Wearne S J, Creighton T E
Medical Research Council, Laboratory of Molecular Biology, Cambridge, England.
Proteins. 1989;5(1):8-12. doi: 10.1002/prot.340050103.
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.
已确定蛋白质的折叠构象对特定天冬酰胺残基脱酰胺化速率的影响。由于通过不可逆地断裂蛋白质二硫键可生成稳定的未折叠蛋白，因此可在相同条件下比较天然和未折叠的核糖核酸酶A（RNase A）。通过电泳和色谱法追踪RNase A不稳定的Asn-67残基的脱酰胺化过程。在80℃时，观察到热未折叠的二硫键结合形式和还原形式的脱酰胺化速率相似。然而，在37℃和pH 8条件下，天然RNase A的脱酰胺化速率可忽略不计，比还原的未折叠RNase A慢30多倍。这表明Asn-67残基位于天然蛋白质的局部构象中，该构象极大地抑制了脱酰胺化。这种构象是66 - 68位残基的β-转角。
