1. Volume 24, Issue 6, Pages 946-955 (June 2016)
A De Novo Designed Coiled-Coil Peptide with a Reversible pH-Induced Oligomerization Switch 
Robert Lizatović, Oskar Aurelius, Olof Stenström, Torbjörn Drakenberg, Mikael Akke, Derek T. Logan, Ingemar André 
Structure 
Volume 24, Issue 6, Pages (June 2016)
DOI: /j.str
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2. Structure 2016 24, 946-955DOI: (10.1016/j.str.2016.03.027)
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3. Figure 1 Structural Modeling and Computational Design
(A) Schematic presentation of the computational protocol for the de novo design of self-assembling peptides used to engineer pHios.
(B) The results of the fold-and-dock simulations of the pHios sequence in various symmetry groups. The relative stability of the oligomers is displayed on the y axis. The state with lowest energy is predicted to be the native state. REU, Rosetta energy units.
(C) Sequence of the pHios peptide with the Gln15 residue in green. (D) and (E) Structures of the predicted lowest energy pHios hexamer and pentamer, respectively. The top views of the two oligomers are shown with the putative hydrogen-bonding network being formed only between Gln15 residues in the C6 hexamer.
(F) The distribution of glutamates in the pentamer model of pHios with Glu24 (predicted to have a highly upshifted pKa) encircled.
Structure  , DOI: ( /j.str )
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4. Figure 2 AUC Characterization of the pHios Peptide
(A) Sedimentation equilibrium (SE) of the pHios peptide at pH 6. The computed partial-specific volume and monomer molecular weight (MW) of pHios were  cm3/g and 3,722 Da respectively. Top: overlays between the model (black) and experimental data (green). Bottom: the residuals of the fit.
(B and C) SE data at pH 7 and 8, respectively. In all the SE experiments, the peptide concentration was 190 μM, and the rotor speeds used were 16, 24.5, and 32 krpm.
(D) Top: the c(s) analysis of pHios at pH 6 obtained by analyzing the sedimentation velocity (SV) data with Sedfit (Schuck, 2000). Bottom: residual bitmap of the fit.
(E and F) c(s) analysis and residual bitmap of pHios at pH 7 and 8, respectively. In all the SV experiments, the samples were spun at 50 krpm and the peptide concentration was 190 μM.
See also Table S1.
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5. Figure 3 CD Spectra and Thermal Denaturation of the pHios Peptides
(A) Far UV CD spectra of pHios at pH 6 and 8.
(B) Thermal denaturation of pHios at pH 6 and 8 as monitored by the R2 value.
See also Figures S3 and S5 and Table S2.
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6. Figure 4 Crystal Structure of pHiosYI
(A and B) The side and top views of the oligomer, respectively. Chains A are colored red, whereas chains B are blue. The position of Gln15 is shown in panel (B).
See also Figures S1 and S2 and Table 1.
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7. Figure 5 Effect of pH on Assembly Structure
(A) pH titration of pHios monitored by NMR. HCCO spectra at different pH values.
(B) Carbonyl chemical shift as a function of pH for the upshifted Glu in the pHios pentamer (green circles). Fit to an ideal Henderson-Hasselbalch equation is shown as a solid black line.
(C) Thermodynamic model for the pH-dependent oligomerization switching. Concentrations of the pentamer (blue line) and the hexamer (red line) are shown as a function of pH for a model with the following set of parameters: ΔG°pentamer, unprotonated = −60 kcal/mol, ΔG°hexamer, unprotonated = −75 kcal/mol, pKa,pentamer = 7.0, pKa,hexamer1 = 7.5, pKa,hexamer2 = 7.5, see Experimental Procedures for model definition).
(D) Fit of the thermodynamic model to the NMR peak intensity data from the pentameric (blue circles, 10 peaks) and the hexameric (red triangles, 7 peaks) resonances. The intensity data are normalized to a range between 0 and 1. Blue and red lines represent the best fitted model with the following set of parameters: ΔG°pentamer, unprotonated = −60.1 kcal/mol, ΔG°hexamer, unprotonated = −74.4 kcal/mol, pKa,pentamer = 7.0, pKa,hexamer1 = 7.5, pKa,hexamer2 = 7.4.
See also Figures S4 and S6 and Table S3.
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8. Figure 6 Biophysical Characterization of the pHiosA15 Peptide
(A) SE of the pHiosA15 peptide at pH 7. The computed partial-specific volume and monomer MW of pHiosA15 were  cm3/g and 3,666 Da, respectively. Top: overlays between the fitted model (black) and experimental data (green). Bottom: the residuals of the fit.
(B) Same SE experiment at pH 8.
(C) Top: the c(s) analysis of pHiosA15 at pH 7 obtained by analyzing the SV data with Sedfit. Bottom: residual bitmap of the fit.
(D) The same c(s) analysis and the corresponding residual bitmap of pHiosA15 at pH 8.
(E) The far UV CD spectra of pHiosA15 at pH 8.
(F) The comparison of pHios and pHiosA15 thermal melts monitored by R2 at pH 8.
Structure  , DOI: ( /j.str )
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