Directing peptide crystallization through curvature control of nanotubes ‡

International audienceIn 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

Liquid crystalline properties of type I collagen: Perspectives in tissue morphogenesis

Collagen molecules form the major part of tissues like bone, cornea or tendon where they organize into ordered fibrillar networks. The acid-soluble protein spontaneously assembles in liquid crystalline phases, characterized in polarized light microscopy and X-ray diffraction. Collagen fibrillogenesis obtained in condensed media establishes a link between the fibrillar networks described in vivo and the mesomorphic states obtained in vitro. Cellematrix interactions on these biomimetic materials are currently analysed with perspectives in tissue engineering. In a morphogenetic context, we propose the hypothesis of a liquid crystalline order, between soluble precursor molecules, preceding fibrillogenesis

PHASES DENSES DE COLLAGÈNE DE TYPE I :<br>TRANSITION ISOTROPE/CHOLESTÉRIQUE, FIBRILLOGENÈSE ET MINÉRALISATION

The aim of this work is the in vitro study the self-assembly properties of collagen molecules in dense phases. First, we focused on acidic colloidal suspensions of collagen. We studied the isotropic/cholesteric (I/N*) transition as well as the structure of the liquid-crystalline phase thus obtained. For this we used polarized light optical microscopy and small angle X-ray scattering. Moreover, we described the rheological properties of these solutions through oscillating experiments with a cone-plane rheometer. Then we studied the influence of simple physico-chemical parameters on the formation of fibrillar gels resulting from the neutralization of acidic solution (« fibrillogenesis »). These gels were characterized by X-ray scattering and Transmission Electron Microscopy on ultrathin sections. Finally, we present some results concerning the mineralization of such ordered fibrillar matrices ; in particular, we highlight the coalignment of a hydroxyapatite phase along with the organic one. This work takes place in a wider context of tissue morphogenesis comprehension and of ordered biomaterials synthesis.L'objectif de ce travail consiste en l'étude in vitro des phénomènes d'autoassemblage des molécules de collagène de type I en phases denses. Dans un premier temps nous nous sommes consacrés à l'étude de solutions colloïdales de collagène solubilisé en milieu acide. Nous avons étudié la transition isotrope/cholestérique ainsi que la structure de la phase cristal-liquide obtenue à l'aide de la microscopie optique à lumière polarisée et de la diffusion des rayons X aux petits angles. De plus, nous avons décrit les propriétés rhéologiques de ces solutions à l'aide d'expériences en régime oscillant en géométrie cône-plan. Ensuite, nous avons cherché à déterminer l'influence de quelques paramètres physicochimiques simples sur la formation de gels fibrillaires à partir des solutions acides (la « fibrillogenèse »). Ces gels ont été caractérisés par diffusion des rayons X et microscopie électronique à transmission sur coupes ultrafines. Enfin, nous présentons quelques expériences de minéralisation de ces matrices fibrillaires ordonnées ; nous y montrons comment nous avons réussi à obtenir une phase minérale coalignée avec la phase organique. Ce travail s'inscrit dans un contexte plus large de compréhension de la morphogenèse tissulaire et de la synthèse de biomatériaux ordonnés

PHASES DENSES DE COLLAGÈNE DE TYPE I :<br>TRANSITION ISOTROPE/CHOLESTÉRIQUE, FIBRILLOGENÈSE ET MINÉRALISATION

The aim of this work is the in vitro study the self-assembly properties of collagen molecules in dense phases. First, we focused on acidic colloidal suspensions of collagen. We studied the isotropic/cholesteric (I/N*) transition as well as the structure of the liquid-crystalline phase thus obtained. For this we used polarized light optical microscopy and small angle X-ray scattering. Moreover, we described the rheological properties of these solutions through oscillating experiments with a cone-plane rheometer. Then we studied the influence of simple physico-chemical parameters on the formation of fibrillar gels resulting from the neutralization of acidic solution (« fibrillogenesis »). These gels were characterized by X-ray scattering and Transmission Electron Microscopy on ultrathin sections. Finally, we present some results concerning the mineralization of such ordered fibrillar matrices ; in particular, we highlight the coalignment of a hydroxyapatite phase along with the organic one. This work takes place in a wider context of tissue morphogenesis comprehension and of ordered biomaterials synthesis.L'objectif de ce travail consiste en l'étude in vitro des phénomènes d'autoassemblage des molécules de collagène de type I en phases denses. Dans un premier temps nous nous sommes consacrés à l'étude de solutions colloïdales de collagène solubilisé en milieu acide. Nous avons étudié la transition isotrope/cholestérique ainsi que la structure de la phase cristal-liquide obtenue à l'aide de la microscopie optique à lumière polarisée et de la diffusion des rayons X aux petits angles. De plus, nous avons décrit les propriétés rhéologiques de ces solutions à l'aide d'expériences en régime oscillant en géométrie cône-plan. Ensuite, nous avons cherché à déterminer l'influence de quelques paramètres physicochimiques simples sur la formation de gels fibrillaires à partir des solutions acides (la « fibrillogenèse »). Ces gels ont été caractérisés par diffusion des rayons X et microscopie électronique à transmission sur coupes ultrafines. Enfin, nous présentons quelques expériences de minéralisation de ces matrices fibrillaires ordonnées ; nous y montrons comment nous avons réussi à obtenir une phase minérale coalignée avec la phase organique. Ce travail s'inscrit dans un contexte plus large de compréhension de la morphogenèse tissulaire et de la synthèse de biomatériaux ordonnés

Phases denses de collagène de type I (transition isotrope/cholestérique, fibrollogenèse et minéralisation)

L objectif de ce travail consiste en l étude in vitro des phénomènes d auto-assemblage des molécules de collagène de type I en phases denses. Dans un premier temps nous nous sommes consacrés à l étude de solutions colloïdales de collagène solubilisé en milieu acide. Nous avons considéré la formation d une phase cristal-liquide nématique chirale (cholestérique) sous l effet de la concentration et nous nous sommes donc proposés de caractériser la transition isotrope/cholestérique (I/N*) et la structure de la phase cristal-liquide obtenue à l aide de la microscopie optique à lumière polarisée et de la diffusion des rayons X aux petits angles. Les concentrations critiques à la transition et le paramètre d ordre de la phase nématique sont cohérents avec les prédictions théoriques pour des macromolécules semi-flexibles. De plus, nous avons décrit les propriétés rhéologiques de ces solutions à l aide d expériences en régime oscillant en géométrie cône-plan.Ensuite, nous avons cherché à déterminer l influence de quelques paramètres physico-chimiques simples sur la formation de gels fibrillaires (la fibrillogenèse ). Ces gels ont été caractérisés par diffusion des rayons X et microscopie électronique à transmission sur coupes ultrafines. Enfin, nous présentons quelques tentatives de minéralisation de ces matrices fibrillaires ordonnées ; nous y montrons comment nous avons réussi à obtenir une phase minérale (hydroxyapatite) coalignée avec la phase organique. Ce travail s inscrit dans un contexte plus large de compréhension de la morphogenèse tissulaire et de la synthèse de biomatériaux ordonnés.The aim of this work is the in vitro study the self-assembly properties of collagen molecules in dense phases. First, we focused on acidic colloidal suspensions of collagen. We studied the isotropic/cholesteric (I/N*) transition as well as the structure of the liquid-crystalline phase thus obtained. For this we used polarized light optical microscopy and small angle X-ray scattering. Moreover, we described the rheological properties of these solutions through oscillating experiments with a cone-plane rheometer. Then we studied the influence of simple physico-chemical parameters on the formation of fibrillar gels resulting from the neutralization of acidic solution ( fibrillogenesis ). These gels were characterized by X-ray scattering and Transmission Electron Microscopy on ultrathin sections. Finally, we present some results concerning the mineralization of such ordered fibrillar matrices ; in particular, we highlight the coalignment of a hydroxyapatite phase along with the organic one. This work takes place in a wider context of tissue morphogenesis comprehension and of ordered biomaterials synthesis.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Direct monomer-to-fibril assembly of a Beta-hairpin peptide: A dynamic circular dichroism study

International audienceIt is now weIl established that under certain conditions, pro teins and peptides can form amyloid fibrils outside pathological context to fulfill a beneficial role 1 ,2; in particular, peptide hormones form reversible amyloid aggregates for storage purpose. 3 Although the interactions driving the assembly of reversible functional amyloids are similar to those of pathological amyloids (i.e. a balance between hydrogen bonds, hydrophobic and electrostatic interactions), the mechanisms and dynamics of assembly of functional amyloids have been less thoroughly studied and it is not clear in which respect they may differ. Indeed, while the formation of pathological amyloids is kinetically controlled, the formation of reversible functional amyloids is clearly resulting from thermodynamic equilibriums. In the present work, we study the self-assembly properties of Atosiban, a nonapeptide drug whose sequence is very close to those of the oxytocin and vasopressin hormones. We show that this very soluble peptide consistently self-assembles into 7-nm wide amyloid fibrils above a critica1 aggregation concentration (2-10%w/w depending on the buffer conditions). The peptide system is fully characterized from the monomer to the assembled form with osmotic concentration measurements, transmission electron microscopy, small angle x-ray scattering, infrared and fluorescence spectroscopy, and synchrotron radiation circu1ar dichroism. We have monitored the fibrils assembly in situ with fluorescence spectroscopy and synchrotron radiation circular dichroism and noticed that the peptide undergoes conformational changes during the process. However, aIl evidences point towards a one-step, direct association of monomers into fibrils without passing through oligomeric intermediate species contrary to what is usually reported for pathogenic amyloids. The native ~-hairpin conformation of the mono mer could explain the straightforward assembly. The tyrosine stacking is also shown to play an important role. We propose a packing) model that accounts for the experimental observations

Reversible assembly of a drug peptide into amyloid fibrils: a dynamic circular dichroism study

International audienceThe common view on the amyloid fibril formation is that it is a multistep process that involves many oligomeric intermediate species, which leads to a high degree of polymorphism. This view derives from numerous kinetic studies whose vast majority was carried out with amyloid β fragments or other pathological amyloidogenic sequences. Yet, it is not clear whether the mechanisms inferred from these studies are universal and also apply to functional amyloids, in particular to peptide hormones which form reversible amyloid structures. In the present work, we study the self-assembly properties of Atosiban, a nonapeptide drug whose sequence is very close to those of the oxytocin and vasopressin hormones. We show that this very soluble peptide consistently self-assembles into 7-nm wide amyloid fibrils above a critical aggregation concentration (2-10%w/w depending on the buffer conditions). The peptide system is characterized in details, from the monomeric to the assembled form, with osmotic concentration measurements, transmission electron microscopy, small angle x-ray scattering, infrared and fluorescence spectroscopy, and circular dichroism. We have followed in-situ the fibrils assembly with fluorescence and synchrotron radiation circular dichroism and noticed that the peptide undergoes conformational changes during the process. However, all evidences point towards the association of monomers and dimers into fibrils without passing through oligomeric intermediate species contrary to what is usually reported for pathogenic amyloids. The native β-hairpin conformation of the monomer could explain the straightforward assembly. The tyrosine stacking is also shown to play an important role. We propose a preliminary packing model that accounts for the experimental observations

Reversible Morphological Control of Cholecystokinin Tetrapeptide Amyloid Assemblies as a Function of pH

International audienceMost amyloid assemblies are seen as irreversible and exhibit polymorphism because their assembly is kinetically controlled and different structures are trapped during the aggregation process. However, in the specific case of peptide hormones, formation of amyloid assemblies for storage purposes has been reported. This suggests a strict control of assembly and the ability to disassemble upon hormone secretion. In the present work, we have sought to test these assertions with a short peptide, the cholecys-tokinin (or gastrin) tetrapeptide (CCK-4), that has been found in both gastrointestinal tract and central nervous system, and whose sequence is shared by a large number of hormones. We have thus studied in vitro this peptide's self-assembling properties in dense phases at different pH levels, thus mimicking in vivo storage conditions. The solubility and morphology of the supramolecular assemblies have been shown to vary with the pH. At low pH, the tetrapeptide exhibits a low solubility and forms microcrystals. At higher pH levels, peptide solubility increases and above a high enough concentration, peptide monomers self-assemble into typical amyloid fibrils of 10-20 nm diameter. The physical network formed by these fibrils results in a birefringent hydrogel phase. Despite the different morphological features exhibited at different pH, structural analysis shows strong similarities. Both supramolecular assemblies – microcrystals and fibrils – are structured by β-sheets. We also show that all these morphologies are reversible and can be either dissolved or changed into one another by switching the pH. In addition, we demonstrate that a modification in the charge sequence of the peptide by amino acid mutation modifies its self-assembly properties. In conclusion, just as the CCK-4 sequence is the minimal sequence required for a complete biological activity at CCK B receptors in the brain, it is also sufficient to form amyloid fibers whose properties can be related to hormone storage and release purposes in vivo

Dual internal functionalization of imogolite nanotubes as evidenced by optical properties of Nile red

International audienceHybrid imogolites have been prepared by combining methyltrimethoxysilane with various organosilanes (Si-R) to obtain nanotubes with a bi-functional internal cavity. With an initial doping ratio Si-R/Si of 5%, nanotubes remain the major product in the samples and their diameter is not modified compared to pristine methyl imogolite (Imo–CH3). The nanotube wall’s internal polarity and reactivity is significantly changed as evidenced by using a solvatochromic dye (Nile red). This research opens many possibilities to bring selectivity to the internal cavity or to manipulate the properties of trapped organic molecules

SAXS-WAXS monitoring of the amyloid fibril assembly of a hormone peptide analogue upon pH change.

Amyloid structures represent one of the most widespread and stable protein folding in nature. This particular folding can be either beneficial or detrimental to its host organism depending on the conditions and places of aggregation. Consequently, the space and time control of their formation mechanisms of amyloid fibril formation is crucial. In the present work, we have investigated the assembly properties of the terlipressin (TLP), a peptide analogue of the vasopressin hormone that is known – like many other peptide hormones – to be stored under the form of reversible amyloid fibrils, which suggests a finely tuned aggregation process. In particular, the assembly kinetics has been monitored by small and wide-angle x-ray scattering (SAXS-WAXS). In combination with circular dichroism, ATR-FTIR and cryoTEM characterization, this analysis reveals that the TLP assembly goes through the formation of on-pathway transitory amorphous aggregates in which the amyloid fibrils nucleate. At the end of the process, the “amyloid phase” (i.e., the fibrils) is in dynamic equilibrium with the liquid phase (peptide monomers). The proportion between the two phases depends on the pH and peptide concentration. Finally, the reversibility of the assembly is assessed