Figure 1.
Folding Stabilities and Properties of CI2 and Barnase Fragments
(A) Plot of free energy, G, versus Q at 300 K for six fragments of CI2.
(B) Same as (A), but showing results for barnase.
(C) Mean value of Q computed from simulations for CI2 compared with fluorescence intensities taken from Figure 10 of [34].
(D) Same as (C), but for barnase, and compared with fluorescence intensities taken from Table 1 of [36].
(E) Fractional population of correct secondary structure at all residues in CI2 fragments at 278 K.
(F) Same as (E), but for barnase.
(G) Mean values of Qres at all residues in CI2 fragments at 278 K.
(H) Same as (G), but for barnase.
Figure 2.
Folding Free Energy Surfaces for CI2 Fragments
(A–D) Free energy surfaces for fragments comprising residues 1–40, 1–50, 1–60, and 1–64 (full length), respectively. Free energy (G) is shown on a continuous color scale from 0 (red) to +5 kcal/mol (white); symbols show the mean value of Q in 5-K intervals. Cartoons in bottom-left corner of each panel indicate the “native” structure of each fragment generated using RasMol [94].
Figure 3.
A Coupled Synthesis–Folding Simulation of CI2
Snapshots from a typical de novo folding simulation of CI2 in the presence of the ribosome (grey surface). The nascent chain is colored from blue (N-terminus) to red (C-terminus). Snapshots (A–F) are for timepoints 3.6 μs, 4 μs, 6 μs, 6.04 μs, 6.08 μs, and 6.23 μs, respectively; the length of the nascent protein in (A) and (B) is 40 and 44 residues, respectively, in all other panels, it is full length. This figure was prepared with PyMOL [95].
Figure 4.
Fifty Coupled Synthesis–Folding Simulations of CI2
(A) Snapshots showing all 50 coupled synthesis–folding simulations of CI2 at the moment at which the final residue is released.
(B) and (C) Snapshots for the same system at the moment that the native conformations are achieved; the two views are related by a 90° rotation around the vertical axis. Note that in this simulation model, the proteins do not diffuse far from the ribosome before completing folding.
Figure 5.
Effect of Confinement on Folding of CI2 and Barnase
(A) Mean values of Q during coupled synthesis–folding of CI2 in the presence (filled circles) and absence (open circles) of the ribosome.
(B) Same as (A), but for barnase.
(C) Mean values of the residue-specific Qres value during coupled synthesis–folding of CI2 in the presence of the ribosome; separate lines are plotted for each residue, colored from blue to red. Arrows indicate periods in which several of the Qres values suddenly decrease in concert.
(D) Same as (C), but for barnase; note that for clarity, only the period of synthesis of the first 60 residues is shown.
Figure 6.
Simulated Kinetics of Refolding and De Novo Folding for CI2 and Barnase
(A) Histograms of folding times obtained from refolding and de novo folding trajectories for CI2.
(B) Same as (A), but for barnase.
Figure 7.
Simulated Mechanisms of Refolding and De Novo Folding for CI2 and Barnase
(A) Comparison of computed and experimental Φ values for CI2.
(B) Same as (A), but for for barnase.
(C) Plot of Qres versus Q for refolding of CI2.
(D) Same as (C), but for refolding of barnase.
(E) Same as (C), but for the post-synthesis stage of de novo folding of CI2.
(F) Same as (C), but for the post-synthesis stage of de novo folding of barnase.
In panels (C–F), Qres values are shown on a continuous color scale between 0.0 (blue) and 0.75 (red) for CI2, and between 0.0 (blue) and 0.9 (red) for barnase.
Figure 8.
Consequences of Excessively Stabilizing CI2
(A) Histograms of folding times obtained from refolding and de novo folding trajectories of CI2 with ɛ = 0.80 kcal/mol.
(B) Snapshot of a “trapped” trajectory.
(C) Plots of Q versus time for ten previously trapped trajectories when restarted without the ribosome.
(D) Same as (C), but restarted with the ribosome and ɛ = 0.60 kcal/mol.
Figure 9.
Folding of the Two-Domain Protein SFVP
(A) Evolution of QN, QC, and QINT during a typical refolding trajectory of the serine protease module of SFVP.
(B) The most populated points in (QN, QC, QINT) space during 100 refolding trajectories. Each point in the (QN, QC, QINT) space is counted only once per trajectory in order to prevent repeated revisiting of the same region in a trajectory from biasing the results. Symbol size and color reflect relative population (large, red symbols being populated in 100% of simulations).
(C) Same as (B), but for ten coupled synthesis–folding simulations.
(D) Snapshot of the moment that native structure is cotranslationally formed; the green chain extending back to the peptidyltransferase active site represents the approximately 28 residues synthesized after the protease module's final residue.
Figure 10.
A Typical Coupled Synthesis–Folding Simulation of SFVP
Snapshots from a typical de novo folding simulation of SFVP in the presence of the ribosome (grey surface). The nascent chain is colored from blue (N-terminus) to red (C-terminus), with its length (in amino acids) indicated in each panel. This figure was prepared with PyMOL[95].
Figure 11.
Parameter Sensitivity of Coupled Synthesis–Folding Simulations of CI2
(A) Mean values of Q during coupled synthesis–folding of CI2 comparing the effects of using different timescales for amino acid synthesis (τsyn).
(B) Same as (A), but comparing the effects of assigning larger radii to ribosome pseudo-atoms.
(C) Same as (A), but comparing the effects of including energetically favorable interactions between the nascent protein and the ribosome.
(D) Distribution of mean Qres values obtained during the synthesis of the final amino acid, comparing the effects of assigning larger radii to ribosome pseudo-atoms; error bars indicate the standard deviation of values obtained from 30 independent trajectories.
(E) Same as (D), but comparing the effects of including energetically favorable interactions between the nascent protein and the ribosome.