Photo-renaturation de protéines par des macromolécules chaperonnes

We wanted to design macromolecules that behave like artificial chaperones with different proteins and form which the motor will be light-stimulation. Hydrophobic interactions are proved to be a key point of the chaperone effect both in biological and artificial chaperones. We synthesized polymers with azobenzene moities that have a light-triggered amphiphily. These polymers were shown to be able to photo-associate and photo-dissociate with colloidal partners like micelles or proteins. The parameters that can modulate these associations are ionic strength, hydrophobic modification rate, nature of azobenzene moities... These polymers have exhibited some features of artificial chaperones: they destabilize a model protein like cythochrome~C, they prevent from aggregation and improve the efficiency of the renaturation process of carbonic anhydrase and of an antibody fragment overexpressed in bacteria. The refolding was monitored by following the evolution of secondary structures by circular dichroism and the evolution of the compacity of the protein by fluorescence. The polymer/protein association was studied by capillary electrophoresis and light scattering.Nous avons cherché à concevoir des macromolécules ayant un comportement de chaperonne vis à vis de diverses protéines et notamment en fondant leur activité sur des propriétés stimulables par la lumière. Les interactions hydrophobes constituent un paramètre clé de l'effet chaperonne qui a été mis en évidence dans les chaperonnes biologiques aussi bien que dans les chaperonnes artificielles. Nous avons synthétisé des polymères à amphiphilie photo-stimulable portant des chaînes pendantes azobenzènes. Ces polymères sont capables d'association/dissociation photo-stimulables avec des particules colloïdales à coeur hydrophobe. Différents paramètres peuvent moduler ces associations comme la force ionique, le taux de modification hydrophobe, la nature du greffon... Ces polymères ont montré plusieurs propriétés caractéristiques de chaperonnes artificielles : ils déstabilisent une protéine modèle, le cytochrome C, protègent de l'agrégation et augmentent l'efficacité des procédés de renaturation de l'anhydrase carbonique et d'un fragment d'anticorps surexprimé en bactérie. L'évolution du repliement a été caractérisée par suivi des structures secondaires en dichroïsme circulaire et par suivi de la compacité des protéines en fluorescence. L'association polymère/protéine a été étudiée par électrophorèse capillaire et par diffusion de la lumière

Photo-renaturation de protéines par des macromolécules chaperonnes

Nous avons cherché à concevoir des macromolécules ayant un comportement de chaperonne vis à vis de diverses protéines et notamment en fondant leur activité sur des propriétés stimulables par la lumière. Les interactions hydrophobes constituent un paramètre clé de l'effet chaperonne qui a été mis en évidence dans les chaperonnes biologiques aussi bien que dans les chaperonnes artificielles. Nous avons synthétisé des polymères à amphiphilie photo-stimulable portant des chaînes pendantes azobenzènes. Ces polymères sont capables d'association/dissociation photo-stimulables avec des particules colloïdales à coeur hydrophobe. Différents paramètres peuvent moduler ces associations comme la force ionique, le taux de modification hydrophobe, la nature du greffon... Ces polymères ont montré plusieurs propriétés caractéristiques de chaperonnes artificielles : ils déstabilisent une protéine modèle, le cytochrome C, protègent de l'agrégation et augmentent l'efficacité des procédés de renaturation de l'anhydrase carbonique et d'un fragment d'anticorps surexprimé en bactérie. L'évolution du repliement a été caractérisée par suivi des structures secondaires en dichroïsme circulaire et par suivi de la compacité des protéines en fluorescence. L'association polymère/protéine a été étudiée par électrophorèse capillaire et par diffusion de la lumièrePARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Prevention of Aggregation and Renaturation of Carbonic Anhydrase via Weak Association with Octadecyl- or Azobenzene-Modified Poly(acrylate) Derivatives

The prevention of aggregation during renaturation of urea-denatured carbonic anhydrase B (CAB) via hydrophobic and Coulomb association with anionic polymers was studied in mixed solutions of CAB and amphiphilic poly­(acrylate) copolymers. The polymers were derivatives of a parent poly­(acrylic acid) randomly grafted with hydrophobic side groups (either 3 mol % octadecyl group, or 1–5 mol % alkylamidoazobenzene photoresponsive groups). CAB:polymer complexes were characterized by light scattering and fluorescence correlation spectroscopy in aqueous buffers (pH 7.75 or 5.9). Circular dichroism and enzyme activity assays enabled us to study the kinetics of renaturation. All copolymers, including the hydrophilic PAA parent chain, provided a remarkable protective effect against CAB aggregation during renaturation, and most of them (but not the octadecyl-modified one) markedly enhanced the regain of activity as compared to CAB alone. The significant role of Coulomb binding in renaturation and comparatively the lack of efficacy of hydrophobic association was highlighted by measurements of activity regain before and after in situ dissociation of hydrophobic complexes (achieved by phototriggering the polarity of azobenzene-modified polymers under exposure to UV light). In the presence of polymers (CAB:polymer of 1:1 w/w ratio) at concentration ∼0.6 g L^–1, the radii of the largest complexes were similar to the radii of the copolymers alone, suggesting that the binding of CAB involves one or a few polymer chain(s). These complexes dissociated by dilution (0.01 g L^–1). It is concluded that prevention of irreversible aggregation and activity recovery were achieved when marginally stable complexes are formed. Reaching a balanced stability of the complex plays the main role in CAB renaturation, irrespective of the nature of the binding (by Coulomb association, with or without contribution of hydrophobic association)

Photoresponse of Complexes between Surfactants and Azobenzene-Modified Polymers Accounting for the Random Distribution of Hydrophobic Side Groups

The design of photoresponsive macromolecules has opened the route to many applications, in particular to trigger macroscopic responses induced by light irradiation in complex fluids and polymer−surfactant formulations. In this report, we studied the association of three sets of azobenzene modified polymer (AMPs) derived from poly(acrylic)acid with varying integration levels of azobenzene and various azobenzene hydrophobic moieties, with the neutral surfactant Triton X 100 (TX 100). Binding isotherms in dilute aqueous solutions were determined by spectrophotometry (to measure the fraction of bound azobenzene) and capillary electrophoresis (to measure the amount of bound TX 100). The degree of binding of TX 100 to AMPs increases markedly with increasing azobenzene hydrophobicity and density in AMPs. A noticeable and reversible photoresponse of the associates was observed upon exposure to UV/visible lights, although the magnitude of the UV-triggered photodissociation and blue-triggered association depends on the chemical structure of both the azobenzene and AMPs. We introduce a critical distance <i>l_c</i> that accounts as single parameter for the balance between energy gain of hydrophobic binding and energy loss due to chain conformational constraints. Only segments of chains flanked with two azobenzene groups at their ends and shorter than <i>l_c</i> are assumed to bind tightly. <i>l_c</i> is used to fit both the maximum fraction of azobenzene transferred into TX 100 micelles (with saturation well below 100% despite the presence of excess free TX 100) and the amount of bound TX 100 as a function of the density of azobenzene in the chains. The model includes the effect of random distribution of azobenzene moieties along the chains. From this analysis, we find criterions for optimization of the photoresponse as a function of the azobenzene hydrophobicity and density in the chain, and the chain length

Unfolding of Cytochrome c upon Interaction with Azobenzene-Modified Copolymers

International audienceHydrophilic or amphiphilic macromolecules are common organic matrices used to encapsulate and protect fragile drugs such as proteins. Polymer cargoes are in addition designed for remote control of,protein delivery, upon imparting the macromolecules with stimuli-responsive properties, such as light-triggered :polarity switches. The effect of interaction between polymers and proteins on the Stability of the proteins is, however, rarely Investigated. Here we studied the unfolding/folding equilibrium of. cytochrome c.(cyt c) under its oxidized or reduced forms, in the presence of various,amphiphilic copolymers (by circular dichroism and;intrinsic fluorescence measurements), As models of stimuli-responsive amphiphilic chains, we considered Poly(acrylic acid) derivatives, modified to contain hydrophobic,, light-responsive azobenzene moieties.. These copolymers are, thus, capable to develop both ionic (Under their sodium forms at pH > 8) and hydrophobic associations with the basic protein cyt c (isoelectric point of 10.0). In aqueous buffer upon increasing urea concentrations, cyt c underwent unfolding, at [urea] Of 9-10 M, which was analyzed under the framework Of the equilibrium between two States (native unfolded). In the presence of polymers, the native folding of cyt c was preserved at low Concentrations Of urea (typically 20 degrees C as compared to thermal unfolding in the absence of polymers. Upon exposure to UV light, properties of the polymers chains were perturbed in situ, upon cis/trans isomerization of the azobenzene groups. In polymers displaying a photoresponsive polarity and hydrophobicity switch (conventional azobenzene), the stability of cyt c was not affected by the exposure to light. In contrast, when photoionization occurred (using an hydroxyl-azobenzene whose pK(a) can be photoshifted); unfolding was initiated upon exposure to light. Altogether, these results show that coulombic binding is a predominant driving force that facilitates unfolding in water/urea solutions. In regard to the design of light-responsive systems for protein handling and control of folding, we, conclude that control of the coulombic interaction upon photoionization of chromophores can be more efficient than the more conventional photomodulation of polarit

Kinetic behavior of a-amido trifluoromethyl xanthate Madix agents: Structure-reactivity relationship

A new series of xanthate MADIX agents bearing a-amido trifluoromethyl leaving R groups and exhibiting a good ability for fragmentation is kinetically investigated. Quantitative measurements of both Ctr for xanthates and Cex faithfully supported the experimental observations for Mn and PDIs

α-Amido Trifluoromethyl Xanthates: A New Class of RAFT/MADIX Agents

International audienceXanthates have long been described as poor RAFT/MADIX agents for styrene polymerization. Through the determination of chain transfer constants to xanthates, this work demonstrated beneficial capto-dative substituent effects for the leaving group of a new series of α-amido trifluoromethyl xanthates, with the best effect observed with trifluoroacetyl group. The previously observed Z-group activation with a O-trifluoroethyl group compared to the O-ethyl counterpart was quantitatively established with Cex = 2.7 (3–4 fold increase) using the SEC peak resolution method. This study further confirmed the advantageous incorporation of trifluoromethyl substituents to activate xanthates in radical chain transfer processes and contributed to identify the most reactive xanthate reported to date for RAFT/MADIX polymerization of styrene

Unfolding of Cytochrome <i>c</i> upon Interaction with Azobenzene-Modified Copolymers

Hydrophilic or amphiphilic macromolecules are common organic matrices used to encapsulate and protect fragile drugs such as proteins. Polymer cargoes are in addition designed for remote control of protein delivery, upon imparting the macromolecules with stimuli-responsive properties, such as light-triggered polarity switches. The effect of interaction between polymers and proteins on the stability of the proteins is, however, rarely investigated. Here we studied the unfolding/folding equilibrium of cytochrome <i>c</i> (cyt <i>c</i>) under its oxidized or reduced forms, in the presence of various amphiphilic copolymers (by circular dichroism and intrinsic fluorescence measurements). As models of stimuli-responsive amphiphilic chains, we considered poly­(acrylic acid) derivatives, modified to contain hydrophobic, light-responsive azobenzene moieties. These copolymers are, thus, capable to develop both ionic (under their sodium forms at pH > 8) and hydrophobic associations with the basic protein cyt <i>c</i> (isoelectric point of 10.0). In aqueous buffer upon increasing urea concentrations, cyt <i>c</i> underwent unfolding, at [urea] of 9–10 M, which was analyzed under the framework of the equilibrium between two states (native–unfolded). In the presence of polymers, the native folding of cyt <i>c</i> was preserved at low concentrations of urea (typically <4M). However, the presence of polymers facilitated unfolding, which occurred at urea concentrations lowered by 2–4 M as compared to unfolding in the absence of polymers (polymer/cyt <i>c</i> ratio of 1:1 g/g). The predominant contribution of coulombic interactions was shown by both the lack of significant impact of the amount of (neutral) azobenzene moieties in the copolymers and the disappearance of destabilization at ionic strength higher than 150 mM. In addition, stability was similar to that of an isolated cyt <i>c</i>, in the presence of a neutral chain bearing acryloyl­(oligoethyleneoxide) units instead of the ionized sodium acrylate moieties. DSC measurements showed that in the presence of polymers, cyt <i>c</i> is thermally unfolded in aqueous buffer at temperatures lowered by >20 °C as compared to thermal unfolding in the absence of polymers. Upon exposure to UV light, properties of the polymers chains were perturbed in situ, upon cis/trans isomerization of the azobenzene groups. In polymers displaying a photoresponsive polarity and hydrophobicity switch (conventional azobenzene), the stability of cyt <i>c</i> was not affected by the exposure to light. In contrast, when photoionization occurred (using an hydroxyl-azobenzene whose p<i>K</i>_a can be photoshifted), unfolding was initiated upon exposure to light. Altogether, these results show that coulombic binding is a predominant driving force that facilitates unfolding in water/urea solutions. In regard to the design of light-responsive systems for protein handling and control of folding, we conclude that remote control of the coulombic interaction upon photoionization of chromophores can be more efficient than the more conventional photomodulation of polarity