G kinetics is observed, the folding mechanism is more complex than a two state reaction. However, to distinguish among different reaction schemes is very difficult. The three simplest reaction schemes that result in double exponential folding kinetics are (i) a two-step folding with an on-pathway intermediate, (ii) a two-step folding with an off-pathway intermediate, and (iii) a triangular scheme with an on-pathway intermediate as well as a direct formation of the native state from the denatured state [25?28]. Haq et al. [18] suggested that the data for pwtSAP97 PDZ2 were best fitted to a two-step folding scenario with an off-pathway intermediate at 25uC, or an on-pathway or triangular scheme at 37uC [21]. The (un)folding rate constants for cpSAP97 PDZ2 can be nicely fitted to all of the three above suggested reaction schemes (on-pathway fit shown in Figure 3). Therefore, this data set was not sufficient to distinguish between different potential folding pathways for the cpSAP97 PDZ2.106.9, 43.6, 51.7 90, 90, 103.7 41.3?.3 (2.35?.30) 8.5 (64.7) 13.1 (2.2) 99.5 (98.9) 3.71 (3.74)10415 (521) 21.9/26.81386 5340.9 36.2 44.8 0.008 1.94.5 5.5 0.Values in parentheses represent the highest resolution bin. doi:10.1371/journal.pone.0050055.tFolding of a Circularly Permuted PDZ DomainFigure 2. AZ 876 web Equilibrium and peptide binding data for cp- and pwtSAP97PDZ2. A. Far-UV circular dichroism spectra of the cp- and pwtSAP97PDZ2. B. Urea denaturation curve of cp- and pwtSAP97 PDZ2, respectively. The mD-N -value (1.2 kcal mol21M21) was shared in the curve 370-86-5 Fitting to illustrate the similarities between the curves. See Table 2 for fitted parameters. C. cpSAP97 PDZ2 binding trace at 10 mM peptide fitted to a single exponential function. Residuals are shown below the trace. D. Observed rate constants for the binding of the peptide LQRRRETQV to cp- and pwtSAP97 PDZ2 in 50 mM potassium phosphate, pH 7.5, plotted against peptide concentration. Fitting was done with the general equation for a second order bimolecular association [50]. The association rate constant, kon (slope of the linear region of the curve), decreased from 8.760.3 to 2.960.02 mM21s21 upon circular permutation, while the dissociation rate constant remained basically the same. doi:10.1371/journal.pone.0050055.gThe cpSAP97 PDZ2 24272870 Folds with Two Compact and Two Denatured Like SpeciesMore information on the folding pathway of cpSAP97 PDZ2 was obtained by performing interrupted refolding and interrupted unfolding experiments. In these experiments refolding or unfolding is interrupted after various delay times and then the protein is unfolded/refolded again. This powerful technique allows detection of populations of individual species with time after mixing. Weobserved two kinetic phases at long delay times in the interrupted refolding experiment (thus, at equilibrium) showing that there are two distinct states at equilibrium (Figures 4A and 4D), which are denoted I and N in the scheme in Figure 5. The state that unfolds faster (I) is formed in a clear double exponential fashion. Similarly, the interrupted unfolding experiments revealed two distinct states at high urea concentration (Figure 4E), denoted D and Dcis-P in Fig. 5. Here, the rapidly refolding species unfolds in a clearlyFolding of a Circularly Permuted PDZ DomainTable 2. Equilibrium parameters for the stability of cpSAP97 PDZ2 and kinetic parameters for its association with the peptide LQRRRETQV.pwtSAP97PDZ2 mD-N alue (kcal mol mD-N alue (kc.G kinetics is observed, the folding mechanism is more complex than a two state reaction. However, to distinguish among different reaction schemes is very difficult. The three simplest reaction schemes that result in double exponential folding kinetics are (i) a two-step folding with an on-pathway intermediate, (ii) a two-step folding with an off-pathway intermediate, and (iii) a triangular scheme with an on-pathway intermediate as well as a direct formation of the native state from the denatured state [25?28]. Haq et al. [18] suggested that the data for pwtSAP97 PDZ2 were best fitted to a two-step folding scenario with an off-pathway intermediate at 25uC, or an on-pathway or triangular scheme at 37uC [21]. The (un)folding rate constants for cpSAP97 PDZ2 can be nicely fitted to all of the three above suggested reaction schemes (on-pathway fit shown in Figure 3). Therefore, this data set was not sufficient to distinguish between different potential folding pathways for the cpSAP97 PDZ2.106.9, 43.6, 51.7 90, 90, 103.7 41.3?.3 (2.35?.30) 8.5 (64.7) 13.1 (2.2) 99.5 (98.9) 3.71 (3.74)10415 (521) 21.9/26.81386 5340.9 36.2 44.8 0.008 1.94.5 5.5 0.Values in parentheses represent the highest resolution bin. doi:10.1371/journal.pone.0050055.tFolding of a Circularly Permuted PDZ DomainFigure 2. Equilibrium and peptide binding data for cp- and pwtSAP97PDZ2. A. Far-UV circular dichroism spectra of the cp- and pwtSAP97PDZ2. B. Urea denaturation curve of cp- and pwtSAP97 PDZ2, respectively. The mD-N -value (1.2 kcal mol21M21) was shared in the curve fitting to illustrate the similarities between the curves. See Table 2 for fitted parameters. C. cpSAP97 PDZ2 binding trace at 10 mM peptide fitted to a single exponential function. Residuals are shown below the trace. D. Observed rate constants for the binding of the peptide LQRRRETQV to cp- and pwtSAP97 PDZ2 in 50 mM potassium phosphate, pH 7.5, plotted against peptide concentration. Fitting was done with the general equation for a second order bimolecular association [50]. The association rate constant, kon (slope of the linear region of the curve), decreased from 8.760.3 to 2.960.02 mM21s21 upon circular permutation, while the dissociation rate constant remained basically the same. doi:10.1371/journal.pone.0050055.gThe cpSAP97 PDZ2 24272870 Folds with Two Compact and Two Denatured Like SpeciesMore information on the folding pathway of cpSAP97 PDZ2 was obtained by performing interrupted refolding and interrupted unfolding experiments. In these experiments refolding or unfolding is interrupted after various delay times and then the protein is unfolded/refolded again. This powerful technique allows detection of populations of individual species with time after mixing. Weobserved two kinetic phases at long delay times in the interrupted refolding experiment (thus, at equilibrium) showing that there are two distinct states at equilibrium (Figures 4A and 4D), which are denoted I and N in the scheme in Figure 5. The state that unfolds faster (I) is formed in a clear double exponential fashion. Similarly, the interrupted unfolding experiments revealed two distinct states at high urea concentration (Figure 4E), denoted D and Dcis-P in Fig. 5. Here, the rapidly refolding species unfolds in a clearlyFolding of a Circularly Permuted PDZ DomainTable 2. Equilibrium parameters for the stability of cpSAP97 PDZ2 and kinetic parameters for its association with the peptide LQRRRETQV.pwtSAP97PDZ2 mD-N alue (kcal mol mD-N alue (kc.
