TCEP inducing zinc acetate to
precipitate. This paper mentioned this.
The Crystal Structure of the Olfactory Marker Protein at 2.3 Å Resolution
*Paul C. Smith, Stuart Firestein and John F. Hunt*
*Journal of Molecular Biology
<http://www.sciencedirect.com/science/journal/00222836>
Volume 319, Issue 3
<http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%236899%232002%23996809996%23319471%23FLA%23&_cdi=6899&_pubType=J&view=c&_auth=y&_acct=C000049198&_version=1&_urlVersion=0&_userid=952835&md5=2d1212952cf958e128dad1358783edf4>,
7 June 2002, Pages 807-821 *
I saw if protein sample containing TCEP would either form
precipitation or weird and rounded shape of small molecule crystal but
not salt. I have tried to crush that rounded shape crystal. It's quite
soft, very similar to the way protein crystals got crushed.
Does anyone have this experience? Or actually this kind precipitation
could get nice crystal after optimization?
Thanks,
Jennifer Tsai
On Thu, Sep 25, 2008 at 4:53 PM, Eric <[EMAIL PROTECTED]
<mailto:[EMAIL PROTECTED]>> wrote:
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"
<[EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>>
To: <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>>
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
[EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>
