BioSAXS can also tell you if the protein is folded or not, but in either case, you may want to purify on site since degradation is so fast. Many BioSAXS beamlines (including ours at MacCHESS) now have SEC systems on site.
Richard Gillilan MacCHESS Cornell University On Aug 19, 2011, at 3:55 AM, <herman.schreu...@sanofi-aventis.com<mailto:herman.schreu...@sanofi-aventis.com>> <herman.schreu...@sanofi-aventis.com<mailto:herman.schreu...@sanofi-aventis.com>> wrote: Dear Bei, The first question to ask is not whether there is spontaneous disulfide bond breakage, but whether the correct disulfide bonds have been made in the first place. Extreme protease sensitivity could point to an unfolded/misfolded protein. If you know some protein NMR people, you could ask them to check. Even a 1-D NMR spectrum could give some information whether the protein is correctly folded or not. Another way to check is to see if your protein has proper enzymatic/biological activity. If this activity is ok, the folding is probably ok as well. You may have a protease contaminant, so you may want to check the purification protocol. The least you could do is to add some protease inhibitors to your crystallization setups. I once added PMSF for this purpose. Good luck! Herman ________________________________ From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of joybeiyang Sent: Friday, August 19, 2011 6:28 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] Off topic_protein degradation. Dear all, I am trying to crystallize a protein for which the yield and solubility were both fine. However, this protein has a severe problem of degradation. When stored at RT, the protein will degrade madly into pieces, while stored at 4 degree, the degradation is much slower and a relatively stable truncate form can be get. I am going to try to crystallize the protein at 4 degree, however I still want to understand what's going on there at RT because this protein was supposed to be very stable, it is Cys rich, and the 6 Cys were predicted to form 3 disulfide bonds which hold the protein as a globule, how can a protein with 3 stabilizing disulfide bond be fully degraded like this? Is there a possibility of spontaneous disulfide bond breakage at pH 8 ? Another question is I tried limited proteolysis with this protein, however even at 1:1000(w/w, chymotrypsin), the protein is degraded into pieces in about 2 hrs (again, a relatively stable truncated form can be get between 10 min and 30 min). I am wondering how is the crystallization probability correlated with proteolysis stability? Does this phenomenon indicate that the crystallization probability of my protein is pretty low? Any comments would be greatly appreciated. Bei 2011-08-15
