19 research outputs found

    Neutron Reflectometry Elucidates Density Profiles of Deuterated Proteins Adsorbed onto Surfaces Displaying Poly(ethylene glycol) Brushes: Evidence for Primary Adsorption

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    The concentration profile of deuterated myoglobin (Mb) adsorbed onto polystyrene substrates displaying poly­(ethylene glycol) (PEG) brushes is characterized by neutron reflectometry (NR). The method allows to directly distinguish among primary adsorption at the grafting surface, ternary adsorption within the brush, and secondary adsorption at the brush outer edge. It complements depth-insensitive standard techniques, such as ellipsometry, radioactive labeling, and quartz crystal microbalance. The study explores the effect of the PEG polymerization degree, <i>N</i>, and the grafting density, σ, on Mb adsorption. In the studied systems there is no indication of secondary or ternary adsorption, but there is evidence of primary adsorption involving a dense inner layer at the polystyrene surface. For sparsely grafted brushes the primary adsorption involves an additional dilute outer protein layer on top of the inner layer. The amount of protein adsorbed in the inner layer is independent of <i>N</i> but varies with σ, while for the outer layer it is correlated to the amount of grafted PEG and is thus sensitive to both <i>N</i> and σ. The use of deuterated proteins enhances the sensitivity of NR and enables monitoring exchange between deuterated and hydrogenated species

    Water Collective Dynamics in Whole Photosynthetic Green Algae as Affected by Protein Single Mutation

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    In the context of the importance of water molecules for protein function/dynamics relationship, the role of water collective dynamics in Chlamydomonas green algae carrying both native and mutated photosynthetic proteins has been investigated by neutron Brillouin scattering spectroscopy. Results show that single point genetic mutation may notably affect collective density fluctuations in hydrating water providing important insight on the transmission of information possibly correlated to biological functionality. In particular, we highlight that the damping factor of the excitations is larger in the native compared to the mutant algae as a signature of a different plasticity and structure of the hydrogen bond network

    Neutron reflection intensity as a function of scattering vector, Q.

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    <p>(A) hEcoli deposited at 50°C, (B) dEcoli deposited at 50°C and (C) dEcoli deposited at 25°C. The contrasts used are: H<sub>2</sub>O (blue squares), 40% D<sub>2</sub>O (yellow triangle pointing up) and 60% D<sub>2</sub>O (green down-pointing triangle) and pure D<sub>2</sub>O (pink circles). All reflectivity profiles were measured at 25°C regardless of deposition temperature. Panel D-F give the SLD profiles and bilayer sketches corresponding to the reflection curve above it. In E and F the small additional patch of bilayer to the right represents the extra bilayer of very low coverage, which is used as a model for a small amount of co-adsorbed vesicles. In E, this extra layer was only present in the first contrast (H<sub>2</sub>O) measured and, thus, only the blue SLD profile represents the extended model.</p

    AFM image of a supported lipid membrane of <i>E</i>. <i>coli</i> lipids.

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    <p>A: hEcoli membrane of low coverage. B: a height profile corresponding to the white line in A. Inset in B shows the overall height distribution.</p

    Selected QCM-D traces for bilayer formation, which were part of the optimization process.

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    <p>A: frequency changes. B: dissipation changes. Several overtones are shown for each experiment; 5<sup>th</sup>, 7<sup>th</sup>, 9<sup>th</sup> and 11<sup>th</sup>. The 5<sup>th</sup> overtone carries the marker. SUVs made from Avanti <i>E</i>. <i>coli</i> total lipid extract were added to clean silica under different experimental conditions: blue down-pointing triangle: 100 μg/ml lipid, 1 mM CaCl<sub>2</sub>, orange square: 100 μg/ml lipid, 2 mM CaCl<sub>2</sub>, purple circle: 50 μg/ml lipid, 2 mM CaCl<sub>2</sub>, gray diamond: 250 μg/ml lipid, 2 mM CaCl<sub>2</sub>, pink up-pointing triangle: 100 μg/ml lipid, 2 mM CaCl<sub>2</sub>,–pump stopped after initial adsorption. The * indicates the point where trace number 4 was rinsed with buffer. The others were rinsed at the point marked with a #.</p

    Formation and Characterization of Supported Lipid Bilayers Composed of Hydrogenated and Deuterated <i>Escherichia coli</i> Lipids

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    <div><p>Supported lipid bilayers are widely used for sensing and deciphering biomolecular interactions with model cell membranes. In this paper, we present a method to form supported lipid bilayers from total lipid extracts of <i>Escherichia coli</i> by vesicle fusion. We show the validity of this method for different types of extracts including those from deuterated biomass using a combination of complementary surface sensitive techniques; quartz crystal microbalance, neutron reflection and atomic force microscopy. We find that the head group composition of the deuterated and the hydrogenated lipid extracts is similar (approximately 75% phosphatidylethanolamine, 13% phosphatidylglycerol and 12% cardiolipin) and that both samples can be used to reconstitute high-coverage supported lipid bilayers with a total thickness of 41 ± 3 Å, common for fluid membranes. The formation of supported lipid bilayers composed of natural extracts of <i>Escherichia coli</i> allow for following biomolecular interactions, thus advancing the field towards bacterial-specific membrane biomimics.</p></div

    Phospholipid and fatty acid composition of <i>Pichia Pastoris</i> cells.

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    <p>(A) Phospholipid composition and (B) total fatty acid distribution of <i>Pichia pastoris</i> cells grown in a hydrogenated medium (red) and in a deuterated environment (blue) at 30°C. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PG, phosphatidylglycerol; CL, cardiolipin or diphosphatidyglycerol. Data represent mean values ± s.d (n = 3). In histograms, *<i>P</i><0.05 from Student's <i>t</i>-test, assuming equal variance.</p

    Growth curves of <i>Pichia Pastoris</i> cells.

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    <p>(A) Growth of <i>Pichia pastoris</i> cells in a hydrogenated medium at 30°C (red circle) and 18°C (green circle). (B) Growth of <i>P. pastoris</i> cells in a deuterated medium at 30°C (blue square) and 18°C (cyan square). Errors bars represent the standard deviation from three independent growth curves.</p
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