Folding of bovine pancreatic ribonuclease A (RNase A) is a sequential process which involves the formation of well-populated structural intermediates under suitable conditions. Two intermediates have been detected on the major slow-refolding pathway of RNase A: a late intermediate (IN) which already resembles the native protein in a number of properties and a rapidly formed early intermediate (I1) which shows extensive hydrogen-bonded secondary structure. Here competition experiments between refolding and proteolytic cleavage of the peptide chain are described which yield information about the decrease in accessibility of particular proteolytic cleavage sites during the folding process. Results obtained with pepsin as a proteolytic probe of folding indicate that the primary cleavage site for pepsin, Phe-120-Asp-121, becomes inaccessible early in the course of refolding, if folding is carried out under conditions which effectively stabilize the native state. Under marginally stable conditions, folding is very slow, and protection against peptic cleavage is not detectable prior to the final formation of native protein. The comparison with amide proton exchange experiments suggests that the protection against peptic cleavage occurs during the formation and/or stabilization of hydrogen-bonded secondary structure in the early intermediate (I1). We conclude that the carboxy-terminal region of the RNase peptide chain, which is known to be important for the stability of the folded protein, may also be relevant for early steps of refolding.

A folded intermediate Iy has been detected in the slow refolding reaction of ribonuclease A, US -+ N, where Us is an unfolded species which contains non-native isomers of X-Pro peptide bonds and N is the native enzyme. IN has been characterized previously as a structured folding intermediate : it shows a tyrosine absorbance like native ribonuclease, but it still contains at least one non-native proline peptide bond and its tyrosine fluorescence differs from the native enzyme.Here we have used three probes to characterize further the structure of I N in comparison to native ribonuclease A.(1) The accessibility of the tyrosine hydroxyl groups to ionization has been investigated in IN. (2) The binding of the specific inhibitor cytidine 2'-phosphate (2'CMP) to IN was measured. (3) The regain of enzymatic activity during folding was monitored to decide whether IN is an enzymatically active intermediate.The results indicate that I N possesses a compact folded structure which shields part of the tyrosine residues from the aqueous environment like native ribonuclease A. The active-site region of IN has folded to the native conformation. IN binds 2'CMP as well as native ribonuclease A does and it shows enzymatic activity similar to the native enzyme.We conclude that unfolded molecules, which have only proline-93 in the non-native trans configuration, can refold to the native-like intermediate IN before proline isomerization takes place. Proline-93 is located far away from the active-site region in an external bend and therefore does not block folding. It isomerizes in the folded molecule to the native cis configuration.Unfolded RNase A consists of 20% fast-folding species, UF, and 80% slow-folding species, Us, which coexist in a slow UF -+=-Us equilibrium [I -31. US folds slowly because it contains non-native proline isomers [4-61. The kinetic pathway of refolding of US depends on the final folding conditions ; under strongly native conditions kinetic intermediates have been observed in the course of the US --f N reaction. An early hydrogen-bonded intermediate was identified by measuring competition between amide proton exchange and refolding [7], and a native-like intermediate I N was detected by Cook et al.[8] via a comparison of the absorption changes during the Us + N reaction with the time course of proline isomerization during refolding of US. They concluded that IN is a folded intermediate which shows a tyrosine absorption like native RNase A and which is capable of binding 2'CMP, a specific inhibitor of RNase A. However, IN still contains at least one essential proline in a non-native configuration. Contrary to the changes in tyrosine absorption, the native tyrosine Dedicated to Professor Rainer Jaenicke on the occasion of his fiftieth birthday.Abhreviutions. RNase A, bovine pancreatic ribonuclease with disulfide bonds intact; guanidine . HCI, guanidine hydrochloride; Z'CMP, cytidine ;!'-phosphate; 2',3'CMP cytidine cyclic 2',3'-phosphate; T, time constant of a reaction or kinetic phase (reciprocal of the apparen...