Dr. Artem Evdokimov
eudokima at mail.ncifcrf.gov
Wed Aug 8 11:30:50 EST 2001
> Like you yourself frequently notice, there are no hard rules with
> proteins. It all depends...
Yup :) I just have this irrational fear or organic solvents + proteins
for crystallization. It is not entirely justified, I know.
> concentration kept protein active and soluble. On another extreme
> is a case where acetone pption of 32K protein in the end produced
> xtals scattering to 2.3A in area detector.
Lucky ! We have a score of proteins that are (according to the methods
we used) folded, very pure, soluble, homogenous - but - no crystals :(
We're trying surface residue mutations now...
> P.S. A question: protein from inclusion bodies spontaneopusly
> misfolds into something 100% inactive but 100% soluble. What is the
> best way to see if resulting sup is homogenous or not? More
> filosophical Q: what are the chances of this conformation being
> relevant to anything? (In other words, did Nature missed it or went
> for a trouble of using alternative that does not fold spontaneously?)
Ummm... So I am not sure about 'what's the best way' but here is what I
would have done:
1) Size exclusion
2) Static light scattering (if you don't have static, use dynamic but -
3) Native gels at pH 6.5 and 8.5
If the protein appears homogenous with these techniques, chances are
that it really is.
If you are masochistic, you can try NMR on your sample, see if you can
get some sort of a spectrum, check how broad are the lines, see if
there's anything in the 'fingreprint' region etc. Kind of high price to
pay for just a yes or no answer, though.
Can you try refolding your protein around a substrate-mimic or inhibitor
|Dr. Artem Evdokimov Protein Engineering |
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