Hi Xiaopeng
To add to Artem's comments:
Does the presumed gsh make a mixed disulfide in the active site?i.e. is it
covalently bonded to the active site via a s-s bond?
If yes then MS on your purified sample should easily give you the answer.
If a mixed s-s is indeed the scenario then purifying the enzyme in the presence
of reducing agents and playing with the pH a bit should yield a gsh free
preparation.
Best regards
Savvas
On 16 Mar 2011, at 02:19, Artem Evdokimov artem.evdoki...@gmail.com wrote:
Tightly bound ligands commonly survive purification :) Several unexpected
discoveries have been made this way!
If you think your stuff is GSH, soak some 'real' GSH or co-crystallize with
it, and see if density shape changes from what you had before. This is not
guaranteed to work because sometimes the ligand may be bound so
tightly/exchange slowly that the original ligand just won't budge. This was a
significant issue with some of the kinase inhibitors and nuclear hormone
receptor antagonists that I've had a chance to work with - the binding was
almost 'one way' in real-time situation, and (partial) denaturation was
required to get the ligands out.
If your ligand is indeed GSH, in equimolar amount with the protein, then you
could also try MS as detection technique. The ligand should come off when
protein is subjected to the normal MS environment (typically 0.1% TFA or
formic acid, in a mixture of water and acetonitrile, etc.). To detect it,
don't forget to expand the mass window to get the mass, and set the
ionization mode to positive - you should see a clear 308.3 Da peak, with a
lovely isotope splitting pattern (assuming you have access to MS). In
negative mode the mass will be 1/2 of the already low m.w. of 307, since GSH
has two negative charges. Notably, GSH should also accept e.g. an
iodoacetamide group on the -SH, meaning that you should be able to treat
crystals with iodoacetamide and observe the addition of -CH2CONH2 to the
sulfur. Naturally, the S atom should be pretty prominent in the density
anyway. Ditto mercurials, but they may wreck the crystal. Since your enzyme
may be a GST (assumption on my part here) it also may present GSH to be
reactive with whatever substrates that yor GST is targeting, so you may be
able to identify conjugation products.
Artem
2011/3/14 Xiaopeng Hu huxp...@mail.sysu.edu.cn
Dear all,
Sorry for this off topic question.
We are working on protein/inhibitor complex structure although we can not get
our inhibitors in. However, we did find a strange density at the active site,
it looks really like GSH, the natural co-enzyme of thiis protein.We tried to
use very simple solution to get crystal then exclude the possiblity of buffer
moleculors,but that density is aways there.
I am wondering how this ligand (if it is GSH) can survive all purification
steps and want to indentify it. Are there any methodes to do this work? Let's
say to pick up a crystal and do some analysis?
Many many thanks!!!
Xiaopeng
