Hi Supratim, if both the crystal and Mass spec tell you that you have 6k and 13k fragments, proteolysis definitively took place. There are several possible explanations for the phenomenom you observe:
-time: processes that do not occur during the few hours of a normal biochemical experiments, may occur during a long (8 months!) crystallization experiments. In this time, also sites which are not official (auto)proteolysis sites may get cleaved. -concentration: proteolysis is usually a bimolecular event, which means that the reaction speed goes up with the square of the concentration. So when nothing may happen in a dilute solution, degradation can get very fast once you concentrate to crystallographic relevant (10 mg/ml) concentrations. -selection: if your fragments readily crystallize, but the parent protein does not, crystals will selectively accumulate the cleaved fragments. -contaminating protease: After purification, minute amounts of a contaminating protease may still be present, which over a long time in concentrated solutions, can wreak havoc with you protein. Also you may not have added the right inhibitor for this contaminating protease, or the inhibitor may get inactivated over time. The long time it took for your crystals to appear tells me that your parent protein does not want to crystallize and after 8 months, enough fragments had accumulated to crystallize instead. In your case, I would try the following: -add ligands to stabilize the protein and speed up crystallization -try different protein constructs. -try adding trypsin or chymotrypsin to do limited proteolysis during crystallization. Maybe you get larger fragments that also crystallize. -If you are really desperate: try antibodies or nanobodies. Best, Herman ________________________________ Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von supratim dey Gesendet: Sonntag, 16. Juni 2013 15:22 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] protein degradation Hi i have setup a crystallization of a complex formed by two different proteins of molecular weights 53kD and 13kD. During purification of this complex there was slight degradation band of 53KD protein as observed from SDS PAGE but it did not effect complex formation. Crystals appeared after 8 months and on solving the structure i could find only 6kD fragment of the 53kD protein associated in complex form with 13kD protein and the rest of the fragment remains absent. Mass spec analysis with the crystals gave the same result. I couldn't explain this anomalous behavior. I have used different types of protease inhibitors and there is no autodegradation or autoproteolysis site. Can anyone please suggest what accounts for such unusual phenomenon. How to identify if any autoproteolysis event is taking place.
