Js. Reader et al., A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding, PROTEIN SCI, 10(6), 2001, pp. 1216-1224
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.