Abstract 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.
The native state of human muscle acylphosphatase (AcP) presents two alpha-helices. In this study we have investigated folding and aggregation of a number of protein variants having mutations aimed at changing the propensity of these helical regions. Equilibrium and kinetic measurements of folding indicate that only helix-2, spanning residues 55-67, is largely stabilized in the transition state for folding therefore playing a relevant role in this process. On the contrary, the aggregation rate appears to vary only for the variants in which the propensity of the region corresponding to helix-1, spanning residues 22-32, is changed. Mutations that stabilize the first helix slow down the aggregation process while those that destabilize it increase the aggregation rate. AcP variants with the first helix destabilized aggregate with rates increased to different extents depending on whether the introduced mutations also alter the propensity to form beta-sheet structure. The fact that the first alpha-helix is important for aggregation and the second helix is important for folding indicates that these processes are highly specific. This partitioning does not reflect the difference in intrinsic alpha-helical propensities of the two helices, because helix-1 is the one presenting the highest propensity. Both processes of folding and aggregation do not therefore initiate from regions that have simply secondary structure propensities favorable for such processes. The identification of the regions involved in aggregation and the understanding of the factors that promote such a process are of fundamental importance to elucidate the principles by which proteins have evolved and for successful protein design.
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