Aimed at isolating the residues that reflect mainly effect (d). Effect

Aimed at isolating the residues that reflect mainly effect (d). Effect (d) is residue independent, but effects (a-c) lead to residue-dependent variations in the Hesperidin fractional shifts. The effect (d) is best represented by the fractional activation (X) measured for the residue with cosine H absolute values ,1 (Figure 3C). In the case of de312(apo), the majority of such residues exhibit positive fractional activation values (Fig. 3B, red bars). These regions are also subject to the largest chemical shift changes caused by cAMP (Fig. 3, grey zones)[10,21], suggesting de312(apo) mutation shifts the pre-equilibrium toward apo/active conformations. The CHESPA analysis of de310(apo) and de305(apo) mutants leads to results similar to those obtained for de312(apo), but with overall larger chemical shift differences and fractional activation values (Figure 3A ), indicating that these mutations further destabilize the C-terminal hinge helix. The de310(apo) and de305(apo) constructs appear therefore to mimic the apo/active state more closely than de312(apo). However, due to structural distortions introduced by these mutations, the fractional activation values appear to be somewhat residue dependent (Fig. 3B) and based on the projection analysis alone it is not possible to obtain a reliable quantitative estimate of the overall relative shift towards the active state caused by the C-terminal truncation. In order to circumvent this limitation of the projection analysis, we utilized a recently introduced alternative approach based on singular value CB 5083 supplier decomposition (SVD) of NMR chemical shifts [26], which provides an improved isolation of the ppm changes that exclusively reflect variations in the position of the inactive vs. active equilibrium.The Singular Value Decomposition (SVD) analysis of the C-terminal truncation mutant indicates that the hinge helix residues 305?10 contribute to auto-inhibitionIn the previously outlined SVD analysis of chemical shifts [26], HSQC spectra for the Wt EPAC1 construct were acquired and assigned in five different states: the Wt(apo) as well as four Wtbound states, saturated with cAMP, Sp-cAMPS, 2′-OMe-cAMP and Rp-cAMPS. The Sp-cAMPS and 2′-OMe-cAMP analogs are both EPAC activators, while Rp-cAMPS functions as an EPAC antagonist, i.e. it binds the EPAC1 CBD without causing activation and is therefore used as a chemical shift reference state in the SVD protocol [26]. Here, we use a similar SVD analysis, but we replace the 2′-OMe-cAMP-bound state with one of the mutants underAuto-Inhibitory Hinge HelixFigure 3. Chemical shift projection analysis to map the effects of the apo truncation mutants de312 (red), de310 (blue) and de305 (green) relative to Wt(apo). The dashed lines represent the secondary structure of the apo-EPAC (PDB ID: 2BYV). The grey highlights are regions subject to some of the most significant cAMP-dependent changes on the Wt(apo). (a) The compounded chemical shift profile 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 w.Aimed at isolating the residues that reflect mainly effect (d). Effect (d) is residue independent, but effects (a-c) lead to residue-dependent variations in the fractional shifts. The effect (d) is best represented by the fractional activation (X) measured for the residue with cosine H absolute values ,1 (Figure 3C). In the case of de312(apo), the majority of such residues exhibit positive fractional activation values (Fig. 3B, red bars). These regions are also subject to the largest chemical shift changes caused by cAMP (Fig. 3, grey zones)[10,21], suggesting de312(apo) mutation shifts the pre-equilibrium toward apo/active conformations. The CHESPA analysis of de310(apo) and de305(apo) mutants leads to results similar to those obtained for de312(apo), but with overall larger chemical shift differences and fractional activation values (Figure 3A ), indicating that these mutations further destabilize the C-terminal hinge helix. The de310(apo) and de305(apo) constructs appear therefore to mimic the apo/active state more closely than de312(apo). However, due to structural distortions introduced by these mutations, the fractional activation values appear to be somewhat residue dependent (Fig. 3B) and based on the projection analysis alone it is not possible to obtain a reliable quantitative estimate of the overall relative shift towards the active state caused by the C-terminal truncation. In order to circumvent this limitation of the projection analysis, we utilized a recently introduced alternative approach based on singular value decomposition (SVD) of NMR chemical shifts [26], which provides an improved isolation of the ppm changes that exclusively reflect variations in the position of the inactive vs. active equilibrium.The Singular Value Decomposition (SVD) analysis of the C-terminal truncation mutant indicates that the hinge helix residues 305?10 contribute to auto-inhibitionIn the previously outlined SVD analysis of chemical shifts [26], HSQC spectra for the Wt EPAC1 construct were acquired and assigned in five different states: the Wt(apo) as well as four Wtbound states, saturated with cAMP, Sp-cAMPS, 2′-OMe-cAMP and Rp-cAMPS. The Sp-cAMPS and 2′-OMe-cAMP analogs are both EPAC activators, while Rp-cAMPS functions as an EPAC antagonist, i.e. it binds the EPAC1 CBD without causing activation and is therefore used as a chemical shift reference state in the SVD protocol [26]. Here, we use a similar SVD analysis, but we replace the 2′-OMe-cAMP-bound state with one of the mutants underAuto-Inhibitory Hinge HelixFigure 3. Chemical shift projection analysis to map the effects of the apo truncation mutants de312 (red), de310 (blue) and de305 (green) relative to Wt(apo). The dashed lines represent the secondary structure of the apo-EPAC (PDB ID: 2BYV). The grey highlights are regions subject to some of the most significant cAMP-dependent changes on the Wt(apo). (a) The compounded chemical shift profile 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 w.