[ccp4bb]: SUMMARY: Selenomethionine oxidation during RP-HPLC

[Axel Innis <axel@cryst.bioc.cam.ac.uk>]

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Here's a summary of the suggestions I got for the following question:

I'd like to purify a small disulphide-rich protein containing
selenomethionine for MAD on a C8 reverse phase column. The buffers I
normally use contain 0.1% TFA and Acetonitrile and are purged with helium,
but the disulphide bonds in my protein don't allow me to use a reducing
agent such as DTT or beta-mercaptoethanol. If anyone has had to deal with
a similar case before, could they please let me know whether the
selenomethionine became chemically modified during this purification step.

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SUGGESTIONS:

- Why not do the structure with oxidised SeMet protein. Oxidised selenium
gives a stronger MAD signal than reduced selenium. The problem is when you
get mixed oxidation states, because then you don't get any absorption peak
at all. That's why most people add DTT to their crystallisation buffers.
You could try crystallising in the presence of an oxidising agent instead.
If your problem is not that huge (i.e. relatively few seleniums [20-ish],
relatively small protein [30 kDa-ish]), then you can probably even get
away with using the remote wavelength, where there is no absorption peak
and a relatively weak anomalous signal.  But then you have to collect your
data properly.

- If your protein's folded and disulphided, and you then add DTT, does it
unfold?  Because if not, it's often okay to reduce the SeMet only just
before you freeze the crystal, because the SeMet oxidation is reversible.
So, you purify and crystalize the thing without DTT, and in the minutes or
hours before mounting, you add the DTT.

- Have you considered using the sulphur anomalous scattering to solve the
phases? May be possible with sufficient resolution (e.g. using Acorn).



Thank you all for your suugestions,

Axel