Hello Eric,
Way back in the '80s as a rookie graduate student, I took on a similar
task. The crystal structures of RNase A all had a negatively
charged ion (sulfate, phosphate or a nucleotide) bound between the two
active site His residues, but we wanted to know if the His side chains
maintained their position without an anion there.
I don't know if this description will help, but ...
The crystals were grown at pH 5.5 (in 40% methanol at 4 degrees C) and
we were able to raise the pH to 9 without destroying the crystals. The
pH maintained at pH 9 for a week and then was reduced again to about
6.8 without the addition of a phosphate- or sulfate-containing buffer.
All of this was done at 4 C. Admittedly this was RNase A and the
crystals grew to millimeter size in about a week, which makes
manipulation somewhat easier. It did take a few attempts with different
rates of changing the pH to succeed in not destroying the crystals.
In the end we found a water had replaced the sulfate ion:
http://pubs.acs.org/doi/pdf/10.1021/bi00400a013
Cheers,
Rob
On Mon, 2016-07-18 13:08 +0000, "Lewandowski, Eric"
<[log in to unmask]> wrote:
> Hello All,
>
> I am looking for advice about how to remove a phosphate from my
> protein active site. Every time I solve the structure of my protein
> (which is a phosphatase), there is a phosphate in the active site,
> and what is curious is that no phosphate is present in any of the
> purification buffers or crystallization condition (there is no
> sulfate present either). We have confirmed with Mass Spec that the
> phosphate is not covalently linked to the protein. Both soaking and
> co-crystallization with compounds fail to remove the phosphate. Does
> anyone have any experience/suggestions about how to get rid of
> phosphate in a situation like this?
>
>
> Thank You,
>
> Eric Lewandowski
>
>
>
> ----------------------------------------------------
> Eric M. Lewandowski, Ph.D.
> University of South Florida
> Morsani College of Medicine
> Department of Molecular Medicine
> 12901 Bruce B. Downs Blvd. MDC 07
> Tampa, FL 33612-4799
--
Robert L. Campbell, Ph.D.
Adjunct Assistant Professor
Dept. of Biomedical & Molecular Sciences, Botterell Hall Rm 644
Queen's University,
Kingston, ON K7L 3N6 Canada
Tel: 613-533-6821
<[log in to unmask]> http://pldserver1.biochem.queensu.ca/~rlc
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