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

¹ School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
² Oxford Centre of the Molecular Sciences, University of Oxford, New Chemistry Laboratory, Oxford OX1 3QT, United Kingdom
³ Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

The folding of apo-pseudoazurin, a 123-residue, predominantly ß-sheet protein with a complex Greek key topology, has been investigated using several 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, respectively, at pH 7.0 and at 15°C. This process has an activation enthalpy of 90 kJ/mole and is catalyzed by cyclophilin A, indicating that 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 proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.

Keywords: Pseudoazurin; proline isomerization; ß-sheet; protein folding

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