Figure 6. The effect of prolyl isomerization on the kinetics of refolding.

(A) The effect of cyclophilin, a peptidyl-prolyl isomerase, on the refolding rate constants of the terminal extensions series. Rate constants for the fast (filled symbols) and slow (open symbols) phases of Nank1-5C₁ (red, circles) and Nank1-5C₂ (blue, squares) in the presence of a range of cyclophilin concentrations. Refolding was initiated by the dilution of urea to final concentrations of 0.83 and 1.17 M urea, respectively. (B) Interrupted unfolding double-jump assay to test for heterogeneity in the unfolded state. Nank1-5C₁ (red, ^) and Nank1-5C₂ (blue, ▪) were rapidly unfolded by the addition of urea to final concentrations of 5 and 7 M urea, respectively. Following a variable delay time, refolding was initiated by the rapid dilution of urea to a final concentration of 0.83 M and 1.16 M urea, respectively. Amplitudes were obtained by fitting the resulting fluorescence-monitored refolding traces with a double-exponential function. The relative amplitude is the fitted amplitude of the slow phase to the total amplitude change. Solid lines represent single exponential fits. Conditions: 25 mM Tris·HCl, 150 mM NaCl, pH 8, 20 °C.