CA in spider silk glands.

<p>CA activity and Azure blue staining of histological sections from (A) the sac of a <i>Tegenaria sp</i>. major ampullate gland, (B) <i>E. australis</i> minor ampullate gland, (C) <i>A. diadematus</i> aggregate gland duct, (D) <i>Tegenaria sp</i>. tubuliform gland, and (E) the third limb of the duct of an <i>A. diadematus</i> major ampullate gland. Black precipitates represent CA activity (arrow heads). In (A) the glandular lumen is labeled and the dotted arrow points towards the duct. Nuclei are indicated by (N) and the lumen by (Lu). Scale bar, (A) 50 µm and (B–E) 20 µm.</p

NT and CT respond differently to lowered pH.

<p>Stability of NT and CT (from <i>A. ventricosus</i>) in (A) 20 mM HEPES/MES buffer with 154 mM NaCl and (B) the same buffer without NaCl, measured with Trp fluorescence and CD spectroscopy at 222 nm, respectively, presented as urea concentrations for apparent half-denaturation ([den]^50%, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001921#s3" target="_blank">Materials and Methods</a> for details on how [den]^50% was determined) as a function of pH. The pH region in which CA activity is found in major ampullate glands is indicated by a shaded area in (A).</p

CD spectra of NT and CT.

<p>The residual molar ellipticity was measured at 25, 45, 65, 85, and 95°C and at 25°C after cooling for (A) NT and (B) CT at pH 7.5, 6.5, and 5.5 from top to bottom.</p

pH, bicarbonate, and carbon dioxide in major ampullate glands.

<p>Photograph of a major ampullate gland in which measured pH and HCO₃⁻ values and calculated pCO2 values at different locations are indicated. See Table 1 for details of ISM measurements. Scale bar, 1 mm.</p

CS₂ effects on NT.

<p>Chemical shift perturbations of MaSp NT backbone amides at pH 7.2 and 300 mM NaCl (upper panel) and pH 5.5 (lower panel) upon addition of CS₂ (0 to 200 mM). The most perturbed residues are labeled, and positions of helices 1–5 are indicated above the plot.</p

CT forms amyloid-like fibrils at low pH.

<p>(A) ThT fluorescence for CT at pH 6.0, 5.7, 5.5, 5.3, and 5.0, and NT at pH 5.0, in sodium acetate buffer. (B) Transmission electron micrograph showing fibrils formed from CT incubated at pH 5.5. Scale bar, 200 nm. (C) Congo red stained fibrils formed by CT at pH 5.5, viewed under crossed polarizers. Green birefringence is visible. Scale bar, 50 µm.</p

CA in spider silk glands.

<p>CA activity and Azure blue staining of histological sections from (A) the sac of a <i>Tegenaria sp</i>. major ampullate gland, (B) <i>E. australis</i> minor ampullate gland, (C) <i>A. diadematus</i> aggregate gland duct, (D) <i>Tegenaria sp</i>. tubuliform gland, and (E) the third limb of the duct of an <i>A. diadematus</i> major ampullate gland. Black precipitates represent CA activity (arrow heads). In (A) the glandular lumen is labeled and the dotted arrow points towards the duct. Nuclei are indicated by (N) and the lumen by (Lu). Scale bar, (A) 50 µm and (B–E) 20 µm.</p

Summary of HDX ESI MS data for CT.

<p>(A) Deuterium incorporation in <i>A. ventricosus</i> MiSp CT at each pH. The degree of deuteration of the peptic peptides at pH 7.5, 6.5, and 5.5 is indicated according to the color code on the right. (B) Deuterium uptake graphs for the major peptic peptide species at pH 7.5, 6.5, and 5.5. Graphs show the average of three repeats. The error bars indicate the standard deviations. The corresponding peptic peptide sequences are given above each graph.</p

2D [¹⁵N-¹H]-HSQC NMR spectra of CT at different pH.

<p>2D [¹⁵N-¹H]-HSQC NMR spectra of CT at different pH.</p

Effect of methazolamide (MTZ) on the pH gradient.

<p>pH values in <i>N. clavipes</i> major ampullate glands before MTZ treatment (pH – MTZ), during MTZ treatment (pH + MTZ, in the presence of 0.1 mM MTZ for 1 h) and after washing for 30 min (pH after washing). Locations are indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001921#pbio-1001921-g001" target="_blank">Figure 1</a>.</p