ROLE OF THE CYS-2 CYS-10 DISULFIDE BOND FOR THE STRUCTURE, STABILITY,AND FOLDING KINETICS OF RIBONUCLEASE-T1

The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by subst ituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in r ibonuclease F1. This C2S/C1ON variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substitu ted amino acids only. The thermodynamic stability of ribonuclease T1 i s strongly reduced by breaking the Cys 2-Cys 10 bond, and the free ene rgy of denaturation is decreased by about 10 kJ/mol. The folding mecha nism is not affected, and the trans to cis isomerizations of Pro 39 an d Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correl ated with the decrease in stability: the folding kinetics of the wild- type protein and the C2S/C1ON variant become indistinguishable when th ey are compared under conditions of identical stability. Apparently, t he Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bon d has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.