FIGURE 3.

Pin1 catalyzes conformational changes in phosphorylated p27^Kip1. A, selected region of a two-dimensional ROESY spectrum of a nonphosphorylated Thr-187 peptide (AGSVEGTPKKPGLR) at a concentration of 2.4 mm and in the presence of 0.03 mm Pin1 (mixing time 110 ms) is shown. There is no cross-peak found (as indicated by arrows). B and C, selected regions of a two-dimensional ROESY spectrum of a phosphorylated Thr-187 peptide (GSVEGpTPKKPGA, where boldface indicates positions 6 and 8, respectively) at a concentration of 2.0 mm are shown in the absence (B) or in the presence (C) of 0.03 mm Pin1 (mixing time 110 ms). Negative and positive peaks are indicated by arrows. Diagonal peaks from cis and trans conformers are indicated by cc and tt, respectively. Exchange peaks resulting from Pin1-catalyzed isomerization are labeled ct and tc. Note that rotating frame nuclear Overhauser enhancement and exchange cross-peaks are indicated by arrows. Diagonal peaks of Thr (T6) and Lys (K8) amide protons from the phosphorylated Thr-187 peptides are identified by arrows. D and E, ratios of cross-peak and diagonal peak intensities for the cis and trans conformations of Thr-6 on rotating frame nuclear Overhauser enhancement mixing times and its isomerization rates are indicated; D, from the cis to trans (k_ct^cat) conformation; E, from the trans to cis (k_tc^cat) conformation. F, illustration of the conformational change of phosphorylated Thr-187 peptides by Pin1. The peptide models were generated using PyMOL. In the absence of Pin1, the isomerization rates between the cis and trans conformations are very slow; however, in the presence of Pin1, the isomerization rates are greatly enhanced by Pin1. The rates of Thr-6 are presented.