Figure 7.

The effect of prolines on CARP folding kinetics. (A) Chevron plots of NR₂ with (▵) and without (▴) prolines. The box highlights the cis-trans proline isomerization phase observed in NR₂(Pro) folding traces. Similar phases are seen for other CARP(Pro) constructs. The area of each point marker is proportional to its fitted kinetic amplitude. (B) Parallel-Ising kinetic reaction net for the folding of NR₂C(Pro). Unlike the reaction net for CARP(Ala), for CARP(Pro) constructs, a cis-trans proline isomerization reaction in the unfolded state precedes the nucleation step. (C) Global fit of the parallel-Ising kinetic model to CARP(Pro) stopped-flow fluorescence data (see Fig. S9, B–D, and Fig. S12). Only the rate constants with relative amplitude higher than 10% are displayed. Lines and symbols are as described in Fig. 6. (D) Relative amplitudes of the proline isomerization phase of NR_x (where x = 1–3) do not depend on the number of prolines. Lines indicate expected folding amplitudes for constructs of different lengths, assuming that all prolines must be trans to fold, and assuming a trans/cis equilibrium constant of 8.9 (26). Our measured amplitudes for the prolyl isomerization phase do not depend on the number of repeats, suggesting that only one proline needs to be in trans to initiate folding. To see this figure in color, go online.