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

SDS-PAGE analysis of S-MB and MB peptides.

<p><u>Lane 1 (Ln 1)</u>: Molecular mass markers (in kDa). <u>Lane 2 (Ln 2)</u>: MB, 4 µg. <u>Lane 3 (Ln 3</u>): S-MB, 4 µg. Arrows to the right of Lane 3 indicate the predicted, approximate positions of the respective monomer and dimer bands for MB and/or S-MB. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008672#s2" target="_blank"><u>Methods</u></a> for additional details.</p

Primary sequences and disulfide bonding patterns for Super Mini-B (S-MB) and Mini-B (MB).

<p><u>Panel A</u>: S-MB (41 amino-acid residues; 1-letter amino-acid notation), with Phe-1, Cys-8 and Ser-41 indicated. <u>Panel B</u>: MB (34 residues), with its numbering based on the parent S-MB, and Cys-8 and Ser-41 indicated. In Panels A and B, the disulfide-linkages are shown between Cys-8 and Cys-41 and between Cys-11 and Cys-34, with the positively-charged arginines (R+) and lysines (K+) denoted. The N-terminal insertion sequence SP-B(1–8) corresponds to NH2-FPIPLPYC-CONH2 (see text).</p

The evolving 3D model of monomeric Super Mini-B (S-MB) in 40% HFIP/60% water at the starting (“0 nsec”) and ending (“100 nsec”) times of the MD simulation.

<p><u>Plate A</u>: Snapshot of S-MB at “0 nsec”. DSSP analysis indicated the following secondary conformation map (residues in parentheses): coil (1–8, 22, 26, 29, 39–41); turn (18–21, 23–25, 34–38); bend (27–28); and α-helix (9–17, 31–33) (see text). <u>Plate B</u>: Snapshot of S-MB at “100 nsec”. DSSP analysis indicated the following secondary conformation map (residues in parentheses): coil (1–6, 23–25, 28–29, 39–41); turn (7–8, 18–21, 36–38); bend (22, 26–27); and α-helix (9–17, 30–35). In Plates A and B, MD simulations were performed in the GROMACS version 3.3.3 environment (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008672#s2" target="_blank"><u>Methods</u></a>). The protein backbone structure is shown with color-coded ribbons denoting the following domains: N-terminal insertion sequence (green), N-terminal helix (red), turn-loop (green), and C-terminal helix (red) rendered with PyMOL v0.99. Appropriately colored side-chains are shown as stick figures attached to the N-terminal insertion sequence (green), helix (red) or loop (green) ribbon backbones. The orientations of S-MB in Plates A and B are the same as that for S-MB in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008672#pone-0008672-g002" target="_blank">Fig. 2A</a>, with the N-terminal Phe-1 at the far-left. Disulfide linkages between the N-terminal helix in the foreground and C-terminal helix in the background are highlighted in yellow.</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

Propensity for β-sheet aggregation determined for MB (----) and S-MB (^____) from PASTA and AGGRESCAN analyses.

<p><u>Plate A</u>: Plot of the aggregation propensity [i.e., h(k)], calculated from the PASTA algorithm for the relative energies of the various antiparallel and parallel β-sheet pairings <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008672#pone.0008672-Trovato2" target="_blank">[62]</a>. The h(k) values are plotted for each peptide residue (k), and normalized so that the summation of all h(k)s for a peptide equals 1.0. <u>Plate B</u>: Plot of the normalized “hot spot” area as a function of each peptide residue (k), determined from the AGGRESCAN algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008672#pone.0008672-ConchilloSole1" target="_blank">[64]</a> for the relative propensity of local peptide domains to form aggregates. The sequence and numbering for MB and S-MB are in Fig. 2.</p

ATR-FTIR spectra of MB and S-MB in the structure-promoting solvents hexafluoroisopropanol (HFIP) and methanol (MeOH), the detergent lipid SDS and the phospholipids POPG and DPPC.

<p><u>Panel A</u>: Stacked FTIR spectra of MB in 40% HFIP (i.e., 40% HFIP/60% deuterated sodium phosphate buffer, pH 7.4), 100% MeOH (i.e., 100% methanol), deuterated SDS, POPG and DPPC. <u>Panel B</u>: FTIR spectra of S-MB in 40% HFIP, 100% MeOH, SDS, POPG and DPPC. In Panels A and B, the IR spectra for MB and S-MB each show dominant α-helical components centered at 1657–1651 cm⁻¹ (arrows), with minor bands at ∼1637–1613 cm⁻¹ (arrow at 1620 cm⁻¹ ) denoting β-sheet. Peptide concentrations were 470 µM for solvent spectra and 10:1 lipid:peptide (mole:mole) for lipid spectra. The abscissa (left to right) is 1740–1560 cm⁻¹, while the ordinate represents absorption (in arbitrary units). See text for discussion.</p

Arterial oxygenation and dynamic compliance in surfactant-treated, ventilated rats with ARDS induced by <i>in vivo</i> lavage.

<p>Arterial partial pressure of oxygen (PaO₂ in torr) and dynamic compliance (mL/kg/cm H₂O) are shown as a function of time for groups of 8–10 rats treated with synthetic lung surfactants (synthetic lipids +1.5 mol% S-MB, MB, or SP-B(1–8)), synthetic lipids +1.5% porcine SP-B (positive control), or synthetic lipids alone as a negative control. Data are shown as mean ± SEM.</p

Molecular graphics representations of the 3D structure of dimer S-MB after 10-nsec of MD simulation in a SDS/water environment.

<p>The 3D-structure of dimer S-MB the open conformation was determined from the 10-nsec MD simulation of a ZDOCK/RossettaDock structure, with 28 SDS molecules in a water box and rendered using Rasmol version 2.7.4.2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008672#s2" target="_blank"><u>Methods</u></a>). <u>Plate A</u>: The protein backbone structure is shown with color-coded ribbons denoting helix (red) or non-helix (green), with appropriately colored sidechains indicated as stick figures. The four disulfide linkages are highlighted in yellow. The two N-terminal insertion sequences (residues 1–7) are represented by space-filling backbones and sidechains colored in purple. These N-terminal sequences adopt extended conformations that are centered just over the N- and C-terminal helices, with each having its N-terminal Phe-1 near the loop region (Gly-25 and Gly-26). The 28 SDS lipids are shown as wireframe molecules that are colored according to the cpk convention. The approximate two-fold axis relating the two monomers is shown by an arrow. <u>Plate B</u>: The space-filling model of dimer S-MB is shown in the same orientation as in Plate A, but without the SDS detergent. Polar/charged and nonpolar/hydrophobic residues are colored in blue and red, using the Wimley-White ranking system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008672#pone.0008672-Wimley1" target="_blank">[117]</a> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008672#pone-0008672-g001" target="_blank">Fig. 1B</a>).</p