Maité Paternostre - Academia.edu (original) (raw)
Papers by Maité Paternostre
Biochemistry, Apr 19, 1988
A method has been developed for identifying the step in a detergent-mediated reconstitution proce... more A method has been developed for identifying the step in a detergent-mediated reconstitution procedure at which an integral membrane protein can be associated with phospholipids to give functional proteoliposomes. Large liposomes prepared by reverse-phase evaporation were treated with various amounts of the detergents Triton X-100, octyl glucoside, or sodium cholate as described in the preceding paper [Paternostre, M.-T., Roux, M., & Rigaud, J. L. (1988) Biochemistry (preceding paper in this issue)]. At each step of the solubilization process, we added bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. The protein-phospholipid detergent mixtures were then subjected to SM2 Bio-Beads treatments to remove the detergent, and the resulting vesicles were analyzed with respect to protein insertion and orientation in the membrane by freeze-fracture electron microscopy, sucrose density gradients, and proton pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With sodium cholate, proteoliposomes were formed only from ternary phospholipid-protein-detergent micelles. With octyl glucoside, besides proteoliposome formation from ternary mixed micelles, direct incorporation of bacteriorhodopsin into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes with optimal proton pumping activity. With Triton X-100, protein insertion into destabilized liposomes was also observed but involved a transfer of the protein initially present in phospholipid-Triton X-1 00-protein micelles into Triton X-100 saturated liposomes. Our results further demonstrated that protein orientation in the resulting proteoliposomes was critically dependent upon the mechanism by which the protein was incorporated. 'This work was supported in part by grants from CNRS (ATP * Author to whom correspondence should be addressed. 901 445). starts and lipid-detergent mixed micelles begin to form. The end point of solubilization is reached when all the membrane material is converted into mixed micelles. It is generally assumed that this solubilization process represents the reverse of detergent-mediated reconstitution of pure liposomes or proteoliposomes (Helenius & Simons, 1975; Eytan, 1982). One possible sequence of events during detergent-mediated reconstitution is the following: Initially, lipid-detergent and lipid-protein-detergent micelles are formed and the detergent is then removed. At a certain point, the micelles are no longer soluble, and structures containing lipids and proteins are formed that, upon reorganization, are transformed into vesicles. The last phase of detergent removal corresponds to the removal of residual detergent from detergent-saturated proteoliposomes.
The miniaturization, integration, and analysis of chemical and biological processes on the nanoli... more The miniaturization, integration, and analysis of chemical and biological processes on the nanoliter-scale have currently led to remarkable progress in biotechnology, protein crystallization, and combinatorial chemistry. Aside from having advantages such as reduction of sample volumes and shorter reaction and analysis times, microfluidics is a powerful tool for investigations of soft condensed matter and biological systems. The marriage of microfluidics with spatially resolved microdiffraction provides new opportunities to study time-resolved reaction dynamics as well as non-equilibrium dynamics of biomaterials. The characterization of these complex materials, which are typically liquid-crystalline at ambient conditions, is significantly improved owing to a concurrent orientation during the self assembly process. Moreover, the influence of external stress on the biological materials can be analyzed. Here, we present experiments on the dynamics of the compaction of DNA by linker-histone proteins and artificial polyimine dendrimers.
Etude des protocoles de reconstitution utilisant les detergents et developpement d'une nouvel... more Etude des protocoles de reconstitution utilisant les detergents et developpement d'une nouvelle methode permettant de determiner les conditions optimales d'incorporation d'une proteine membranaire dans des liposomes
HAL (Le Centre pour la Communication Scientifique Directe), 2007
We investigated the spectroscopic properties of the aromatic residues in a set of octapeptides wi... more We investigated the spectroscopic properties of the aromatic residues in a set of octapeptides with various self-assembly properties. These octapeptides are based on lanreotide, a cyclic peptide analogue of somatostatin-14 that spontaneously self-assembles into very long and monodisperse hollow nanotubes. A previous study on these lanreotide-based derivatives has shown that the disulfide bridge, the peptide hairpin conformation and the aromatic residues are involved in the self-assembly process and that modification of these properties either decreases the self-assembly propensity or modifies the molecular packing resulting in different self-assembled architectures. In this study we probed the local environment of the aromatic residues, naphthyl-alanine, tryptophan and tyrosine, by Raman and fluorescence spectroscopy, comparing nonassembled peptides at low concentrations with the self-assembled ones at high concentrations. As expected, the spectroscopic characteristics of the aromatic residues were found to be sensitive to the peptide-peptide interactions. Among the most remarkable features we could record a very unusual Raman spectrum for the tyrosine of lanreotide in relation to its propensity to form H-bonds within the assemblies. In Lanreotide nanotubes, and also in the supramolecular architectures formed by its derivatives, the tryptophan side chain is water-exposed. Finally, the low fluorescence polarization of the peptide aggregates suggests that fluorescence energy transfer occurs within the nanotubes.
Liposome Methods and Protocols
... 5 Lipids in Viral Fusion Anu Puri, Maite Paternostre, and Robert Blumenthal 1. Introduction 1... more ... 5 Lipids in Viral Fusion Anu Puri, Maite Paternostre, and Robert Blumenthal 1. Introduction 1.1. ... 151173. 5. Blumenthal, R., Puri, A., Sarkar, DP, Chen, Y., Eidelman, O., and Morris, SJ (1989) Membrane fusion mediated by viral spike glycoproteins, in Cell Biology ...
Manual on Membrane Lipids, 1996
Liposomes are hollow microspheres, their membrane is composed of one or several lipid bilayers an... more Liposomes are hollow microspheres, their membrane is composed of one or several lipid bilayers and encapsulates a small volume of the medium in which they have been prepared. This morphology enables them to encapsulate water-soluble compounds in their internal volume and liposoluble ones in the bilayers.
Langmuir, 2013
Zeta-potential measurements. Different fits for the SAXS profiles of (A) Lanreotide-sulfate (1:2)... more Zeta-potential measurements. Different fits for the SAXS profiles of (A) Lanreotide-sulfate (1:2) and (B) Lanreotide-tartrate (1:2). Characterization of lanreotide-tartrate sample prepared with a 1:1 peptide-counterion ratio. SAXS profiles of competition experiments. Electron micrographs of an aged solution. Detailed calculations for the model of the multi-walled NTs free energy. *** Zeta-potential measurements. Sample Zeta potential (mV) Conductivity (mS/cm)
Journal of Thermal Analysis and Calorimetry, 2009
Journal of Peptide Science, 2008
We investigated the spectroscopic properties of the aromatic residues in a set of octapeptides wi... more We investigated the spectroscopic properties of the aromatic residues in a set of octapeptides with various self-assembly properties. These octapeptides are based on lanreotide, a cyclic peptide analogue of somatostatin-14 that spontaneously selfassembles into very long and monodisperse hollow nanotubes. A previous study on these lanreotide-based derivatives has shown that the disulfide bridge, the peptide hairpin conformation and the aromatic residues are involved in the self-assembly process and that modification of these properties either decreases the self-assembly propensity or modifies the molecular packing resulting in different self-assembled architectures. In this study we probed the local environment of the aromatic residues, naphthyl-alanine, tryptophan and tyrosine, by Raman and fluorescence spectroscopy, comparing nonassembled peptides at low concentrations with the self-assembled ones at high concentrations. As expected, the spectroscopic characteristics of the aromatic residues were found to be sensitive to the peptide-peptide interactions. Among the most remarkable features we could record a very unusual Raman spectrum for the tyrosine of lanreotide in relation to its propensity to form H-bonds within the assemblies. In Lanreotide nanotubes, and also in the supramolecular architectures formed by its derivatives, the tryptophan side chain is water-exposed. Finally, the low fluorescence polarization of the peptide aggregates suggests that fluorescence energy transfer occurs within the nanotubes.
European Biophysics Journal, 1998
To investigate whether lipid solubilization is of relevance in describing the interaction between... more To investigate whether lipid solubilization is of relevance in describing the interaction between melittin and biological membranes, we studied melittin-induced polymorphism using model membranes composed of the biological lipid sphingomyelin (bovine brain). The behavior of the system was monitored by solid state 31 P-NMR and turbidity measurements and compared to the peptides well-characterized action on the synthetic lipid dipalmitoylphosphatidylcholine. It was found that melittin-induced macroscopic changes of sphingomyelin membranes are qualitatively the same as in the case of dipalmitoylphosphatidylcholine bilayers. The sphingomyelin/melittin system is thus proposed to show a reversible vesicle-todisc transition (fluid-to-gel phase) through an intermediate fusion or aggregation event centered at the main transition temperature, T m , as reported in the case of saturated phosphatidylcholine. In the case of spontaneous disc formation at 37°C, the lipid-to-peptide molar ratio in the discoidal objects was determined to be approximately 20 for dipalmitoylphosphatidylcholine and about 12 in the case of natural sphingomyelin. Melittin partition coefficients between membranes and the aqueous medium at 37°C were found to be 6.1±0.8 mM-1 and 3.7±0.4 mM-1 for sphingomyelin and dipalmitoylphosphatidylcholine, respectively. For very high peptide quantities (lipid-to-peptide molar ratio, R i ≤5) mixed micelles are formed over the entire temperature range (20°to 60°C) for both kinds of lipids.
Biophysical Journal, 2004
Lanreotide is a synthetic octapeptide used in the therapy against acromegaly. When mixed with pur... more Lanreotide is a synthetic octapeptide used in the therapy against acromegaly. When mixed with pure water at 10% (w/w), Lanreotide (acetate salt) forms liquid crystalline and monodisperse nanotubes with a radius of 120 Å. The molecular and supramolecular organization of these structures has been determined in a previous work as relying on the lateral association of 26 b-sheet filaments made of peptide noncovalent dimers, the basic building blocks. The work presented here has been devoted to the corresponding self-association mechanisms, through the characterization of the Lanreotide structures formed in water, as a function of peptide (acetate salt) concentration (from 2% to 70% (w/w)) and temperature (from 158C to 708C). The corresponding states of water were also identified and quantified from the thermal behavior of water in the Lanreotide mixtures. At room temperature and below 3% (w/w) Lanreotide acetate in water, soluble aggregates were detected. From 3% to 20% (w/w) long individual and monodisperse nanotubes crystallized in a hexagonal lattice were evidenced. Their molecular and supramolecular organizations are identical to the ones characterized for the 10% (w/w) sample. Heating induces the dissolution of the nanotubes into soluble aggregates of the same structural characteristics as the room temperature ones. The solubilization temperature increases from 208C to 708C with the peptide concentration and reaches a plateau between 15% and 25% (w/w) in peptide. These aggregates are proposed to be the b-sheet filaments that self-associate to build the walls of the nanotubes. Above 20% (w/w) of Lanreotide acetate in water, polydisperse embedded nanotubes are formed and the hexagonal lattice is lost. These embedded nanotubes exhibit the same molecular and supramolecular organizations as the individual monodisperse nanotubes formed at lower peptide concentration. The embedded nanotubes do not melt in the range of temperature studied indicating a higher thermodynamic stability than individual nanotubes. In parallel, the thermal behaviors of water in mixtures containing 2-80% (w/w) in peptide have been studied by differential scanning calorimetry, and three different types of water were characterized: 1), bulk water melting at 08C, 2), nonfreezing water, and 3), interfacial water melting below 08C. The domains of existence and coexistence of these different water states are related to the different Lanreotide supramolecular structures. All these results were compiled into a binary Lanreotide-water phase diagram and allowed to propose a self-association mechanism of Lanreotide filaments into monodisperse individual nanotubes and embedded nanotubes.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2000
The process of formation of lipid vesicles using the technique of detergent removal from mixed-mi... more The process of formation of lipid vesicles using the technique of detergent removal from mixed-micelles is examined. Recent studies on the solubilization and reconstitution of liposomes participated to our knowledge of the structure and properties of mixed lipid^detergent systems. The mechanisms involved in both the lipid self assembly and the micelle^vesicle transition are first reviewed. The simplistic three step minimum scheme is described and criticized in relation with isothermal as well as a function of the [det]/[lip] ratio, phase diagram explorations. The techniques of detergent elimination are reviewed and criticized for advantages and disadvantages. New methods inducing micelle^vesicle transition using enzymatic reaction and T-jump are also described and compared to more classical ones. Future developments of these techniques and improvements resulting of their combinations are also considered. Proper reconstitution of membrane constituents such as proteins and drugs into liposomes are examined in the light of our actual understanding of the micelle^vesicle transition.
Journal of Peptide Science, 2014
In the absence of efficient crystallization methods, the molecular structures of fibrous assembli... more In the absence of efficient crystallization methods, the molecular structures of fibrous assemblies have so far remained rather elusive. In this paper, we present a rational method to crystallize the lanreotide octapeptide by modification of a residue involved in a close contact. Indeed, we show that it is possible to modify the curvature of the lanreotide nanotubes and hence their diameter. This fine tuning leads to crystallization because the radius of curvature of the initially bidimensional peptide wall can be increased up to a point where the wall is essentially flat and a crystal is allowed to grow along a third dimension. By comparing X-ray diffraction data and Fourier transform Raman spectra, we show that the nanotubes and the crystals share similar cell parameters and molecular conformations, proving that there is indeed a structural continuum between these two morphologies. These results illustrate a novel approach to crystallization and represent the first step towards the acquisition of an Å-resolution structure of the lanreotide nanotubes β-sheet assembly.
Pharmaceutical Research, 2004
Purpose. To get a continuous description of the insertion and partition processes of sodium tauro... more Purpose. To get a continuous description of the insertion and partition processes of sodium taurocholate (TC) into the lipid bilayers of vesicles that can serve as a model for understanding the mechanism of destabilization by the bile salts of liposomes used as drug carriers for oral administration. Methods. The progressive solubilization of egg phosphatidylcholine vesicles during TC addition at controlled rates was followed by continuous turbidity (OD) and resonance energy transfer (RET) between two fluorescent probes. The influence of the lipid and TC concentrations as well as the rate of TC addition on the processes were examined. Results. Continuous turbidity recordings allowed following of the size and composition evolutions of the mixed TC/lipid aggregates formed at different steps of the vesicle-micelle transition. The solubilization mechanism is governed by complex kinetics that depend on the surfactant concentration and its addition rate. A two-step process characterizes the evolution of the vesicular state: interaction of TC molecules with the external monolayer of the vesicles first occurs. The homogeneous distribution of TC within the lipid matrix after its insertion is a very slow process. A micellar structural reorganization is observed when TC is added rapidly. Conclusions. This work provides detailed information on the slow insertion and diffusion kinetics of TC in liposomal bilayers by using a dynamic study which mimics physiological phenomena of digestion.
Proceedings of the National Academy of Sciences
Functional and versatile nano- and microassemblies formed by biological molecules are found at al... more Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different f...
Journal of Thermal Analysis and Calorimetry, 1998
ABSTRACT The thermotropic transitions of 1,2-dipalmitoylphosphatidylcholine (DPPC) and the struct... more ABSTRACT The thermotropic transitions of 1,2-dipalmitoylphosphatidylcholine (DPPC) and the structural changes of its lamellar phases have been studied between 0 and 50°C by both DSC and synchrotron small angle X-ray diffraction/scattering as a function of temperature (XRDT) and sodium taurocholate concentration [TC] in the 0–40 mM range ([DPPC]=50 mM) at pH 7.4. The existence of multiple phase transitions (up to 5 peaks within a 5°C interval) in a narrow domain of temperature between 25 and 42°C depending on the [TC]/[lipid] ratio was observed in the DSC curves. XRDT showed that at low ratios they might correspond to transitions between lamellar phases, the structural characteristics of which are given. At higher ratios a lamellar to micellar transition was observed, and the temperature at which it was observed decreased as a function of the TC content. The relationships with DPPC vesicle bilayer permeabilization and solubilization are discussed.
European Journal of Pharmaceutics and Biopharmaceutics, 2009
In order to better understand the mechanism of destabilization of liposomes used as drug carriers... more In order to better understand the mechanism of destabilization of liposomes used as drug carriers for oral administration by bile salts, the insertion and partition of sodium taurocholate (TC) into small unilamellar vesicles (SUV) and multilayers (ML) of dipalmitoylphosphatidylcholine (DPPC) were examined by continuous turbidity analysis and DSC. Optical density was recorded during the progressive solubilisation of DPPC SUV and ML into DPPC/TC mixed micelles by varying the rate of TC addition and the temperature. The results show that the insertion and diffusion of TC in the DPPC membrane is a slow process influenced by the polymorphism of the lipid, independently of its organisation. This dynamic study mimics physiological phenomena of the digestion of liposomes. In the gastrointestinal tract, DPPC SUV would be more resistant to TC than egg phosphatidylcholine (EPC) SUV [K. Andrieux, L. Forte, S. Lesieur, M. Paternostre, M. Ollivon, C. Grabielle-Madelmont, Insertion and partition of sodium taurocholate into egg phosphatidylcholine vesicles, Pharm. Res. 21 (2004) 1505-1516] because of the lower insertion of TC into DPPC bilayer at 37°C at low TC concentration in the medium (fasted conditions). At high TC concentration (postprandially or after lipid absorption), the use of DPPC to prepare liposomes will delay or reduce the liberation of a drug encapsulated into liposomes in the gastrointestinal tract. As a conclusion, the addition of DPPC appears an attractive strategy to formulate orally administered liposomes.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1998
Biochemistry, Apr 19, 1988
A method has been developed for identifying the step in a detergent-mediated reconstitution proce... more A method has been developed for identifying the step in a detergent-mediated reconstitution procedure at which an integral membrane protein can be associated with phospholipids to give functional proteoliposomes. Large liposomes prepared by reverse-phase evaporation were treated with various amounts of the detergents Triton X-100, octyl glucoside, or sodium cholate as described in the preceding paper [Paternostre, M.-T., Roux, M., & Rigaud, J. L. (1988) Biochemistry (preceding paper in this issue)]. At each step of the solubilization process, we added bacteriorhodopsin, the light-driven proton pump from Halobacterium halobium. The protein-phospholipid detergent mixtures were then subjected to SM2 Bio-Beads treatments to remove the detergent, and the resulting vesicles were analyzed with respect to protein insertion and orientation in the membrane by freeze-fracture electron microscopy, sucrose density gradients, and proton pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With sodium cholate, proteoliposomes were formed only from ternary phospholipid-protein-detergent micelles. With octyl glucoside, besides proteoliposome formation from ternary mixed micelles, direct incorporation of bacteriorhodopsin into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes with optimal proton pumping activity. With Triton X-100, protein insertion into destabilized liposomes was also observed but involved a transfer of the protein initially present in phospholipid-Triton X-1 00-protein micelles into Triton X-100 saturated liposomes. Our results further demonstrated that protein orientation in the resulting proteoliposomes was critically dependent upon the mechanism by which the protein was incorporated. 'This work was supported in part by grants from CNRS (ATP * Author to whom correspondence should be addressed. 901 445). starts and lipid-detergent mixed micelles begin to form. The end point of solubilization is reached when all the membrane material is converted into mixed micelles. It is generally assumed that this solubilization process represents the reverse of detergent-mediated reconstitution of pure liposomes or proteoliposomes (Helenius & Simons, 1975; Eytan, 1982). One possible sequence of events during detergent-mediated reconstitution is the following: Initially, lipid-detergent and lipid-protein-detergent micelles are formed and the detergent is then removed. At a certain point, the micelles are no longer soluble, and structures containing lipids and proteins are formed that, upon reorganization, are transformed into vesicles. The last phase of detergent removal corresponds to the removal of residual detergent from detergent-saturated proteoliposomes.
The miniaturization, integration, and analysis of chemical and biological processes on the nanoli... more The miniaturization, integration, and analysis of chemical and biological processes on the nanoliter-scale have currently led to remarkable progress in biotechnology, protein crystallization, and combinatorial chemistry. Aside from having advantages such as reduction of sample volumes and shorter reaction and analysis times, microfluidics is a powerful tool for investigations of soft condensed matter and biological systems. The marriage of microfluidics with spatially resolved microdiffraction provides new opportunities to study time-resolved reaction dynamics as well as non-equilibrium dynamics of biomaterials. The characterization of these complex materials, which are typically liquid-crystalline at ambient conditions, is significantly improved owing to a concurrent orientation during the self assembly process. Moreover, the influence of external stress on the biological materials can be analyzed. Here, we present experiments on the dynamics of the compaction of DNA by linker-histone proteins and artificial polyimine dendrimers.
Etude des protocoles de reconstitution utilisant les detergents et developpement d'une nouvel... more Etude des protocoles de reconstitution utilisant les detergents et developpement d'une nouvelle methode permettant de determiner les conditions optimales d'incorporation d'une proteine membranaire dans des liposomes
HAL (Le Centre pour la Communication Scientifique Directe), 2007
We investigated the spectroscopic properties of the aromatic residues in a set of octapeptides wi... more We investigated the spectroscopic properties of the aromatic residues in a set of octapeptides with various self-assembly properties. These octapeptides are based on lanreotide, a cyclic peptide analogue of somatostatin-14 that spontaneously self-assembles into very long and monodisperse hollow nanotubes. A previous study on these lanreotide-based derivatives has shown that the disulfide bridge, the peptide hairpin conformation and the aromatic residues are involved in the self-assembly process and that modification of these properties either decreases the self-assembly propensity or modifies the molecular packing resulting in different self-assembled architectures. In this study we probed the local environment of the aromatic residues, naphthyl-alanine, tryptophan and tyrosine, by Raman and fluorescence spectroscopy, comparing nonassembled peptides at low concentrations with the self-assembled ones at high concentrations. As expected, the spectroscopic characteristics of the aromatic residues were found to be sensitive to the peptide-peptide interactions. Among the most remarkable features we could record a very unusual Raman spectrum for the tyrosine of lanreotide in relation to its propensity to form H-bonds within the assemblies. In Lanreotide nanotubes, and also in the supramolecular architectures formed by its derivatives, the tryptophan side chain is water-exposed. Finally, the low fluorescence polarization of the peptide aggregates suggests that fluorescence energy transfer occurs within the nanotubes.
Liposome Methods and Protocols
... 5 Lipids in Viral Fusion Anu Puri, Maite Paternostre, and Robert Blumenthal 1. Introduction 1... more ... 5 Lipids in Viral Fusion Anu Puri, Maite Paternostre, and Robert Blumenthal 1. Introduction 1.1. ... 151173. 5. Blumenthal, R., Puri, A., Sarkar, DP, Chen, Y., Eidelman, O., and Morris, SJ (1989) Membrane fusion mediated by viral spike glycoproteins, in Cell Biology ...
Manual on Membrane Lipids, 1996
Liposomes are hollow microspheres, their membrane is composed of one or several lipid bilayers an... more Liposomes are hollow microspheres, their membrane is composed of one or several lipid bilayers and encapsulates a small volume of the medium in which they have been prepared. This morphology enables them to encapsulate water-soluble compounds in their internal volume and liposoluble ones in the bilayers.
Langmuir, 2013
Zeta-potential measurements. Different fits for the SAXS profiles of (A) Lanreotide-sulfate (1:2)... more Zeta-potential measurements. Different fits for the SAXS profiles of (A) Lanreotide-sulfate (1:2) and (B) Lanreotide-tartrate (1:2). Characterization of lanreotide-tartrate sample prepared with a 1:1 peptide-counterion ratio. SAXS profiles of competition experiments. Electron micrographs of an aged solution. Detailed calculations for the model of the multi-walled NTs free energy. *** Zeta-potential measurements. Sample Zeta potential (mV) Conductivity (mS/cm)
Journal of Thermal Analysis and Calorimetry, 2009
Journal of Peptide Science, 2008
We investigated the spectroscopic properties of the aromatic residues in a set of octapeptides wi... more We investigated the spectroscopic properties of the aromatic residues in a set of octapeptides with various self-assembly properties. These octapeptides are based on lanreotide, a cyclic peptide analogue of somatostatin-14 that spontaneously selfassembles into very long and monodisperse hollow nanotubes. A previous study on these lanreotide-based derivatives has shown that the disulfide bridge, the peptide hairpin conformation and the aromatic residues are involved in the self-assembly process and that modification of these properties either decreases the self-assembly propensity or modifies the molecular packing resulting in different self-assembled architectures. In this study we probed the local environment of the aromatic residues, naphthyl-alanine, tryptophan and tyrosine, by Raman and fluorescence spectroscopy, comparing nonassembled peptides at low concentrations with the self-assembled ones at high concentrations. As expected, the spectroscopic characteristics of the aromatic residues were found to be sensitive to the peptide-peptide interactions. Among the most remarkable features we could record a very unusual Raman spectrum for the tyrosine of lanreotide in relation to its propensity to form H-bonds within the assemblies. In Lanreotide nanotubes, and also in the supramolecular architectures formed by its derivatives, the tryptophan side chain is water-exposed. Finally, the low fluorescence polarization of the peptide aggregates suggests that fluorescence energy transfer occurs within the nanotubes.
European Biophysics Journal, 1998
To investigate whether lipid solubilization is of relevance in describing the interaction between... more To investigate whether lipid solubilization is of relevance in describing the interaction between melittin and biological membranes, we studied melittin-induced polymorphism using model membranes composed of the biological lipid sphingomyelin (bovine brain). The behavior of the system was monitored by solid state 31 P-NMR and turbidity measurements and compared to the peptides well-characterized action on the synthetic lipid dipalmitoylphosphatidylcholine. It was found that melittin-induced macroscopic changes of sphingomyelin membranes are qualitatively the same as in the case of dipalmitoylphosphatidylcholine bilayers. The sphingomyelin/melittin system is thus proposed to show a reversible vesicle-todisc transition (fluid-to-gel phase) through an intermediate fusion or aggregation event centered at the main transition temperature, T m , as reported in the case of saturated phosphatidylcholine. In the case of spontaneous disc formation at 37°C, the lipid-to-peptide molar ratio in the discoidal objects was determined to be approximately 20 for dipalmitoylphosphatidylcholine and about 12 in the case of natural sphingomyelin. Melittin partition coefficients between membranes and the aqueous medium at 37°C were found to be 6.1±0.8 mM-1 and 3.7±0.4 mM-1 for sphingomyelin and dipalmitoylphosphatidylcholine, respectively. For very high peptide quantities (lipid-to-peptide molar ratio, R i ≤5) mixed micelles are formed over the entire temperature range (20°to 60°C) for both kinds of lipids.
Biophysical Journal, 2004
Lanreotide is a synthetic octapeptide used in the therapy against acromegaly. When mixed with pur... more Lanreotide is a synthetic octapeptide used in the therapy against acromegaly. When mixed with pure water at 10% (w/w), Lanreotide (acetate salt) forms liquid crystalline and monodisperse nanotubes with a radius of 120 Å. The molecular and supramolecular organization of these structures has been determined in a previous work as relying on the lateral association of 26 b-sheet filaments made of peptide noncovalent dimers, the basic building blocks. The work presented here has been devoted to the corresponding self-association mechanisms, through the characterization of the Lanreotide structures formed in water, as a function of peptide (acetate salt) concentration (from 2% to 70% (w/w)) and temperature (from 158C to 708C). The corresponding states of water were also identified and quantified from the thermal behavior of water in the Lanreotide mixtures. At room temperature and below 3% (w/w) Lanreotide acetate in water, soluble aggregates were detected. From 3% to 20% (w/w) long individual and monodisperse nanotubes crystallized in a hexagonal lattice were evidenced. Their molecular and supramolecular organizations are identical to the ones characterized for the 10% (w/w) sample. Heating induces the dissolution of the nanotubes into soluble aggregates of the same structural characteristics as the room temperature ones. The solubilization temperature increases from 208C to 708C with the peptide concentration and reaches a plateau between 15% and 25% (w/w) in peptide. These aggregates are proposed to be the b-sheet filaments that self-associate to build the walls of the nanotubes. Above 20% (w/w) of Lanreotide acetate in water, polydisperse embedded nanotubes are formed and the hexagonal lattice is lost. These embedded nanotubes exhibit the same molecular and supramolecular organizations as the individual monodisperse nanotubes formed at lower peptide concentration. The embedded nanotubes do not melt in the range of temperature studied indicating a higher thermodynamic stability than individual nanotubes. In parallel, the thermal behaviors of water in mixtures containing 2-80% (w/w) in peptide have been studied by differential scanning calorimetry, and three different types of water were characterized: 1), bulk water melting at 08C, 2), nonfreezing water, and 3), interfacial water melting below 08C. The domains of existence and coexistence of these different water states are related to the different Lanreotide supramolecular structures. All these results were compiled into a binary Lanreotide-water phase diagram and allowed to propose a self-association mechanism of Lanreotide filaments into monodisperse individual nanotubes and embedded nanotubes.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2000
The process of formation of lipid vesicles using the technique of detergent removal from mixed-mi... more The process of formation of lipid vesicles using the technique of detergent removal from mixed-micelles is examined. Recent studies on the solubilization and reconstitution of liposomes participated to our knowledge of the structure and properties of mixed lipid^detergent systems. The mechanisms involved in both the lipid self assembly and the micelle^vesicle transition are first reviewed. The simplistic three step minimum scheme is described and criticized in relation with isothermal as well as a function of the [det]/[lip] ratio, phase diagram explorations. The techniques of detergent elimination are reviewed and criticized for advantages and disadvantages. New methods inducing micelle^vesicle transition using enzymatic reaction and T-jump are also described and compared to more classical ones. Future developments of these techniques and improvements resulting of their combinations are also considered. Proper reconstitution of membrane constituents such as proteins and drugs into liposomes are examined in the light of our actual understanding of the micelle^vesicle transition.
Journal of Peptide Science, 2014
In the absence of efficient crystallization methods, the molecular structures of fibrous assembli... more In the absence of efficient crystallization methods, the molecular structures of fibrous assemblies have so far remained rather elusive. In this paper, we present a rational method to crystallize the lanreotide octapeptide by modification of a residue involved in a close contact. Indeed, we show that it is possible to modify the curvature of the lanreotide nanotubes and hence their diameter. This fine tuning leads to crystallization because the radius of curvature of the initially bidimensional peptide wall can be increased up to a point where the wall is essentially flat and a crystal is allowed to grow along a third dimension. By comparing X-ray diffraction data and Fourier transform Raman spectra, we show that the nanotubes and the crystals share similar cell parameters and molecular conformations, proving that there is indeed a structural continuum between these two morphologies. These results illustrate a novel approach to crystallization and represent the first step towards the acquisition of an Å-resolution structure of the lanreotide nanotubes β-sheet assembly.
Pharmaceutical Research, 2004
Purpose. To get a continuous description of the insertion and partition processes of sodium tauro... more Purpose. To get a continuous description of the insertion and partition processes of sodium taurocholate (TC) into the lipid bilayers of vesicles that can serve as a model for understanding the mechanism of destabilization by the bile salts of liposomes used as drug carriers for oral administration. Methods. The progressive solubilization of egg phosphatidylcholine vesicles during TC addition at controlled rates was followed by continuous turbidity (OD) and resonance energy transfer (RET) between two fluorescent probes. The influence of the lipid and TC concentrations as well as the rate of TC addition on the processes were examined. Results. Continuous turbidity recordings allowed following of the size and composition evolutions of the mixed TC/lipid aggregates formed at different steps of the vesicle-micelle transition. The solubilization mechanism is governed by complex kinetics that depend on the surfactant concentration and its addition rate. A two-step process characterizes the evolution of the vesicular state: interaction of TC molecules with the external monolayer of the vesicles first occurs. The homogeneous distribution of TC within the lipid matrix after its insertion is a very slow process. A micellar structural reorganization is observed when TC is added rapidly. Conclusions. This work provides detailed information on the slow insertion and diffusion kinetics of TC in liposomal bilayers by using a dynamic study which mimics physiological phenomena of digestion.
Proceedings of the National Academy of Sciences
Functional and versatile nano- and microassemblies formed by biological molecules are found at al... more Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different f...
Journal of Thermal Analysis and Calorimetry, 1998
ABSTRACT The thermotropic transitions of 1,2-dipalmitoylphosphatidylcholine (DPPC) and the struct... more ABSTRACT The thermotropic transitions of 1,2-dipalmitoylphosphatidylcholine (DPPC) and the structural changes of its lamellar phases have been studied between 0 and 50°C by both DSC and synchrotron small angle X-ray diffraction/scattering as a function of temperature (XRDT) and sodium taurocholate concentration [TC] in the 0–40 mM range ([DPPC]=50 mM) at pH 7.4. The existence of multiple phase transitions (up to 5 peaks within a 5°C interval) in a narrow domain of temperature between 25 and 42°C depending on the [TC]/[lipid] ratio was observed in the DSC curves. XRDT showed that at low ratios they might correspond to transitions between lamellar phases, the structural characteristics of which are given. At higher ratios a lamellar to micellar transition was observed, and the temperature at which it was observed decreased as a function of the TC content. The relationships with DPPC vesicle bilayer permeabilization and solubilization are discussed.
European Journal of Pharmaceutics and Biopharmaceutics, 2009
In order to better understand the mechanism of destabilization of liposomes used as drug carriers... more In order to better understand the mechanism of destabilization of liposomes used as drug carriers for oral administration by bile salts, the insertion and partition of sodium taurocholate (TC) into small unilamellar vesicles (SUV) and multilayers (ML) of dipalmitoylphosphatidylcholine (DPPC) were examined by continuous turbidity analysis and DSC. Optical density was recorded during the progressive solubilisation of DPPC SUV and ML into DPPC/TC mixed micelles by varying the rate of TC addition and the temperature. The results show that the insertion and diffusion of TC in the DPPC membrane is a slow process influenced by the polymorphism of the lipid, independently of its organisation. This dynamic study mimics physiological phenomena of the digestion of liposomes. In the gastrointestinal tract, DPPC SUV would be more resistant to TC than egg phosphatidylcholine (EPC) SUV [K. Andrieux, L. Forte, S. Lesieur, M. Paternostre, M. Ollivon, C. Grabielle-Madelmont, Insertion and partition of sodium taurocholate into egg phosphatidylcholine vesicles, Pharm. Res. 21 (2004) 1505-1516] because of the lower insertion of TC into DPPC bilayer at 37°C at low TC concentration in the medium (fasted conditions). At high TC concentration (postprandially or after lipid absorption), the use of DPPC to prepare liposomes will delay or reduce the liberation of a drug encapsulated into liposomes in the gastrointestinal tract. As a conclusion, the addition of DPPC appears an attractive strategy to formulate orally administered liposomes.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1998