E of the apo-mutants relative to apo-Wt, that is the magnitude of vector A in Figure 2A. (b) Fractional shift toward activation achieved by the mutations in the absence of cAMP and with compounded chemical shifts greater than 0.05 ppm between the Wt(apo) and Wt(holo) state. (c) Cosine values for the projection angle, as in Figure 2A, which is also an indicator of the direction of chemical shift movement along the activation path (vector B in Fig. 2A). doi:10.1371/journal.pone.0048707.ginvestigation, e.g. the de312(apo) mutant. The 2′-OMe-cAMP was selected for this replacement because two other activators are already included in the 301353-96-8 analysis (i.e. cAMP and Sp-cAMPS) and therefore the SVD analysis is meaningful even in the absence of the 2′-OMe-cAMP state. Through this approach, the projection analysis is effectively expanded to include not only the Wt(apo) and cAMP-bound reference states (Fig. 2), but also the Sp-cAMPSand Rp-cAMPS-bound forms, leading to an improved identification of the chemical shift changes that reflect uniquely variations in the activation equilibrium. For instance, when the 2′-OMe-cAMPsaturated state is replaced with the de312(apo) mutant, the first two principal components (PC) computed through SVD (i.e. PC1 and PC2) account for more than 93 of the total variance (Table 1). PC1 reflects activation whereas PC2 is reflective of binding effects, as illustrated in Figure 4A by the Wt(Sp-cAMPS)?Wt(Rp-cAMPS) and Wt(cAMP) t(Rp-cAMPS) loadings aligned with PC1 and the Wt(apo) t(Rp-cAMPS) loading aligned with PC2. The PC1 component of the difference between the Wt(cAMP) t(Rp-cAMPS) and the Wt(apo) t(Rp-cAMPS) loadings provides therefore a measure of the maximal activationcaused by cAMP and is utilized to buy Licochalcone-A normalize the PC1 component of the difference between the mutant(apo) t(Rp-cAMPS) and the Wt(apo) t(Rp-cAMPS) loadings (Fig. 4A, red arrows). This ratio of these PC1 components indicates that the de312(apo) deletion mutant causes a 7 shift towards the apo/active conformers (Fig. 4B). The reliability of this approach was crossvalidated by applying the SVD method to L273W (Figure S1 in Supporting Information), which leads to a 47 shift of the Wt(apo) equilibrium towards the inactive conformers, consistent with previous analyses [27]. A similar approach was also used to analyze the other 23727046 two C-terminal deletion mutants, i.e. de310 and de305 (Fig. 4A, blue and green symbols, respectively), which cause further destabilization of the a6 helix. The percentage shifts towards activation caused by the successively truncating mutations de312, de310 and de305 are summarized in Figure 
4B. Figure 4B shows that the de310 and de305 truncations result in a further dramatic increase in the relative population of the apo/active conformers to 27 and 35 , respectively. Overall, the SVD analyses of Figure 4A indicate that, while deletion of the Cterminal tail in de312 causes only a subtle shift towards activation,Auto-Inhibitory Hinge Helixperturbations in the C-terminal region of the hinge helix, implemented through the de310 and de305 truncations, lead to a more drastic stabilization of the active conformation in the absence of cAMP. These results are in agreement with the overall findings of CHESPA (Fig. 3B, 3C) and together consistently point to a significant and previously unanticipated auto-inhibitory role for residues 305?10 of the EPAC hinge helix.The covariance analysis of chemical shifts reveals that the hinge-helix is coupled to t.E of the apo-mutants relative to apo-Wt, that is the magnitude of vector A in Figure 2A. (b) Fractional shift toward activation achieved by the mutations in the absence of cAMP and with compounded chemical shifts greater than 0.05 ppm between the Wt(apo) and Wt(holo) state. (c) Cosine values for the projection angle, as in 
Figure 2A, which is also an indicator of the direction of chemical shift movement along the activation path (vector B in Fig. 2A). doi:10.1371/journal.pone.0048707.ginvestigation, e.g. the de312(apo) mutant. The 2′-OMe-cAMP was selected for this replacement because two other activators are already included in the analysis (i.e. cAMP and Sp-cAMPS) and therefore the SVD analysis is meaningful even in the absence of the 2′-OMe-cAMP state. Through this approach, the projection analysis is effectively expanded to include not only the Wt(apo) and cAMP-bound reference states (Fig. 2), but also the Sp-cAMPSand Rp-cAMPS-bound forms, leading to an improved identification of the chemical shift changes that reflect uniquely variations in the activation equilibrium. For instance, when the 2′-OMe-cAMPsaturated state is replaced with the de312(apo) mutant, the first two principal components (PC) computed through SVD (i.e. PC1 and PC2) account for more than 93 of the total variance (Table 1). PC1 reflects activation whereas PC2 is reflective of binding effects, as illustrated in Figure 4A by the Wt(Sp-cAMPS)?Wt(Rp-cAMPS) and Wt(cAMP) t(Rp-cAMPS) loadings aligned with PC1 and the Wt(apo) t(Rp-cAMPS) loading aligned with PC2. The PC1 component of the difference between the Wt(cAMP) t(Rp-cAMPS) and the Wt(apo) t(Rp-cAMPS) loadings provides therefore a measure of the maximal activationcaused by cAMP and is utilized to normalize the PC1 component of the difference between the mutant(apo) t(Rp-cAMPS) and the Wt(apo) t(Rp-cAMPS) loadings (Fig. 4A, red arrows). This ratio of these PC1 components indicates that the de312(apo) deletion mutant causes a 7 shift towards the apo/active conformers (Fig. 4B). The reliability of this approach was crossvalidated by applying the SVD method to L273W (Figure S1 in Supporting Information), which leads to a 47 shift of the Wt(apo) equilibrium towards the inactive conformers, consistent with previous analyses [27]. A similar approach was also used to analyze the other 23727046 two C-terminal deletion mutants, i.e. de310 and de305 (Fig. 4A, blue and green symbols, respectively), which cause further destabilization of the a6 helix. The percentage shifts towards activation caused by the successively truncating mutations de312, de310 and de305 are summarized in Figure 4B. Figure 4B shows that the de310 and de305 truncations result in a further dramatic increase in the relative population of the apo/active conformers to 27 and 35 , respectively. Overall, the SVD analyses of Figure 4A indicate that, while deletion of the Cterminal tail in de312 causes only a subtle shift towards activation,Auto-Inhibitory Hinge Helixperturbations in the C-terminal region of the hinge helix, implemented through the de310 and de305 truncations, lead to a more drastic stabilization of the active conformation in the absence of cAMP. These results are in agreement with the overall findings of CHESPA (Fig. 3B, 3C) and together consistently point to a significant and previously unanticipated auto-inhibitory role for residues 305?10 of the EPAC hinge helix.The covariance analysis of chemical shifts reveals that the hinge-helix is coupled to t.