A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding

The folding of apo-pseudoazurin, a 123-residue, predominantly beta -sheet p rotein with a complex Greek key topology, has been investigated using sever al biophysical techniques. Kinetic analysis of refolding using far and near -ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respecti vely, at pH 7.0 and at 15 degreesC. This process has an activation enthalpy of similar to 90 kJ/mole and is catalyzed by cyclophilin A, indicating tha t folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before p roline isomerization, an intermediate accumulates during folding. This spec ies has a substantial signal in the far-UV CD, a nonnative signal in the ne ar-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulp honate binding), a noncooperative denaturation transition, and a dynamic st ructure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially fold ed states of other proteins and discuss its role in folding of this complex Greek key protein.