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A partially folded intermediate species of the β‐sheet protein apo‐pseudoazurin is trapped during proline‐limited folding
Author(s) -
Reader John S.,
Van Nuland Nico A. J.,
Thompson Gary S.,
Ferguson Stuart J.,
Dobson Christopher M.,
Radford Sheena E.
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.52801
Subject(s) - chemistry , circular dichroism , protein folding , phi value analysis , isomerization , protein secondary structure , crystallography , denaturation (fissile materials) , downhill folding , folding (dsp implementation) , native state , stereochemistry , biophysics , biochemistry , catalysis , electrical engineering , nuclear chemistry , engineering , biology
The folding of apo‐pseudoazurin, a 123‐residue, predominantly β‐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.