Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin *

doi: 10.1074/jbc.M113.508838

Figure Lengend Snippet: P454 membrane partition followed by fluorescence. A, ratio of fluorescence intensity at 340 and 380 nm as a function of lipid concentration. The negatively charged small unilamellar vesicles (squares) or large unilamellar vesicles (circles) were made of DOPC/DOPE/DOPG/Chol (4:3:2:1). The P454 fluorescence intensity ratio values were fitted as described under “Materials and Methods,” providing partition coefficient KX and the Hill number (KX = 2.9 × 105, nH = 1.35 for SUVs; KX = 3.9 × 104, nH = 1.04 for LUVs). B, partition coefficient KX as a function of peptide/lipid ratio. Thermodynamic parameters are reported in supplemental Table S1. Error bars correspond to S.D.

Article Snippet: Coarse Grained Molecular Dynamics of the P454 Peptide Coarse grained simulations were performed with the two peptides P414 and P454 in the presence of neutral and negatively charged lipid bilayers.

Techniques: Membrane, Fluorescence, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin *

doi: 10.1074/jbc.M113.508838

Figure Lengend Snippet: Isothermal titration calorimetry of peptide membrane partition. The heat flow traces show the calorimetric titrations of P454 (A) and P414 (B, gray traces) by consecutive injections of negatively charged SUVs (DOPC/DOPE/DOPG/Chol, 4:3:2:1). B also shows the heat flow produced by injections of neutral SUVs (DOPC/DOPE/Chol, 6:3:1) into P454 (black traces; offset −0.5 μcal/s in the figure for a better visualization). Each peak corresponds to the injection of 5 μl of 20 mm SUVs into the ITC reaction cell containing 1.41 ml of 42 μm peptide in buffer B at 25 °C. C, integrated binding isotherm of P454 with negative SUVs (filled circles) or with neutral SUVs (empty circles) and P414 with negative SUVs (filled squares). D, heat flow trace of anionic SUVs titrated by a solution of P454. Each peak corresponds to the injection of 5 μl of 85 μm P454 into the ITC reaction cell containing 1.41 ml of 5 mm anionic SUVs in buffer B at 25 °C. Thermodynamic parameters are reported in supplemental Table S2.

Article Snippet: Coarse Grained Molecular Dynamics of the P454 Peptide Coarse grained simulations were performed with the two peptides P414 and P454 in the presence of neutral and negatively charged lipid bilayers.

Techniques: Isothermal Titration Calorimetry, Membrane, Produced, Injection, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin *

doi: 10.1074/jbc.M113.508838

Figure Lengend Snippet: Circular dichroism and FTIR spectra of P454. A, far-UV CD spectra of P454 (40 μm) in the absence (solid line) or in the presence (dashed lines) of increasing concentrations (0.5, 1, and 2 mm from top to bottom) of anionic SUVs made of DOPC/DOPE/DOPG/cholesterol (4:3:2:1) in buffer B at 25 °C. B, synchrotron radiation CD spectra of P454 (200 μm) in the absence (solid line) or in the presence (dashed line) of anionic SUVs (10 mm) in buffer B at 25 °C. C and D, transmission FTIR spectra and band deconvolutions of P454 (150 μm) in D2O buffer B (C) or in the presence (D) of anionic SUVs (5 mm lipids) in D2O buffer B at 25 °C. The S.D. value is within 5%. Secondary structure content analysis is reported in supplemental Table S3.

Article Snippet: Coarse Grained Molecular Dynamics of the P454 Peptide Coarse grained simulations were performed with the two peptides P414 and P454 in the presence of neutral and negatively charged lipid bilayers.

Techniques: Circular Dichroism, Transmission Assay

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin *

doi: 10.1074/jbc.M113.508838

Figure Lengend Snippet: ATR-FTIR spectra of P454. The ATR-FTIR spectra of P454 mixed with lipids (DOPC/DOPE/DOPG/cholesterol, 4:3:2:1) were recorded with perpendicular (top) or parallel (middle) polarization of the infrared light. The resulting dichroic spectrum of P454 in membranes (bottom) is multiplied by a scaling factor of 8 for convenience (see “Materials and Methods”). The S.D. value is within 5%. Secondary structure content analysis is reported in supplemental Table S4.

Article Snippet: Coarse Grained Molecular Dynamics of the P454 Peptide Coarse grained simulations were performed with the two peptides P414 and P454 in the presence of neutral and negatively charged lipid bilayers.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin *

doi: 10.1074/jbc.M113.508838

Figure Lengend Snippet: Molecular dynamics of P454 inserted into an anionic lipid bilayer. Snapshot of the system after 1 μs of molecular dynamics simulation at constant temperature (323.15 K) and pressure (1 bar). Red, P454 peptides; light blue, DOPC; green, cholesterol; yellow-orange, DOPG. For clarity, water is not depicted.

Article Snippet: Coarse Grained Molecular Dynamics of the P454 Peptide Coarse grained simulations were performed with the two peptides P414 and P454 in the presence of neutral and negatively charged lipid bilayers.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin *

doi: 10.1074/jbc.M113.508838

Figure Lengend Snippet: Radial distribution function of the lipids around the center of mass of P454 in the presence or absence of DOPG in the lipid bilayer. The radial distribution function was calculated using the phosphate beads for DOPC and DOPG or the hydroxyl beads for cholesterol. The dashed line indicates the first shell of lipids around the P454 mass center. The small and wide peak at ∼4 nm corresponds to lipids on the opposite side of the bilayer.

Article Snippet: Coarse Grained Molecular Dynamics of the P454 Peptide Coarse grained simulations were performed with the two peptides P414 and P454 in the presence of neutral and negatively charged lipid bilayers.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Characterization of a Membrane-active Peptide from the Bordetella pertussis CyaA Toxin *

doi: 10.1074/jbc.M113.508838

Figure Lengend Snippet: Membrane permeabilization by P454. A, time course of ANTS and DPX efflux from negatively charged LUVs (0.45 mm lipids) made of DOPC/DOPE/DOPG/Chol (4:3:2:1 lipid ratio) in the presence of the indicated concentrations of P454 (solid lines). Dashed line, permeabilization of neutral LUVs (DOPC/DOPE/Chol at a 6:3:1 ratio) in the presence of 1 μm P454. B, time course of ANTS and DPX efflux from negatively charged LUVs (0.45 mm lipids) made of DOPC/DOPE/DOPG/Chol (4:3:2:1) in the presence of 0.3 μm P454, AC384, or AC489.

Article Snippet: Coarse Grained Molecular Dynamics of the P454 Peptide Coarse grained simulations were performed with the two peptides P414 and P454 in the presence of neutral and negatively charged lipid bilayers.

Techniques: Membrane