Re: [ccp4bb] Off topic_protein degradation
To dear Herman, Thank you very much for your comments, I do include protease inhibitor in the protein sample (I use Roche, which always works fine for me), and that's why I am so surprised to see that it degrades madly. On the other hand, since the protein behaves quite well on SEC --- nice symmetrical peak, resonable oligomeric state (dimer), I never thought of misfolding problem, but you're right, I should check the proteins' secondary structure first. Thanks again. To dear Chris, Thank you very much for your advise of adding GSSH, and thank you so much for telling me a working concentration. Best, Bei 2011/8/19 Chris Meier crystallogra...@christophmeier.com Bei: My experience confirms Herman's comment -- your protein may be unfolded or misfolded. In addition to protease inhibitors, you could also add an oxidising agent (e.g. 10mM GSSH) to the protein buffer. In my experience this keeps many disulphide-bonded proteins happy and stable. Hope this helps. Best wishes, Chris From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi-aventis.com Sent: Fri, August 19,2011 8:55 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off topic_protein degradation. 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 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
Re: [ccp4bb] Off topic_protein degradation.
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 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
Re: [ccp4bb] Off topic_protein degradation.
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.commailto:herman.schreu...@sanofi-aventis.com herman.schreu...@sanofi-aventis.commailto: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.UKmailto: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
Re: [ccp4bb] Off topic_protein degradation.
If you are unsure about whether the disulfides have formed treat a small amount of protein with N-ethylmaleimide. If the disulfides have not formed, when you perform mass spec on the protein you should see an increase of mass of 125Da for each exposed cysteine.
Re: [ccp4bb] Off topic_protein degradation.
Another option is DTNB (aka Ellman's Reagent). This compound reacts with free thiols and produces a yellow color. The reaction is stoichiometric, so if you have access to a very basic spectrophotometer and know your protein concentration you can quantify the free thiols quite easily. Basic pH doesn't break disulfides, if anything it stabilizes them. I would be more concerned that they are incorrectly formed as described by Herman. Mike - Original Message - From: D Bonsor dbon...@ihv.umaryland.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, August 19, 2011 6:21:41 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] Off topic_protein degradation. If you are unsure about whether the disulfides have formed treat a small amount of protein with N-ethylmaleimide. If the disulfides have not formed, when you perform mass spec on the protein you should see an increase of mass of 125Da for each exposed cysteine. -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
[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
