Dear bb,
I have a high resolution dataset showing that the N-terminal threonine
of my protein has been modified and cyclized, with the main chain N and
side chain OG1 now involved in a non-planar 5 membered ring sugar-like
structure. The molecule seems to be 5-6 atoms long with some 'head group'.
http://studwww.ugent.be/~jeleghee/ccp4/coot_front.png
http://studwww.ugent.be/~jeleghee/ccp4/coot_side.png
http://studwww.ugent.be/~jeleghee/ccp4/coot_angle.png
Modification must have occurred in the drop since N-terminal sequencing
through Edman degradation after it came from the column gave a signal,
i.e. the N-terminal Thr amine was free. This also confirmed that the
initiator Met is definitely cleaved off.
The crystallization condition is as follows;
- 0.1 M Tris pH 8.2
- 1.65 M (NH_4 )_2 SO_4
- 2 % PEG400
- 0.25 % (v/v) ?-octyl glucoside
It is worthy to mention that in the periphery of the protein,
ethylacetate is bound (the reaction product of the ethanol and acetate
impurities/breakdown products of the PEG400 stock solution). I don't
know whether any PEG400 breakdown products or others derived from some
of my crystallization components could show that sort of reactivity.
Many thanks in advance if anyone has any pointers,
Jonathan
--
Jonathan Elegheert
Ph.D. Student
Ghent University
