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
