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 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 degrees C. This process has an activation enthalpy of approximately 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.
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J S
JS
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
Van Nuland
N A
NA
Thompson
G S
GS
Ferguson
S J
SJ
Dobson
C M
CM
Radford
S E
SE
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry*; D002942:Circular Dichroism; D021984:Cyclophilin A / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011392:Proline / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012964:Sodium / Q000494:pharmacology; D013237:Stereoisomerism; D013997:Time Factors; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C047163:pseudoazurin; D001400:Azurin; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D011392:Proline; D012964:Sodium; D021984:Cyclophilin A