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:[email protected]] On
Behalf Of joybeiyang
Sent: Friday, August 19, 2011 6:28 AM
To: [email protected]
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
