peotein Structural variation - ..plz help (Apr/20/2007 )

Hi

Which is the best method to identify structural variations in a protein?
I am working with a recombinant protein which I believe to have a variation in folding pattern from the standards available.
But when I did CD spectra of my protein with respect to std. they matched.

So is there any other method to investigate the difference?
Also how do you differentiate the difference in 3-D level (what I mean is that is it a possibility that my secondary structures matches but my tertiary Don’t how do I find this out?)
Plz help

Thanks a lot

If your secondary structure is very similar between the two variants, but they have different tertiary structures, you may have to use use NMR or X-ray crystallography to show atomic level differences. You might get lucky with some gel filtration, or possibly some analytical ultracentrifugation if you can show the protein is rod-shaped or globular etc, compared to the mutant.

What makes you suspect the mutant has a different structure?
If you have something odd happening with domains, you could try some hydrophobic interactions, or mild reverse-phase, but I think you'd be drawing a pretty long bow to get low-resolution data accepted by a reviewer unless there is some gross change in the elution profile...

hi

well i did peptide mapping of my protein in two different ways..when it was done under native condition my in house protein showed variation..while when i opend up my protein by denaturation and reduction they perfectly matched with the profile of std.

well i am hoping that the variation is in 3-d level...
thanks for ur suggestion will try NMR....
any other ides...plz pool in thanks