Crystal structure of So-RNase HI, Ec-RNase HI, and Sto-esterase.

<p>(A) So-RNase HI, (B) Ec-RNase HI and (C) Sto-esterase.</p

GdnHCl-induced kinetic unfolding curves of Sto-RNase HI.

<p>Lines represent the fit of Eq. (4). (A) C58/145A. Curve represents the unfolding trace to a final concentration of 5.8 M GdnHCl. (B) ΔC6. Curve represents the unfolding trace to a final concentration of 5.0 M GdnHCl.</p

Crystal structure of wild-type Sto-RNase HI.

<p>C-terminal seven residues (cyan); hydrophobic side-chains (blue); hydrogen bonds (thin red lines); and disulfide bond (thick red line).</p

Thermodynamic parameters for denaturation of wild-type, C58/145A and ΔC6 Sto-RNase HI.

a<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016226#pone.0016226-You2" target="_blank">[14]</a>.</p>b<p>Errors are standard error values from the data fitting using Eq. (3).</p

GdnHCl-induced equilibrium unfolding curves and thermodynamic stability profiles (temperature dependence of ΔG(H2O)) of Sto-RNase HI.

<p>Wild-type (solid line and circles), C58/145A (dashed line and triangles), and ΔC6 (dot-dashed line and squares). (A) GdnHCl-induced equilibrium unfolding at 20°C. The apparent fraction of unfolded protein is shown as a function of GdnHCl concentration. Lines are best fits to a two-state equation. (B) Thermodynamic stability profiles. Closed symbols are the T_m value from the heat-induced unfolding experiment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016226#pone.0016226-You2" target="_blank">[14]</a>. Lines represent the fit of Eq. (3) using both equilibrium and heat-induced unfolding data.</p

Denaturation temperatures of chimeric proteins.

a<p>Errors are ±0.3°C.</p>b<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016226#pone.0016226-Tadokoro2" target="_blank">[16]</a>.</p>c<p>Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016226#pone.0016226-Haruki1" target="_blank">[28]</a>.</p

Statistics on data processing and structure determination of ΔC6 Sto-RNase HI.

<p>Values in parentheses are the highest-resolution bin of respective data.</p>a<p>R_merge = Σ |I_hkl - hkl>|/Σ I_hkl, where I_hkl is the intensity measurement for reflection with indices hkl and hkl> is the mean intensity for multiply recorded reflections.</p>b<p>R_{work, free} = Σ ||F_obs| - |F_calc||/Σ |F_obs|, where the R-factors are calculated using the working and free reflection sets, respectively. The free reflections comprise a random 10% of the data held aside for unbiased cross-validation throughout refinement.</p

SDS-PAGE and heat-induced unfolding curves of chimeric proteins.

<p>(A) SDS-PAGE. Lanes 1, 3, 5, are a low-molecular weight marker kit (GE Healthcare). Lanes 2, 4, 6 are purified chimeric So-RNase HI, Ec-RNase HI and Sto-esterase. (B) Heat-induced unfolding. The apparent fraction of unfolded protein is shown as a function of temperature. Curves 1, 2 and 3 represent the unfolding traces of chimeric So-RNase HI (closed circles), Ec-RNase HI (open circles) and Sto-esterase (cross). Lines are best fits to a two-state equation.</p

CD spectra and crystal structure of Sto-RNase HI variants.

<p>(A) CD spectra of wild-type (solid line), C58/145A (dashed line), and ΔC6 (dot-dashed line) Sto-RNase HI. (B) Crystal structure of ΔC6 Sto-RNase HI. (C) Crystal structure of wild-type Sto-RNase HI. The C-terminal seven residues are in cyan.</p

A proposed model of rapamycin’s neuroprotective effect during retinal inflammation.

<p>The basal level of activated mTOR promoted NF-κB activation during inflammation, which induced the retinal expression of the inflammatory cytokines, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Activated NF-κB elevated the activated and phosphorylated mTOR (pmTOR) level during inflammation, which may have exacerbated the pathogenesis. The subsequent retinal activation of signal transducer and activator of transcription 3 (STAT3), at least partly, reduced the rhodopsin protein expression in a post-transcriptional manner and impaired rod photoreceptor function. Rapamycin also attenuated inflammatory leukocyte adhesion to the vessel walls, and increased potentially neuroprotective autophagy in the retina during inflammation.</p