Supplemental Experimental Procedures

An extra, fifth disulfide bond was introduced into RNase A, a 124-residue protein containing four native disulfide bonds at positions (26-84), (40-95), (58-110) and (65-72), using recombinant DNA methodology. The five-disulfide-bond-containing protein is referred to as a 5 S-S form. The 5 S-S form is converted to the 4 S-S form by selectively reducing the extra disulfide bond, followed by blocking the free thiols by AEMTS to prevent re-oxidation to the 5 S-S form. The 4 S-S form is essentially pseudo-wild-type; it is possible, however, that substitution of two residues in the wild-type by cysteines, and/or AEMTS-blockage of the two free thiols from the reduced fifth disulfide bond, may introduce slightly different interactions than those present in the wild-type protein, contributing to kinetics that could be slightly different from those of the wild-type protein. Hence, the kinetics of folding (or unfolding) of the 5 S-S (oxidized) form are compared with those of the 4 S-S (reduced) form, and not with the wild-type protein. The site for adding a fifth disulfide crosslink to wild-type RNase A was identified by the following procedure to connect solvent exposed residues in close proximity in the native three-dimensional crystal structure of RNase A (Wlodawer et al., 1988). The three