Effect of protein conformation on rate of deamidation: ribonuclease A.
The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determined. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds. Deamidation of the labile Asn-67 residue of RNase A was followed electrophoretically and chromatographically. At 80 degrees C, similar rates of deamidation were observed for the disulfide-bonded form, which is thermally unfolded, and the reduced form. At 37 degrees C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the beta-turn of residues 66-68.
Wearne
S J
SJ
Medical Research Council, Laboratory of Molecular Biology, Cambridge, England.
Creighton
T E
TE
D000588:Amines / Q000032:analysis; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000032:analysis*; D013696:Temperature
D000588:Amines; D012259:Ribonuclease, Pancreatic; D005998:Glycine