R different conditions. The main phase is the kobs value with

R different conditions. The main phase is the kobs value with the largest amplitude. Rollovers in the refolding and unfolding arm of the chevron plots can be detected when altering between stabilizing and destabilizing buffers, respectively. These rollovers illustrate switches between the rate limiting transition states of the (un)folding reaction. Fitting was done using bT alues obtained from a curve fit with 6 different PDZ domains in a previous study [19] and the good fit to the data for the circular permutant illustrates that the positions of the folding transition states along the reaction coordinate is similar for all PDZ domains, including the circular permutant. See Table S1 for the best fit parameters. The 0.6 M Na2SO4 buffer also contained 50 mM potassium phosphate, pH 7.5, while the 50 mM potassium acetate buffer, pH 5.6, contained KCl to keep the ionic strength at the same value for all experiments. doi:10.1371/journal.pone.0050055.gformed native contacts in the transition state for folding of pwtSAP97 PDZ2.DiscussionFolding pathways of circularly permuted proteins have been studied in a limited number of cases [2,4?,38,39] and in only one of these has the folding pathway remained the same as for the native protein [9]. It has been argued that changes in folding pathway due to circular permutation depend on the folding nucleus; a diffuse folding nucleus covering most of the protein is less likely to change the folding pathway compared to a regional 1313429 compact nucleus [9,40]. In agreement with this notion, Gianni and co-workers demonstrated that circular permutation of PTPBL PDZ2 resulted in stabilization of an intermediate [7]. The folding mechanism of PTP-BL PDZ2 has been thoroughly investigated by W-value analysis and constrained molecular dynamics simulations [19], to estimate the extent of formation of native contacts in the transition state for folding. PTP-BL PDZ2 folds with an early rather compact regional nucleus and a late, very native like transition state [19,20]. Its early folding nucleus consists of b-strands 1, 4 and 6. For PTP-BL PDZ2, the same circular permutation was made as the one in the present study (i.e., based on the naturally TA 02 cost occurring circularly permuted PDZ domain D1pPDZ [17]), but with a different outcome. Thus, by linking b1 and b6 in PTP-BL PDZ2, this early nucleus is stabilized, which is reflected in a higher folding rate constant but also significant stabilization of an intermediate, which is likely to be off-pathway[6]. It is believed that such intermediates are dis-favoured by natural selection because of the increased risk for misfolding [10]. It was recently suggested that the 11089-65-9 chemical information relation between the position of the cleavage site and active site in circular permutants is important for whether the folding pathways change due to the permutation [41]. The site of our permutation is one amino acid away from the GLGF site, which is conserved among all PDZ domains and involved in binding of the backbone and C-terminus of the protein ligand [42]. However, while our data do not directly address the effect of permutation in the binding site, we note that SAP97 PDZ2 is not affected by the circular permutation but its homolog PTP BL PDZ2 displays a dramatic change in kinetic folding mechanism. For SAP97 PDZ2, circular permutation increased the unfolding rate constant but the folding rate constant (D to N transition, Figure 5) remained unchanged. Effectively, this corresponds to a Wvalue of zero, both at the.R different conditions. The main phase is the kobs value with the largest amplitude. Rollovers in the refolding and unfolding arm of the chevron plots can be detected when altering between stabilizing and destabilizing buffers, respectively. These rollovers illustrate switches between the rate limiting transition states of the (un)folding reaction. Fitting was done using bT alues obtained from a curve fit with 6 different PDZ domains in a previous study [19] and the good fit to the data for the circular permutant illustrates that the positions of the folding transition states along the reaction coordinate is similar for all PDZ domains, including the circular permutant. See Table S1 for the best fit parameters. The 0.6 M Na2SO4 buffer also contained 50 mM potassium phosphate, pH 7.5, while the 50 mM potassium acetate buffer, pH 5.6, contained KCl to keep the ionic strength at the same value for all experiments. doi:10.1371/journal.pone.0050055.gformed native contacts in the transition state for folding of pwtSAP97 PDZ2.DiscussionFolding pathways of circularly permuted proteins have been studied in a limited number of cases [2,4?,38,39] and in only one of these has the folding pathway remained the same as for the native protein [9]. It has been argued that changes in folding pathway due to circular permutation depend on the folding nucleus; a diffuse folding nucleus covering most of the protein is less likely to change the folding pathway compared to a regional 1313429 compact nucleus [9,40]. In agreement with this notion, Gianni and co-workers demonstrated that circular permutation of PTPBL PDZ2 resulted in stabilization of an intermediate [7]. The folding mechanism of PTP-BL PDZ2 has been thoroughly investigated by W-value analysis and constrained molecular dynamics simulations [19], to estimate the extent of formation of native contacts in the transition state for folding. PTP-BL PDZ2 folds with an early rather compact regional nucleus and a late, very native like transition state [19,20]. Its early folding nucleus consists of b-strands 1, 4 and 6. For PTP-BL PDZ2, the same circular permutation was made as the one in the present study (i.e., based on the naturally occurring circularly permuted PDZ domain D1pPDZ [17]), but with a different outcome. Thus, by linking b1 and b6 in PTP-BL PDZ2, this early nucleus is stabilized, which is reflected in a higher folding rate constant but also significant stabilization of an intermediate, which is likely to be off-pathway[6]. It is believed that such intermediates are dis-favoured by natural selection because of the increased risk for misfolding [10]. It was recently suggested that the relation between the position of the cleavage site and active site in circular permutants is important for whether the folding pathways change due to the permutation [41]. The site of our permutation is one amino acid away from the GLGF site, which is conserved among all PDZ domains and involved in binding of the backbone and C-terminus of the protein ligand [42]. However, while our data do not directly address the effect of permutation in the binding site, we note that SAP97 PDZ2 is not affected by the circular permutation but its homolog PTP BL PDZ2 displays a dramatic change in kinetic folding mechanism. For SAP97 PDZ2, circular permutation increased the unfolding rate constant but the folding rate constant (D to N transition, Figure 5) remained unchanged. Effectively, this corresponds to a Wvalue of zero, both at the.