Covalent attachment of functionalized lipid bilayers to planar waveguides for measuring protein binding to biomimetic membranes

Horst Vogel
1995
Article

Résumé

A new method is presented for measuring sensitively the interactions between ligands and their membrane-bound receptors in situ by using integrated optics, thus avoiding the need for addnl. labels. Phospholipid bilayers were attached covalently to waveguides by a novel protocol that can in principle be used with any glass-like surface. In a first step, phospholipids carrying head-group thiols were covalently immobilized onto SiO2-TiO2 waveguide surfaces. This was accomplished by acylation of aminated waveguides with the heterobifunctional crosslinker N-succinimidyl-3-maleimidopropionate, followed by the formation of thio ethers between the surface-grafted maleimides and the synthetic thiolipids. The surface-attached thiolipids served as hydrophobic templates and anchors for the deposition of a complete lipid bilayer either by fusion of lipid vesicles or by lipid self-assembly from mixed lipid/detergent micelles. The step-by-step lipid bilayer formation on the waveguide surface was monitored in situ by an integrated optics technique, allowing the simultaneous detn. of optical thickness and one of the two refractive indexes of the adsorbed org. layers. Surface coverages of 50-60% were calcd. for thiolipid layers. Subsequent deposition of POPC resulted in an overall lipid layer thickness of 45-50 .ANG., which corresponds to the thickness of a fluid bilayer membrane. Specific recognition reactions occurring at cell membrane surfaces were modeled by the incorporation of lipid-anchored receptor mols. into the supported bilayer membranes. (1) The outer POPC layer was doped with biotinylated phosphatidylethanolamine. Subsequent specific binding of streptavidin was optically monitored. (2) A lipopeptide was incorporated into the outer POPC monolayer. Membrane binding of monoclonal antibodies, which were directed against the peptide moiety of the lipopeptide, was optically detected. Specific antibody binding correlated well with the lipopeptide concn. in the outer monolayer. [on SciFinder (R)]

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https://infoscience.epfl.ch/record/81024?ln=fr

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Horst Vogel
1995
Article

Résumé

Official source

https://infoscience.epfl.ch/record/81024?ln=fr

À propos de ce résultat

Concepts associés (18)

Concepts associés

Chargement

Publications associées (26)

Publications associées

Chargement

Lipid membranes for the fabrication of functional micro- and nano-structures

Gopakumar Gopalakrishnan

The central goal of this thesis work is to fabricate novel, functional fluorescent nanostructures in confined systems offered by phospholipid membranes, which are known to have highly ordered, thermotropic and lyotropic structures. In separate approaches, we have used three different lipid systems: multilamellar planar lipid membranes, unilamellar vesicular membranes as well as lipid monolayers for the development of functional fluorescent nano-, micro- and meso-scopic structures. Techniques like fluorescence microscopy, single particle imaging, electron microscopy, electron diffraction were used to achieve fundamental understanding of the resulting structures. Multilayer stacks of phospholipid membranes have been used as an effective template for the growth of high aspect ratio fluorescent nanowires. The room temperature synthesis was achieved in the confined nanometer-sized interlamellar water space of lipid multilayers where supersaturating CdCl2 concentrations were induced by acidification leading to controlled unidirectional growth of nanowires. The possibility to render the nanowires fluorescent by doping with CdS quantum dots (QDs) and the light waveguiding along hundreds of micrometers together with the possibility of lateral manipulation make these nanowires attractive candidates for future optoelectronic applications. Novel organic-inorganic functional nanocontainers have been designed and tested by making use of vesicle forming lipid bilayers in combination with semiconductor QDs. Hydrophobic QDs can be integrated into bilayers of lipid vesicles and such lipid/QD hybrid vesicles are capable to fuse with live cells, thereby stain the cell's plasma membrane selectively with fluorescent QDs and transfer the vesicle's cargo into the cell. Modification of the membrane of such hybrid vesicles on the other hand, made them capable to enter the cytoplasm of live cells. Additionally, these hybrid vesicles were found extremely useful for long-term model membrane imaging studies. The results described in this thesis imply that cell and lipid membranes can integrate any kind of hydrophobic nanoparticle whose size matches the membrane thickness, opening novel possibilities to manipulate them as individuals or in ensemble with wide-ranging applications for nanobiotechnology. In a further step, the ability of phospholipid molecules to exhibit lamellar to non-lamellar transition using external stimuli enabled a directed self-assembly of QDs into mesoscale fluorescent structures. An easy and versatile method for the surface modification of TOPO coated CdSe QDs using 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) have been achieved. DPPA predominantly form non-lamellar phases when dispersed in water, for example, upon addition of Ca2+ or at a pH below 6 are known to form hexagonal II phases. This particular property of DPPA has been exploited to form mesoscale self-assemblies of QD based structures both in solution and in confined systems. Potential applications include the detection or removal of Ca2+ ions in attoliter volumes, the construction of functional devices where QDs are reversibly organized in different forms as well as use of fluorescently labeled DPPA molecules for cellular studies using fluorescence microscopy.

EPFL2006

Chargement

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease share common mechanisms characterized by protein misfolding and aggregation, including formation of plaques or inclusion bodies. The rapid growing number of patients has motivated intensive research to understand biological phenomena involved in the pathology and hence developing therapeutic strategies. Here, we will focus on β-amyloid peptide (Aβ) involved in Alzheimer's disease. In the first part of this thesis, tissue transglutaminase (tTGase) has been investigated due to its implication in a number of cellular processes and disease states, where the enzymatic actions of tTGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of tTGase in cell survival or cell death mechanisms still remain elusive. tTGase mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that, tTGase induces intramolecular cross-linking of Aβ peptide, resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that, a large population of Aβ monomers contained an increase in mass of 0.984 Da at a glutamine residue, indicating that glutamine Q15 serves as an indispensable substrate in TGase mediated deamidation to glutamate E15. We provide strong analytical evidence on tTGase mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that, tTGase induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation. In the second part of this work, we explore the idea of using synthetic peptides that can switch conformation in a controlled way as a sensor for β-breakers of Aβ. The concept of switch-peptides allows the controlled onset of polypeptides folding and misfolding at physiological pH by in situ O,N-acyl migration. The synthesized sequence consisted in the core sequence responsible for Aβ aggregation, flanked by two β-sheet-inducing template through ester bonds. The triggering of the acyl migration allows the native sequence to be recovered from the ester bonds and structural change from flexible unordered conformation to β-sheets and subsequently fibrils formation were observed in solution. In a more biological environment made of lipid vesicles, the conformation of the peptide has been shown to act comparably to wild type Aβ peptide by circular dichroism (CD), namely increased negative charges over the surface led to increased helical propensity followed by a transition to β-sheets and accelerated fibrils formation. Thereafter, the conformation of the cysteine analogue of the switch-peptide covalently linked to gold surface was characterized by Fourier-transform infrared spectroscopy (FTIR). In monolayer of switch-peptide tethered to a planar gold surface, the peptide already adopts most likely β-turn conformation and no changes in the infrared spectra were detected after the triggering of the switch. On gold nanoparticles, the switch-peptide folds spontaneously into β-sheet conformation. These unwanted foldings limit the efficiency of the sensor and has driven us to an alternative configuration, where gold nanoparticles were coated with a 95:5 mixture of two peptide sequences able to stabilize the nanoparticles and to act as recognition sequence, respectively. In this configuration, the switch-peptide is added in the gold nanoparticles solution and the aggregation triggered. Here, we want to take advantage of a particular feature of gold nanoparticles that consists in a change of solution color from red to blue as the nanoparticles come closer to each other during aggregation. Preliminary results have shown that a discrimination between aggregated and soluble samples is possible. However, further improvements are necessary before a potential biosensor could be expected.

EPFL2011

Chargement

Perturbations of the Straight Transmembrane alpha-Helical Structure of the Amyloid Precursor Protein Affect Its Processing by gamma-Secretase

Matteo Dal Peraro, Mitko Dimitrov, Patrick Fraering, Thomas Lemmin

Background: Amyloid- neurotoxicity depends on the specificity of the proteolytic cleavage of the amyloid precursor protein (APP) transmembrane domain. Results: The APP transmembrane -helix is straight in a biological membrane bilayer. Conclusion: The flexibility of APP is key for adapting to the lipid environment and modulating proteolytic processing by -secretase. Significance: The dynamic characterization of APP is expected to rationalize the design of -secretase modulators. The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by -secretase of its C-terminal fragment produces amyloid- (A) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight -helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the -secretase enzyme, as suggested by a series of Gly(700) mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of A38 compared with A40 peptides, which is reminiscent of the effect of -secretase modulators inhibitors.

American Society for Biochemistry and Molecular Biology2014

Chargement

Lipid membranes for the fabrication of functional micro- and nano-structures

Gopakumar Gopalakrishnan

EPFL2006

Perturbations of the Straight Transmembrane alpha-Helical Structure of the Amyloid Precursor Protein Affect Its Processing by gamma-Secretase

Matteo Dal Peraro, Mitko Dimitrov, Patrick Fraering, Thomas Lemmin

American Society for Biochemistry and Molecular Biology2014

EPFL2011