From: Caitlyn Claire Yeykal
Date: 5 November 2011 22:02
Hi -- has anyone had crystals that are colored in regular (unpolarized) light? Mine are yellow, and I'm not aware of anything in the buffer conditions that might cause this. I read online that glutaraldehyde can turn protein crystals a golden color, but as far as I know there isn't any of that in the well. Purified in HBS pH 7.2; crystallized in LiCl/PEG4K/Tris pH 8. Any explanations?
Thanks!
Caitlyn
____________________________________
Caitlyn C. Yeykal
Mrksich Group/Adams Group
Dept. of Biochemistry, University of Chicago
929 E. 57th St., Rm 547B
Chicago, IL 60615
caitlyn@uchicago.edu
----------
From: Nat Echols
Many cofactors can cause this - the ones I'm familiar with are LLP and
NAD, but there are quite a few others, and they will often co-purify
with the proteins that bind them. Possibly certain metals as well,
but I'm less certain about that. You may be able to identify the
responsible molecule from the absorbance across the visible light
spectrum, although I found it difficult to track down information on
peak absorbances when I last tried this several years ago.
-Nat
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From: Caitlyn Claire Yeykal
Thanks for all the replies -- there are no suggestions in the literature or in crystallized or predicted domain structures that this protein binds a cofactor, and, although I did purify it in insect cells, PAGE gels and activity assays support the assertion that it's not ferritin. Nobody has seen any metal ions bound, either, but there are a few domains that haven't been crystallized, so maybe. Again, thanks for all the possibilities; will keep them in mind.
Caitlyn
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From: REGINALD MCNULTY
It's also possible that there's oxidation in the buffer causing the yellow color. I'm not sure how common this is with HEPES. But I see it all the time with MOPS. Alternatively, does the yellow color bind the column during purification? If so, then it sounds like a co-purified flavin or protein.
Best regards,
Reginald McNulty
----------
From: Craig A. Bingman
In another thread, you indicated that there were no identifiable cofactor binding sites in your protein, so we are down to less common situations. Some proteins are spontaneously decorated with pyridoxal on surface lysine residues. In some cases, this has absolutely nothing to do with the enzymatic activity of the protein.
On Nov 5, 2011, at 5:02 PM, Caitlyn Claire Yeykal wrote:
----------
From: Artem Evdokimov
----------
From: Adrian Goldman
----------
From: Bosch, Juergen
Date: 5 November 2011 22:02
Hi -- has anyone had crystals that are colored in regular (unpolarized) light? Mine are yellow, and I'm not aware of anything in the buffer conditions that might cause this. I read online that glutaraldehyde can turn protein crystals a golden color, but as far as I know there isn't any of that in the well. Purified in HBS pH 7.2; crystallized in LiCl/PEG4K/Tris pH 8. Any explanations?
Thanks!
Caitlyn
____________________________________
Caitlyn C. Yeykal
Mrksich Group/Adams Group
Dept. of Biochemistry, University of Chicago
929 E. 57th St., Rm 547B
Chicago, IL 60615
caitlyn@uchicago.edu
----------
From: Nat Echols
Many cofactors can cause this - the ones I'm familiar with are LLP and
NAD, but there are quite a few others, and they will often co-purify
with the proteins that bind them. Possibly certain metals as well,
but I'm less certain about that. You may be able to identify the
responsible molecule from the absorbance across the visible light
spectrum, although I found it difficult to track down information on
peak absorbances when I last tried this several years ago.
-Nat
----------
From: Caitlyn Claire Yeykal
Thanks for all the replies -- there are no suggestions in the literature or in crystallized or predicted domain structures that this protein binds a cofactor, and, although I did purify it in insect cells, PAGE gels and activity assays support the assertion that it's not ferritin. Nobody has seen any metal ions bound, either, but there are a few domains that haven't been crystallized, so maybe. Again, thanks for all the possibilities; will keep them in mind.
Caitlyn
----------
From: REGINALD MCNULTY
It's also possible that there's oxidation in the buffer causing the yellow color. I'm not sure how common this is with HEPES. But I see it all the time with MOPS. Alternatively, does the yellow color bind the column during purification? If so, then it sounds like a co-purified flavin or protein.
Best regards,
Reginald McNulty
----------
From: Craig A. Bingman
In another thread, you indicated that there were no identifiable cofactor binding sites in your protein, so we are down to less common situations. Some proteins are spontaneously decorated with pyridoxal on surface lysine residues. In some cases, this has absolutely nothing to do with the enzymatic activity of the protein.
On Nov 5, 2011, at 5:02 PM, Caitlyn Claire Yeykal wrote:
----------
From: Artem Evdokimov
When we were working on PheRS we noticed that our protein preps (and crystals) had shades of color: sometimes they were pinkish and sometimes yellowish, or even blueish (and often colorless)!
We solved the structure eventually and found a new metal-binding microdomain previously not found in these transferases. The funky colors were caused by the microdomain binding various metals, depending on how those particular fermentations were done and how many purification steps were taken :) In the deposited structure luck of the draw had Zn in the site. It just goes to show that proteins have plenty of tricks left up their metaphorical sleeves.
Artem
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From: Adrian Goldman
I agree: light yellow (straw-yellow) proteins often indicate metal-binding - it's typically iron III. Zn salts tend to be colourless, and the Mn-pink is too pale to be visible at protein (mM) concentrations. This can be determined by doing flame-spectroscopy, if you don't mind destroying your protein, or at the beam by scanning the various (first-transition metal) edges - because if it is adventitious metal binding, it's likely to be one of the obvious metals.
Adrian
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From: Bosch, Juergen
Cobalt leaching of TALON resin perhaps ?
Should be more orange type of color, but it depends on the concentration.
In any event if you shoot those crystals run a scan to find out what metal is bound and use it for phasing if you have access to a MAD line.
Jürgen
......................
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
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