1. Interplay between Protein Thermal Flexibility and Kinetic Stability
Andrea G. Quezada, A. Jessica Díaz-Salazar, Nallely Cabrera, Ruy Pérez-Montfort, Ángel Piñeiro, Miguel Costas 
Structure 
Volume 25, Issue 1, Pages (January 2017)
DOI: /j.str
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2. Structure 2017 25, 167-179DOI: (10.1016/j.str.2016.11.018)
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3. Figure 1 Separation of the TIM Structure in Eight Regions
The TIM barrel was divided into eight equivalent regions (r1 to r8), each comprising an α helix, a loop, and a β strand. The dimer structure for TbTIM is shown. The number of residues are: 35, 25, 31, 28, 42, 45, 21, and 23 for regions r1 to r8, respectively. The aligned sequences of TbTIM and TcTIM and the secondary structure are displayed. Residues that are identical for both proteins are in black, while colored ones indicate the differences between them. There are 13, 7, 8, 5, 15, 10, 3, and 4 such differences in regions r1 to r8, respectively. This figure was generated with PyMOL.
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4. Figure 2 Examples of Experimental DSC Thermograms
Results for two chimerical proteins are shown: (A) Tc(1–5)Tb(6–8) and (B) Tc(1–4)Tb(5–8) at the indicated scan rates and at 0.4 mg/mL. Symbols represent the apparent heat capacity and continuous lines represent the fitting to the two-state irreversible model. The profiles have been shifted in the y axis for clarity. Arrhenius plots for the same chimerical proteins are shown in (C) and (D), respectively. The symbols correspond to the different scan rates employed: 0.5, 1, 1.5, 2.0, 2.5, and 3.0 K/min. Straight lines are the best fit to all the points. The values for Tm, widths of the calorimetric transitions at half the maximum height (W) and Eact derived from (C) and (D) for all the chimerical proteins are given in Table 1.
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5. Figure 3 Secondary Structure
The number of residues forming part of the α helices (top panel) and beta strands (bottom panel) along the trajectories for subunits A and B of TbTIM and TcTIM at the temperatures indicated in the plots.
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6. Figure 4
Evolution with the Simulation Time of the TbTIM and TcTIM Structures
(A) Snapshots of the simulation trajectories at 8, 16, and 24 ns for 400, 450, 490, and 510 K. For the simulations at 700 K, snapshots are at 2, 5, and 7 ns. Subunits A and B of TbTIM are colored blue and orange, while those of TcTIM are colored red and cyan.
(B) Projection of the trajectories on a 3D Cα-RMSD space for the dimer and for both subunits of TbTIM and TcTIM at 510 K. The simulation time is represented by the colors, as indicated by the blue-red gradient bar.
(C) Progressive moving dispersion (PMD) plots over sets of 200 points for the trajectories shown in (B). Subunits A and B are colored as in (A), while the dimer is colored in black for both proteins. The minima represent structures that remain relatively stable during a significant part of the trajectory, while the maxima represent trajectory segments where the protein structure is evolving quickly.
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7. Figure 5 Residue Displacements
Difference of the average distance of each Cα atom (Δdj) between the last cluster structure of each trajectory and the crystal structures of TbTIM and TcTIM, using analysis of subunits A and B.
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8. Figure 6 Displacements and Their Temperature Gradients
(A) Region displacements (average for both subunits) of the clusters structures around 2, 8, 14, 20, and 24 ns.
(B) Total Dwt,T at each temperature for TbTIM and TcTIM.
(C) Temperature gradient of the displacement (TGD) for each region of both wild-type proteins. Dwt,T and TGD are the average for both subunits and for all clusters. The error bars in (C) are the uncertainties of the slopes of the straight lines fitted to the Dr,T versus T data for each region of both proteins.
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9. Figure 7
Correlation between the Calorimetric Experimental Results and MD Simulations
Eact (A) and W (B) versus TGD for the wild-type proteins and a series of six chimeras where TbTIM is progressively converted into TcTIM: Tc(1)Tb(2–8), Tc(1–2)Tb(3–8), Tc(1–3)Tb(4–8), Tc(1–4)Tb(5–8), Tc(1–5)Tb(6–8), and Tc(1–6)Tb(7–8). TGD was calculated using Equations 7 and 9, and the Eact values are those reported in Table 1. Red and dark blue symbols are for wild-type TcTIM and TbTIM, respectively. The straight lines (Eact = −660.01   × TGD and W = 11.53–4.33 × TGD) correspond to the best fit of the data and the correlation coefficients (R) are shown in the plots. The orange symbols are Eact (C) and W (D) versus TGD for the remaining 23 chimeras in Table 1. The straight lines in (C) and (D) are the same as those in (A) and (B), respectively, showing that the predicted Eact and W values from the correlation are in reasonable agreement with the experimental ones. Arrows indicate the direction of increasing cooperativity of the unfolding transition, kinetic stability, and thermal flexibility.
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10. Figure 8
Comparison of TGDres Values for Each of the Residues of TcTIM and TbTIM
Regions are separated by dashed lines. Residues with a significant difference in TGD values between TbTIM and TcTIM are highlighted. The secondary structure is depicted at the top of the plot.
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11. Figure 9 Thermal Energy versus the Conformational Space Landscape
Schematic diagram showing the effects of the static and thermal flexibilities on the thermal energy (E) versus the conformational space (CS) landscape for TcTIM and TbTIM, producing different kinetics stabilities (activation energy, Eact) for each protein. The minima corresponding to the native structures have been shifted in the vertical axis to show more clearly the difference in static flexibility between both proteins. At a given temperature (thermal energy) the static flexibility is proportional to the size of the CS available to the protein (dotted horizontal lines). As the temperature increases the static flexibility increases widening the CS available to the protein (see also Figure 6B). The thermal flexibility (TGD) is inversely proportional to the slope of the E versus the CS curve joining the native state (N) and transition state (TS), and represents the increment of the CS available to the protein when energy in provided. As a result of a higher thermal flexibility for TcTIM its kinetic stability is larger than for TbTIM. In this work, these slopes and hence TGD have been taken as temperature independent (see also Figures 6B and 6C).
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