Effect of deamidation on folding of ribonuclease A

The folding of ribonuclease A (RNase A) has been extensively studied by cha racterizing the disulfide containing intermediates using different experime ntal conditions and analytical techniques. So far, some aspects still remai n unclear such as the role of the loop 65-72 in the folding pathway. We hav e studied the oxidative folding of a RNase A derivative containing at posit ion 67 the substitution Asn --> isoAsp where the local structure of the loo p 65-72 has been modified keeping intact the C65-C72 disulfide bond. By com paring the folding behavior of this mutant to that of the wild-type protein , we found that the deamidation significantly decreases the folding rate an d alters the folding pathway of RNase A. Results presented here shed light on the role of the 65-72 region in the folding process of RNase A and also clarifies the effect of the deamidation on the folding/unfolding processes. On a more general ground, this study represents the first characterization of the intermediates produced along the folding of a deamidated protein.