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:[log in to unmask]] *On Behalf
> Of *Jennifer Han-Chun Tsai
> *Sent:* Thursday, September 25, 2008 6:18 PM
> *To:* [log in to unmask]
> *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 <[log in to unmask]
> <mailto:[log in to unmask]>> 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"
> <[log in to unmask] <mailto:[log in to unmask]>>
> To: <[log in to unmask] <mailto:[log in to unmask]>>
> 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
> [log in to unmask] <mailto:[log in to unmask]>
>
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