Mechanical stability analysis.

<p>(a) Optical tweezers setup (b) Force-extension curve of dsDNA showing overstretching at 65 pN, as well as the characteristic step-wise relaxation. The measured DNA stretching curves did not display additional steps that might have arisen from STN unfolding or its detachment from the surface. (c) Fraction of tethers that resisted 60 pN in first and second pull, compared between several commonly used linkage strategies and our proposed linkage strategies based on STN. For the (STN)biotin-DNA-Dig(AntiDig) system, almost all tethers broke at the first pull, and hence the subsequent pulls are not indicated.</p

Corneal thickness measurement by AS-OCT.

<p>(A) En face projection view (green line shows the position of B-Scan). (B and C) Cross-sectional image of the cornea visualizing different corneal layers (B-Scan). Higher magnification is shown (C). Tear: Tear layer, Epi; Corneal epithetium, Sto: Corneal stroma, End: Corneal endothelium. Ref: corneal vertex reflection.</p

Correspondence between corneal thicknesses and the status of corneal epitheliopathy and endothelial cell layers.

<p>The data suggest that, initially, aging affects corneal thickness by changing the boundary layers that maintain the homeostasis of corneal water and electrolytes. Endothelial cell density (ECD) reduces as the mice age from 2M old to 8M old and epithelial layer accumulates damages within this time frame. We have not seen significant changes in corneal epitheliopathy and ECD (contrary to thickness data), when we assessed very old mice (14M). All data were obtained from n = 10 mice/group and representative data from three independent experiments are shown. All data were compared to baseline (2 months). We used female BALB/c mice for this analysis. <i>p</i> values are calculated using the Student’s t-test and error bars represent SEM. (***<0.001).</p

Allometric scaling for corneal thickness.

<p>(A) Allometric scaling analysis of 6-month-old female BALB/c mice. Pearson correlation coefficient was used. (B) Evolutionary allometric scaling of adult mammals (α = 0.2 ± 0.02), calculated from reported data for mice, rabbit, cat, dog, pig, human, cow and elephant. Linear regression analysis and correlation analysis were performed among body weight and corneal thickness. Spearman correlation coefficient was used. Pearson correlation analysis was used for normally distributed data and Spearman correlation analysis was adopted for the abnormally distributed data.</p

Age-related changes in corneal thickness.

<p>(A) Representative OCT images of corneas from female BALB/c mice. (B) Corneal thickness, body weight and corneal thickness/ weight versus age in female BALB/c mice. (C) Corneal thickness depends on sex. Corneal thickness is compared between male and female BALB/c mice. All data were obtained from n = 10 mice/group and representative data from three independent experiments are shown. All data were compared to baseline (1 month). <i>p</i> values are calculated using one-way ANOVA with Bonferroni post hoc test, and error bars represent SEM. (*<0.05, **<0.01, ***<0.001).</p

Cartoon representation of the folding of MBP in complex with TF.

<p>For each folding stage, a representative snapshot is shown, with MBP (and its partial folds) in pale red and TF in cyan. The schematic shows a series of events in the cycle, which starts with the interactions between folding intermediates of substrate proteins (MBP in this case) with TF and results in the fully folded substrate protein released from the TF at the end. This may involve more than one TF molecules.</p

Contacts between N-terminal and Arm2.

<p>List of amino-acid residue pairs involved in inter-domain interactions between N-terminal and Arm2, arranged in descending order of occurrence. Contacts expected to be hydrophobic are italicised.</p

Steered MD simulations of pulling the folded P1 apart to unfold it in isolation (black lines), and in complexes with TF: HC1 (blue lines) and HC2 (red lines).

<p><b>A.</b> Work-extension graphs of each trajectory (dashed lines) and average work (bold solid lines); <b>B.</b> Average number of P1 <i>β</i>–sheet hydrogen bonds in the three systems; <b>C.</b> Average number of TF-P1 contacts. The vertical dashed lines at pulling distance of 8.2 nm marks the end of the energy barrier. <b>D.</b> Plots of second order cumulant expansion (solid lines) and Boltzmann-weighted average work plot (dashed lines) for each system in the first 1.4 ns or over 0.7 nm of pulling. <b>E.</b> The PF-contacts (circles) are broken while TF-P1 contacts (solid lines) remain almost unchanged in the first 1.4 ns (or 0.7 nm) of pulling. <b>F.</b> Cartoon representation of the loss of 4 hydrogen bonds between the first and last <i>β</i>–strands (colored orange) of P1 upon pulling.</p

Crystal structures of.

<p><b>A.</b> Trigger factor (TF, PDB code: 1W26). The surface of the protein is shown as transparent wire-mesh around the backbone. The surfaces of TF domains are colored differently: N-terminal in blue, Linker in indigo, PPIase domain in red, HA1-linker in yellow, Arm1 in orange, and Arm2 in green; <b>B.</b> Maltose binding protein (MBP, PDB code: 1JW4). The surface of MBP is colored gray, while the structure is colored black. Truncate of MBP’s folding intermediates: <b>C.</b> P2 with a backbone in orange and <b>D.</b> P1 with a backbone in red.</p

Four 200 ns long AA-MD simulations of extended TF each with full MBP and P2.

<p><b>A.</b> Contact probabilities of TF residues with MBP (top panel) and P2 (bottom panel). The barcode plots the standard hydrophobicity map of TF: color-scaled from red (hydrophilic) to green (hydrophobic). <b>B.</b> Distribution of the fraction of hydrophilic contacts in TF-substrate binding for different substrates. The dashed lines plot the distribution in the first 10 ns, while solid lines with circles plot the distribution over the whole trajectory. <b>C.</b> Visualization of the most important interaction sites (blue) on TF for MBP and P2. <b>D.</b> Representative final conformations of TF-MBP and TF-P2 complexes. The substrates are shown in transparent white colored wire-mesh.</p