Resistance of DEPN-8+1.5% by weight Mini-B to degradation by phospholipase A₂ (PLA₂) compared to calf lung surfactant extract (CLSE).

<p>Data are mean±SEM for n = 3. DEPN-8+1.5% by weight Mini-B was incubated <i>in vitro</i> with PLA₂ (0.1 Units/ml) for 30 min at 37°C, and degradation was assessed by measuring lipid classes in weight percent based on phosphate analysis of bands on thin layer chromatography. Results for CLSE in the presence and absence of PLA₂ utilized identical methods as reported previously by Wang et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#pone.0001039-Wang3" target="_blank">[30]</a>.</p

Mean association and dissociation kinetic rate constants (k_on, k_off) and equilibrium dissociation constant KD calculated from plasmon resonance measurements for liposomes of DEPN-8 or DPPC flowing past a chip-linked Mini-B monolayer.

*<p>Liposomes of DEPN-8 or DPPC in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4) were flowed past a monolayer of Mini-B linked via Cys4 and Cys27 to a C5M sensor chip in a Biacore 3000 system (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#s4" target="_blank"><u>Methods</u></a>). Mean kinetic rate constants (k_on, k_off) and the equilibrium dissociation constant (KD = k_off/k_on) were determined from curve fitting analyses of plasmon resonance results at six different lipid concentrations (0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 µg/ml for each lipid).</p

Dynamic surface activity of DEPN-8+1.5% or 3% Mini-B compared to CLSE on the captive bubble surfactometer.

<p>Minimum and maximum surface tensions are shown for DEPN-8+1.5% or 3% by weight Mini-B and CLSE on a captive bubble surfactometer during 10 cycles of rapid compression (20 cycles/min) following slow compression as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#pone-0001039-g005" target="_blank">Figure 5</a>. Surface tension values are Mean±SEM for at least three separate experiments. See text for details.</p

Spectroscopic behavior of Mini-B and DEPN-8.

<p><u>Panel A</u>: CD spectrum for Mini-B in trifluoroethanol (TFE); <u>Panel B</u>: FTIR spectrum for DEPN-8; <u>Panel C</u>: FTIR spectral differences for Mini-B in DEPN-8 (dashed line) compared to Mini-B in TFE (solid line). In Panel A, mean residue ellipticity (MRE) averaged over eight scans is plotted against wavelength for Mini-B in 4∶6 (v:v) TFE:10 mM phosphate buffer, pH 7.4. The double minimum at ∼208 and 222 nm is indicative of a high α-helical content. In Panel B, the spectrum for DEPN-8 multilayers (100 µg lipid, arbitrary absorbance units) has a “C-O-C” ether linkage-associated absorption band centered at a wavenumber of 1072 cm⁻¹. In Panel C, the IR spectrum of Mini-B in TFE (solid line) has a peak at 1655 cm⁻¹ indicating high α-helix levels, while the peak at 1658 cm⁻¹ and high-field shoulder at 1678 cm⁻¹ for Mini-B in DEPN-8 (dashed line) indicates an increase in turn/bend conformation with a decreased but still prominent α-helix content. See text for discussion.</p

Dynamic surface activity of DEPN-8+1.5% (by wt) Mini-B compared to CLSE on the pulsating bubble surfactometer.

<p><u>Panel A</u>: 0.5 mg/ml phosphonolipid (phospholipid); <u>Panel B</u>: 2.5 mg/ml phosphonolipid (phospholipid). Surface tension at minimum bubble radius (minimum surface tension) for DEPN-8+1.5% Mini-B and CLSE is graphed as a function of time on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression). Data are Mean±SEM for n = 3–5. See text for details.</p

Adsorption of DEPN-8 with and without 1.5% (by wt) Mini-B compared to calf lung surfactant extract (CLSE).

<p>Adsorption surface tensions are plotted following the addition of a bolus of DEPN-8, DEPN-8+1.5% Mini-B, or CLSE to a stirred subphase (10 mM HEPES with 0.15M NaCl and 1.5 mM CaCl₂ at pH 7.0) in a Teflon® dish at time zero. Final subphase surfactant concentration was uniform at 0.0625 mg lipid/ml. Data are Mean±SEM for n = 3–5. See text for details.</p

Mean proportions of different aspects of secondary structure for Mini-B in structure-promoting TFE solvent or in deuterium-hydrated DEPN-8 multilayers based on CD and FTIR spectroscopic analysis.

*<p>CD spectra for Mini-B in TFE were analyzed for secondary structure using the methods of Sreerama et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#pone.0001039-Sreerama1" target="_blank">[28]</a>, and FTIR spectra were analyzed for secondary conformation based on deconvolution of the amide I band (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001039#s4" target="_blank"><u>Methods</u></a>). FTIR spectra for Mini-B in deuterium-hydrated DEPN-8 multilayers were done at a molar ratio of 10∶1 lipid∶peptide. Tabulated results are means from four closely-reproduced separate determinations for each condition and spectral type.</p

Surface plasmon resonance (SPR) sensorgrams of S-MB or MB binding to immobilized Cys-S-MB or Cys-MB.

<p>S-MB and MB, each with a cysteine added to its N-terminus, were attached to the thiol Biacore chip as described in the text. Solutions of S-MB or MB in HBS-EP buffer (i.e., 10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) were then flowed over the respective chip-linked peptides. Typical SPR responses [Y-axis indicates the relative amount of binding in arbitrary response units (RU)] are shown for either 1 µg S-MB/ml buffer to chipped Cys-S-MB (black line) or chipped Cys-MB (green line), or with 1 µg MB/ml buffer to chipped Cys-S-MB (red line) or Cys-MB (blue line). Relative peptide affinities are: S-MB to S-MB ≫ S-MB to MB∼ MB to S-MB ∼ MB to MB.</p

Mean association and dissociation kinetic rate constants (k_on, k_off) and equilibrium dissociation constants (KD), calculated from surface plasmon resonance (SPR) measurements for aqueous Mini-B (MB) and Super Mini-B (S-MB) flowing past chip-linked Cys-MB and Cys-S-MB monolayers^a.

a<p>MB and S-MB in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4) were flowed past monolayers of N-terminal Cys-MB or N-terminal Cys-S-MB, linked via their respective N-terminal thiol groups to CSM sensor chips in a Biacore 3000 system (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008672#s2" target="_blank">Methods</a>).</p><p>Mean kinetic rate constants (k_on, k_off) and equilibrium dissociation constants (KD = k_off/k_on) were determined from curve fitting analyses of SPR results at 1 µg peptide/ml buffer.</p

Surface activity of synthetic surfactants and native SP-B on the captive bubble surfactometer.

<p>Minimum and maximum surface tension values are plotted for synthetic lipids with 1.5% by weight Mini-B [MB], Super Mini-B [S-MB], SP-B(1-8), or native pig SP-B, and SL alone for 10 successive compression-expansion cycles on a captive bubble surfactometer (20 cycles/min, 37°C). Synthetic lipids = 16:10:6:1:2 (weight ratio) DPPC:POPC:POPG:POPE: cholesterol. Values shown are mean ± SEM for n = 4.</p