Publications (6)13.83 Total impact
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Article: Straight GDP-tubulin protofilaments form in the presence of taxol.
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ABSTRACT: Microtubules exist in dynamic equilibrium, growing and shrinking by the addition or loss of tubulin dimers from the ends of protofilaments. The hydrolysis of GTP in beta-tubulin destabilizes the microtubule lattice by increasing the curvature of protofilaments in the microtubule and putting strain on the lattice. The observation that protofilament curvature depends on GTP hydrolysis suggests that microtubule destabilizers and stabilizers work by modulating the curvature of the microtubule lattice itself. Indeed, the microtubule destabilizer MCAK has been shown to increase the curvature of protofilaments during depolymerization. Here, we show that the atomic force microscopy (AFM) of individual tubulin protofilaments provides sufficient resolution to allow the imaging of single protofilaments in their native environment. By using this assay, we confirm previous results for the effects of GTP hydrolysis and MCAK on the conformation of protofilaments. We go on to show that taxol stabilizes microtubules by straightening the GDP protofilament and slowing down the transition of protofilaments from straight to a curved configuration.Current Biology 11/2007; 17(20):1765-70. · 9.65 Impact Factor -
Article: Self-assembly of solid-supported membranes using a triggered fusion of phospholipid-enriched proteoliposomes prepared from the inner mitochondrial membrane.
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ABSTRACT: A general procedure for the formation ofsolid-supported artificial membranes containing transmembrane proteins is reported. The main objective was to directly use the pool of proteins of the native biomembrane (here the inner membrane from mitochondria of human carcinogenic hepatic cells) and to avoid purification steps with detergent. Proteoliposomes of phospholipid-enriched inner membranes from mitochondria were tethered and fused onto a tailored surface via a streptavidin link. The failure of some preliminary experiments on membrane formation was attributed to strong nonspecific interactions between the solid surface and the protuberant hydrophilic parts of the transmembrane complexes. The correct loading of uniform membranes was performed after optimization of a tailored surface, covered with a grafted short-chain poly(ethylene glycol), so that nonspecific interactions are reduced. Step-by-step assembly of the structure and triggered fusion of the immobilized proteoliposomes were monitored by surface plasmon resonance and fluorescence photobleaching recovery, respectively. The long-range lateral diffusion coefficient (at 22 degrees C) for a fluorescent lipid varies from 2.5 x 10(-8) cm2 s(-1) for a tethered lipid bilayer without protein to 10(-9) cm2 s(-1) for a tethered membrane containing the transmembrane proteins of the respiratory chain at a protein area fraction of about 15%. The decrease in the diffusion coefficient in the tethered membrane with increase in protein area fraction was too pronounced to be fully explained by the theoretical models of obstructed lateral diffusion. Covalent tethering links with the solid are certainly involved in the decrease of the overall lateral mobility of the components in the supported membrane at the highest protein-to-lipid ratios.Langmuir 06/2005; 21(10):4661-8. · 4.19 Impact Factor -
Article: Technological innovation around protein and cell biochip for diagnosis: a translational research from nanoworld to patient
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ABSTRACT: A great challenge in biosensors and diagnosis devices relies on the way to reconstitute relevant biological mechanisms on surface of the biochips and which analytical tools are convenient to provide accurate and rapid information on the structures and function of molecules attached to this surface. A better control in the realization of biochips can be obtained in combining different complementary approaches while always keeping in mind the biological key point. Researches in CLIPP are focused towards this objective. Conception, realization and characterization of protein and cell chips are presented. We detail different strategies of materials engineering1,2,3, chemical functionalizations and biomolecular graftings4, molecular and cellular characterization in physiological conditions5,6, which lead to the optimization of “biorecognitions events” at the surface of the chip. We present herein an original interdisciplinary approach, consisting to carry out in parallel a micro-scale analysis (SPR, fluorescence microscopy) and nano-scale characterizations (AFM, XPS, TOF-SIMS). Concerning protein interfaces, we demonstrated in particular that the molecular orientation in a protein monolayer can be determined based on the specific fragment ions from the protein in TOF-SIMS spectra7. These developments have also contributed to the establishment of a new biomolecular interaction analysis/mass spectrometry (BIA-MS) combination based on an entire “on-a-chip” procedure8. We report a low-cost approach combining Biacore 2000 analysis with homemade chips and MS and MS/MS identification directly onto the chips without elution step. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the “on-a-chip” processing to complete the BIA-MS approach in the discovery and analysis of protein complexes in biological fluids. Our interest is also focused on cell/surface interaction. The cell biochips we are developing consist either of circulating or adherent cells, that we characterized in terms of cell capture on biofunctionalized surface or growth with substrate dependency respectively. Parameters such as cell spreading, growth, morphology, and topography are particularly investigated and controlled by atomic force microscopy in physiological conditions6. With the aim to increase the throughput of analysis, we are currently working on cell and protein micro-arrays. Our expertise in cell and protein biochip preparation, and competences in micro- to nanoscale characterization in liquid conditions, represents precious assets enabling a relevant clinical proteomic research, thanks to deeply controlled steps of biosensor development and use. -
Article: Preparation of flat gold terraces for protein chip developments
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ABSTRACT: A simple method to prepare flat gold terraces on mica for atomic force microscopy biomolecular characterisation is described. The procedure includes preheating of the substrate, metal deposition and an annealing step. All of these steps are at elevated temperatures (300–420°C). This approach allows one to prepare large flat gold terraces (200– 500 nm), which constitute ideal substrates for visualisation and characterisation of a self-assembly monolayer of biomolecules at the nanoscale. The authors illustrated this potential of characterisation with the reconstitution of a protein monolayer. -
Article: Production of microporous aluminum oxide electrodes as supports for tethered lipid bilayers of large surface area
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ABSTRACT: A composite electrode made by association of gold and porous aluminum oxide has been used as template for a supported lipid bilayer. An oxide pore size of 170 nm was chosen so that lipid vesicles or proteoliposomes can enter in the pore and cover the pore wall. Triggered vesicle fusion produced a stable and continuous lipid bilayer supported on the large surface area of the honeycomb structure. Continuity of the supported bilayer was attested by electrochemical measurement of the lateral mobility of ubiquinone, a water-insoluble electroactive marker, dissolved at low concentration in the hydrophobic leaflet of the bilayer. -
Article: Nanobioengineering and Characterization of a Novel Estrogen Receptor Biosensor
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ABSTRACT: We constructed an original supramolecular assembly on a surface of sensor composed of an innovative combination of an engineered cytochrome b5 and a modified nucleic acid bound to a synthetic lipid hemimembrane. The protein/DNA block, called (PDNA) 2, was synthesized and purified before its immobilization onto a hybrid bilayer reconstituted on a gold surface. Surface plasmon resonance (SPR) and atomic force microscopy (AFM) were engaged in parallel on the same substrates in order to better understand dynamic events that occur at the surface of the biosensor. Good correlations were obtained in terms of specificity and reversibility. These findings allow us to present a first application of such biosensor in the study of the interaction processes between nuclear receptor and DNA
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- Langmuir (1)
- Current Biology (1)
Institutions
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2007
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Technische Universität Dresden
Dresden, Saxony, Germany
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2005
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Université de Technologie de Compiègne
Compiègne, Picardie, France
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