Physics and chemistry for life sciences and the environment
Antoine Cossa, Sylvain Trépout, Frank Wien, Johannes Groen, Etienne Le Brun, Florian Turbant, Laetitia Besse, Eva Pereiro, Véronique Arluison
The bacterial chromosomic DNA is packed within a membrane-less structure, the nucleoid, due to the association of DNA with proteins called Nucleoid Associated Proteins (NAPs). Among these NAPs, Hfq is one of the most intriguing as it plays both direct and indirect roles on DNA structure. Indeed, Hfq is best known to mediate post-transcriptional regulation by using small noncoding RNA (sRNA). Although Hfq presence in the nucleoid has been demonstrated for years, its precise role is still unclear. Recently, it has been shown in vitro that Hfq forms amyloid-like structures through its C-terminal region, hence belonging to the bridging family of NAPs. Here, using cryo soft X-ray tomography imaging of native unlabeled cells and using a semi-automatic analysis and segmentation procedure, we show that Hfq significantly remodels the Escherichia coli nucleoid. More specifically, Hfq influences nucleoid density especially during the stationary growth phase when it is more abundant. Our results indicate that Hfq could regulate nucleoid compaction directly via its interaction with DNA, but also at the post-transcriptional level via its interaction with RNAs. Taken together, our findings reveal a new role for this protein in nucleoid remodeling in vivo, that may serve in response to stress conditions and in adapting to changing environments.
Simeon Minic, Burkhard Annighöfer, Milos Milcic, François Maignen, Annie Brûlet, Sophie Combet
Apomyoglobin (apoMb), a model protein in biochemistry, exhibits a strong propensity to bind various ligands, which makes it a good candidate as a carrier of bioactive hydrophobic drugs. The stability of its hydrophobic pocket determines its potential as a carrier of bioactive compounds. High pressure (HP) is a potent tool for studying protein stability, revealing the specific role of hydrophobic cavities in unfolding. We probed the effects of biliverdin (BV) binding and its complex with Zn2+ ions on the structure and HP stability of apoMb. CD spectroscopy and SAXS measurements revealed that BV and BV-Zn2+ complexes make the apoMb structure more compact with higher α-helical content. We performed in situ HP measurements of apoMb intrinsic fluorescence to demonstrate the ability of BV to stabilise apoMb structure at HP conditions. Furthermore, the presence of Zn2+ within the apoMb-BV complex significantly enhances the BV stabilisation effect. In situ visible absorption study of BV chromophore confirmed the ability of Zn2+ to increase the stability of apoMb-BV complex under HP: the onset of complex dissociation is shifted by ∼100 MPa in presence of Zn2+. By combining HP-fluorescence and HP-visible absorption spectroscopy, our strategy highlights the crucial role of tetrapyrrole-metal complexes in stabilising apoMb hydrophobic pocket.
Somia Haouache, Yu Chen, Clara Jimenez-Saelices, Fabrice Cousin, Pan Chen, Yoshiharu Nishiyama, François Jerome, and Isabelle Capron
Nanocelluloses can be used to stabilize oil–water surfaces, forming so-called Pickering emulsions. In this work, we compare the organization of native and mercerized cellulose nanocrystals (CNC-I and CNC-II) adsorbed on the surface of hexadecane droplets dispersed in water at different CNC concentrations. Both types of CNCs have an elongated particle morphology and form a layer strongly adsorbed at the interface. However, while the layer thickness formed with CNC-I is independent of the concentration at 7 nm, CNC-II forms a layer ranging from 9 to 14 nm thick with increasing concentration, as determined using small-angle neutron scattering with contrast-matched experiments. Molecular dynamics (MD) simulations showed a preferred interacting crystallographic plane for both crystalline allomorphs that exposes the CH groups (100 and 010) and is therefore considered hydrophobic. Furthermore, this study suggests that whatever the allomorph, the migration of CNCs to the oil–water interface is spontaneous and irreversible and is driven by both enthalpic and entropic processes.
Céline JAUDOIN, Isabelle GRILLO, Fabrice COUSIN, Maria GEHRKE, Malika OULDALI, Ana-Andreea ARTENI, Luc PICTON, Christophe RIHOUEY, Fanny SIMELIERE, Amélie BOCHOT, Florence AGNELY
Mixtures of hyaluronic acid (HA) with liposomes lead to hybrid colloid–polymer systems with a great interest in drug delivery. However, little is known about their microstructure. Small angle neutron scattering (SANS) is a valuable tool to characterize these systems in the semi-dilute entangled regime (1.5% HA) at high liposome concentration (80 mM lipids). The objective was to elucidate the influence of liposome surface (neutral, cationic, anionic or anionic PEGylated), drug encapsulation and HA concentration in a buffer mimicking biological fluids (37 °C). First, liposomes were characterized by SANS, cryo-electron microscopy, and dynamic light scattering and HA by SANS, size exclusion chromatography, and rheology. Secondly, HA-liposome mixtures were studied by SANS. In HA, liposomes kept their integrity. Anionic and PEGylated liposomes were in close contact within dense clusters with an amorphous organization. The center-to-center distance between liposomes corresponded to twice their diameter. A depletion mechanism could explain these findings. Encapsulation of a corticoid did not modify this organization. Cationic liposomes formed less dense aggregates and were better dispersed due to their complexation with HA. Liposome surface governed the interactions and microstructure of these hybrid systems.
https://doi.org/10.1016/j.jcis.2022.07.146
Florian Turbant, Jehan Waeytens, Camille Campidelli, Marianne Bombled, Denis Martinez, Axelle Grélard, Birgit Habenstein, Vincent Raussens, Marisela Velez, Frank Wien, Véronique Arluison
Hfq is a pleiotropic regulator that mediates several aspects of bacterial RNA metabolism. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, usually via its interaction with small regulatory RNAs. Previously, we showed that the Hfq C-terminal region forms an amyloid-like structure and that these fibrils interact with membranes. The immediate consequence of this interaction is a disruption of the membrane, but the effect on Hfq structure was unknown. To investigate details of the mechanism of interaction, the present work uses different in vitro biophysical approaches. We show that the Hfq C-terminal region influences membrane integrity and, conversely, that the membrane specifically affects the amyloid assembly. The reported effect of this bacterial master regulator on membrane integrity is discussed in light of the possible consequence on small regulatory RNA-based regulation.
Sophie Combet, Françoise Bonneté, Stéphanie Finet, Alexandre Pozza, Christelle Saade, Anne Martel, Alexandros Koutsioubas, Jean-Jacques Lacapère, Biochimie (2022) in press.
The translocator protein (TSPO) is a ubiquitous transmembrane protein of great pharmacological interest thanks to its high affinity to many drug ligands. The only high-resolution 3D-structure known for mammalian TSPO was obtained by NMR for the mouse mTSPO in DPC detergent only in presence of the high-affinity PK 11195 ligand. An atomic structure of free-ligand mTSPO is still missing to better understand the interaction of ligands with mTSPO and their effects on the protein conformation.
Simeon Minić, Burkhard Annighöfer, Arnaud Hélary, Laïla Sago, David Cornu, Annie Brûlet, Sophie Combet
High pressure (HP) is a particularly powerful tool to study protein folding/unfolding, revealing subtle structural rearrangements. Bovine β-lactoglobulin (BLG), a protein of interest in food science, exhibits a strong propensity to bind various bioactive molecules. We probed the effects of the binding of biliverdin (BV), a tetrapyrrole linear chromophore, on the stability of BLG under pressure, by combining in situ HP small-angle neutron scattering (SANS) and HP-UV absorption spectroscopy. Although BV induces a slight destabilization of BLG during HP-induced unfolding, a ligand excess strongly prevents BLG oligomerization. Moreover, at SANS resolution, an excess of BV induces the complete recovery of the protein “native” 3D structure after HP removal, despite the presence of the BV covalently bound adduct. Mass spectrometry highlights the crucial role of cysteine residues in the competitive and protective effects of BV during pressure denaturation of BLG through SH/S-S exchange.
https://doi.org/10.1016/j.bpj.2022.06.003
A presentation in French on Scoop.it! of the Paris-Saclay University.
Simeon Minic, Burkhard Annighofer, Arnaud Helary, Djemel Hamdane, Gaston Hui Bon Hoa, Camille Loupiac, Annie Brulet, and Sophie Combet
To probe intermediate states during unfolding and oligomerization of proteins remains a major challenge. High pressure (HP) is a powerful tool for studying these problems, revealing subtle structural changes in proteins not accessible by other means of denaturation. Bovine b-lactoglobulin (BLG), the main whey protein, has a strong propensity to bind various bioactive molecules such as retinol and resveratrol, two ligands with different affinity and binding sites. By combining in situ HPsmall- angle neutron scattering (SANS) and HP-ultraviolet/visible absorption spectroscopy, we report the specific effects of these ligands on three-dimensional conformational and local changes in BLG induced by HP. Depending on BLG concentration, two different unfolding mechanisms are observed in situ under pressures up to 300 MPa: either a complete protein unfolding, from native dimers to Gaussian chains, or a partial unfolding with oligomerization in tetramers mediated by disulfide bridges. Retinol, which has a high affinity for the BLG hydrophobic cavity, significantly stabilizes BLG both in three-dimensional and local environments by shifting the onset of protein unfolding by 100 MPa. Increasing temperature from 30 to 37C enhances the hydrophobic stabilization effects of retinol. In contrast, resveratrol, which has a low binding affinity for site(s) on the surface of the BLG, does not induce any significant effect on the structural changes of BLG due to pressure. HP treatment back and forth up to 300 MPa causes irreversible covalent oligomerization of BLG. Ab initio modeling of SANS shows that the oligomers formed from the BLG-retinol complex are smaller and more elongated compared to BLG without ligand or in the presence of resveratrol. By combining HP-SANS and HP-ultraviolet/visible absorption spectroscopy, our strategy highlights the crucial role of BLG hydrophobic cavity and opens up new possibilities for the structural determination of HP-induced protein folding intermediates and irreversible oligomerization.
Self-Induced Crystallization in Charged Gold Nanoparticle- Semiflexible Biopolyelectrolyte Complexes
Li Shi, Florent Carn, Arsen Goukassov, Eric Buhler, and François Boué
Mixing negatively charged polyelectrolyte (PEL) with positively charged gold nanoparticles (Au NPs) in aqueous solution results in electrostatics complexes of different shapes and compactness. Here, when complexing with a semirigid PEL hyaluronic acid (HA), we obtain crystals made of nanoparticles in a new region of the phase diagram, as evidenced by small-angle Xray scattering (SAXS). The Au NPs were initially well dispersed in solution; their size distribution is well controlled but does not need to be extremely narrow. The bacterial hyaluronic acid, polydispersed, is commercially available. Such rather simple materials and mixing preparation produce a highly ordered crystalline phase of electrostatic complexes. The details of the interactions between spherical nanoparticles and linear polymer chains remain to be investigated. In practice, it opens a completely new and unexpected method of complexation. It has high potential, in particular because one can take advantage of the versatility of Au NPs associated with the specificity of biopolymers, varied due to natural biodiversity.
Editors : Véronique Arluison and Frank Wien
This volume looks at the different spectroscopic and biophysical methods used by researchers to study the structure and folding of RNA, and to follow their interactions with proteins. The chapters in this book cover topics such as single-molecule spectroscopy of multiple RNA species; surface plasmon resonance, MS or microcalorimetry for investigating molecular interactions with RNA; FTIR, SAXS, SANS and SRCD spectroscopies to analyze RNA structure; use of fluorescent nucleotides to map RNA-binding sites on proteins surfaces or CryoEM; and much more. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and comprehensive, RNA Spectroscopy: Methods and Protocols is a valuable resource for anyone interested in learning more about this developing field.
Aline Maire du Poset, Mikaela Börjesson, Céline Rameau, Claire Madeleine-Perdrillat, Adrien Lerbret, Camille Loupiac, Fabrice Cousin, Ali Assifaoui
We show here how the structure of polygalacturonate (polyGalA) hydrogels cross-linked by Ca2+ cations via external gelation controls the loading and release rate of beta-lactoglobulin (BLG), a globular protein. Hydrogels prepared from a polyGalA/BLG solution are found to be similar to those obtained from a polyGalA solution in our previous study (Maire du Poset et al. Biomacromolecules 2019, 20 (7), 2864–2872): they exhibit similar transparencies and gradients of mechanical properties and polyGalA concentrations. The nominal BLG/polyGalA ratio of the mixtures is almost recovered within the whole mixed hydrogel despite such strong concentration gradients, except in the part of the hydrogels with the largest mesh size, where more BLG proteins are present. This gradient enables one to tune the amount of protein loaded within the hydrogel. At a local scale, the proteins are distributed evenly within the hydrogel network, as shown by small-angle neutron scattering (SANS). The release of proteins from hydrogels is driven by Fickian diffusion, and the release rate increases with the mesh size of the network, with a characteristic time of a few hours. The specific structure of these polysaccharide-based hydrogels allows for control of both the dosage and the release rate of the loaded protein and makes them good candidates for use as oral controlled-delivery systems.
https://dx.doi.org/10.1021/acs.biomac.9b01722
Daniela Russo, Maya Dimova Lambreva, Christiane Alba Simionesco, Pierre Sebban,and Giuseppina Rea
Studies on the dynamical properties of photosynthetic membranes of land plants and purple bacteria have been previously performed by neutron spectroscopy, revealing a tight coupling between specific photochemical reactions and macromolecular dynamics. Here, we probed the intrinsic dynamics of biotechnologically useful mutants of the green alga Chlamydomonas reinhardtii by incoherent neutron scattering coupled with prompt chlorophyll fluorescence experiments. We brought to light that single amino acid replacements in the plastoquinone (PQ)-binding niche of the photosystem II D1 protein impair electron transport (ET) efficiency between quinones and confer increased flexibility to the host membranes, expanding to the entire cells. Hence, a more flexible environment in the PQ-binding niche has been associated to a less efficient ET.Asimilar function/dynamics relationship was also demonstrated in Rhodobacter sphaeroides reaction centers having inhibited ET, indicating that flexibility at the quinones region plays a crucial role in evolutionarily distant organisms. Instead, a different functional/dynamical correlation was observed in algal mutants hosting a single amino acid replacement residing in a D1 domain far from the PQ-binding niche. Noteworthy, this mutant displayed the highest degree of flexibility, and besides having a nativelike ET efficiency in physiological conditions, it acquired novel, to our knowledge, phenotypic traits enabling it to preserve a high maximal quantum yield of photosystem II photochemistry in extreme habitats. Overall, in the nanosecond timescale, the degree of the observed flexibility is related to the mutation site; in the picosecond timescale, we highlighted the presence of a more pronounced dynamic heterogeneity in all mutants compared to the native cells, which could be related to a marked chemically heterogeneous environment.
DOI : https://doi.org/10.1016/j.bpj.2019.03.029
D. Russo, A. De Angelis, A. Paciaroni, B. Frick, N. de Sousa, F. R. Wurm, and J. Teixeira
We investigate the relaxation dynamics of protein−polymer conjugates by neutron scattering spectroscopy to understand to which extent the coating of a protein by a polymer can replace water in promoting thermal structural fluctuations. For this purpose, we compare the dynamics of protein−polymer mixtures to that of conjugates with a variable number of polymers covalently attached to the protein. Results show that the flexibility of the protein is larger in protein−polymer mixtures than in native protein or in conjugates, even in the dry state. Upon hydration, both the native protein and the conjugate show equivalent dynamics, suggesting that the polymer grafted on the protein surface adsorbs all water molecules.
A. Theodoratou, L.-T. Lee, J. Oberdisse and A. Aubert-Pouëssel, Langmuir 35(20) (2019) 6620.
Abstract :
Nanofilms of about 2 nm thickness have been formed at the air–water interface using functionalized castor oil (ICO) with cross-linkable silylated groups. These hybrid films represent excellent candidates for replacing conventional polymeric materials in biomedical applications, but they need to be optimized in terms of biocompatibility, which is highly related to protein adsorption. Neutron reflectivity has been used to study the adsorption of two model proteins, bovine serum albumin and lysozyme, at the silylated oil (ICO)–water interface in the absence and presence of salt at physiologic ionic strength and pH and at different protein concentrations. These measurements are compared to adsorption at the air–water interface. While salt enhances adsorption by a similar degree at the air–water and oil–water interfaces, the impact of the oil film is significant with adsorption at the oil–water interface 3–4-fold higher compared to that at the air–water interface. Under these conditions, the concentration profiles of the adsorbed layers for both proteins indicate multilayer adsorption. The thickness of the outer layer (oil side) is close to the dimension of the minor axis of the protein molecule, ∼30 Å, suggesting a sideway orientation with the long axis parallel to the interface. The inner layer extends to 55–60 Å. Interestingly, in all cases, the composition of the oil film remains intact without significant protein penetration into the film. The optimal adsorption on these nanofilms, 1.7–2.0 mg·m-2, is comparable to the results obtained recently on thick solid cross-linked films using a quartz crystal microbalance and atomic force microscopy, showing in particular that adsorption at these ICO film interfaces under standard physiological conditions is nonspecific. These results furnish useful information toward the elaboration of vegetable oil-based nanofilms in direct nanoscale applications or as precursor films in the fabrication of thicker macroscopic films for biomedical applications.
"Dynamics properties of photosynthetic microorganisms probed by incoherent neutron scattering"
Daniela Russo, Maya Dimova Lambreva, Christiane Alba Simionesco, Pierre Sebban, and Giuseppina Rea
Biophysical Journal, 116 (9) (2019) 1759-1768
Studies on the dynamical properties of photosynthetic membranes of land plants and purple bacteria have been previously performed by neutron spectroscopy, revealing a tight coupling between specific photochemical reactions and macromolecular dynamics. Here, we probed the intrinsic dynamics of biotechnologically useful mutants of the green alga Chlamydomonas reinhardtii by incoherent neutron scattering coupled with prompt chlorophyll fluorescence experiments. We brought to light that single amino acid replacements in the plastoquinone (PQ)-binding niche of the photosystem II D1 protein impair electron transport (ET) efficiency between quinones and confer increased flexibility to the host membranes, expanding to the entire cells. Hence, a more flexible environment in the PQ-binding niche has been associated to a less efficient ET.Asimilar function/dynamics relationship was also demonstrated in Rhodobacter sphaeroides reaction centers having inhibited ET, indicating that flexibility at the quinones region plays a crucial role in evolutionarily distant organisms. Instead, a different functional/dynamical correlation was observed in algal mutants hosting a single amino acid replacement residing in a D1 domain far from the PQ-binding niche. Noteworthy, this mutant displayed the highest degree of flexibility, and besides having a nativelike ET efficiency in physiological conditions, it acquired novel, to our knowledge, phenotypic traits enabling it to preserve a high maximal quantum yield of photosystem II photochemistry in extreme habitats. Overall, in the nanosecond timescale, the degree of the observed flexibility is related to the mutation site; in the picosecond timescale, we highlighted the presence of a more pronounced dynamic heterogeneity in all mutants compared to the native cells, which could be related to a marked chemically heterogeneous environment.
B. Annighöfer , A. Hélary, A. Brulet, A. Colas de la Noue, C. Loupiac and S. Combet
Review of Scientific Instruments 90 (2019) 025106
Abstract :
We report on a high pressure (HP) cell designed for the determination of the structure of molecular solutions by small-angle neutron scattering (SANS). The HP cell is fitted up with two thick metallic windows that make the device very resistant under hydrostatic pressures up to 600 MPa (or 6 kbar). The metallic windows are removable, offering the possibility to adapt the HP cell to a given study with the pressure desired on an appropriate spatial range to study the structure of various molecular solutions by SANS. In this context, we report the absorption, transmission, and scattering properties of different metallic windows. Finally, we describe, as a proof of principle, the solution structure changes of myoglobin, a small globular protein.
Andrés Marcoleta, Frank Wien, Véronique Arluison, Rosalba Lagos, Rafael Giraldo
Bacterial Amyloids (2019)
Amyloids are supramolecular protein assemblies based on fibrillar arrangements of β‐sheets that were first found as linked to neurodegenerative and systemic human diseases. However, there is now overwhelming evidence on alternative roles of amyloids as functional assemblies and as epigenetic determinants of beneficial traits, both in Fungi and Metazoa. Bacteria also use amyloids as functional devices, mainly as extracellular scaffolds in biofilms, but there is increasing evidence for functional roles of amyloids in the bacterial cytosol, and these have enabled to engineer minimal models of a ‘generic’ amyloid disease. Amyloids are thus key players in the physiology of bacteria and versatile building blocks in synthetic biology.
https://doi.org/10.1002/9780470015902.a0028401
Burkhard Annighöfer, Arnaud Hélary, Annie Brûlet, Alexandre Colas de la Noue, Camille Loupiac, and Sophie Combet
We report on a high pressure (HP) cell designed for the determination of the structure of molecular solutions by small-angle neutron scattering (SANS). The HP cell is fitted up with two thick metallic windows that make the device very resistant under hydrostatic pressures up to 600 MPa (or 6 kbar). The metallic windows are removable, offering the possibility to adapt the HP cell to a given study with the pressure desired on an appropriate spatial range to study the structure of various molecular solutions by SANS. In this context, we report the absorption, transmission, and scattering properties of different metallic windows. Finally, we describe, as a proof of principle, the solution structure changes of myoglobin, a small globular protein.
Sophie Combet, Fabrice Cousin, Human Rezaei, Sylvie Noinville
Soluble oligomers of prion proteins (PrP), produced during amyloid aggregation, have emerged as the primary neurotoxic species, instead of the fibrillar end-products, in transmissible spongiform encephalopathies. However, whether the membrane is among their direct targets, that mediate the downstream adverse effects, remains a question of debate. Recently, questions arise from the formation of membrane-active oligomeric species generated during the β-aggregation pathway, either in solution, or in lipid environment. In the present study, we characterized membrane interaction of off-pathway oligomers from recombinant prion protein generated along the amyloid aggregation and compared to lipid-induced intermediates produced during lipid-accelerated fibrillation. Using calcein-leakage assay, we show that the soluble prion oligomers are the most potent in producing leakage with negatively charged vesicles. Binding affinities, conformational states, mode of action of the different PrP assemblies were determined by thioflavin T binding-static light scattering experiments on DOPC/DOPS vesicles, as well as by FTIR-ATR spectroscopy and specular neutron reflectivity onto the corresponding supported lipid bilayers. Our results indicate that the off-pathway PrP oligomers interact with lipid membrane via a distinct mechanism, compared to the inserted lipid-induced intermediates. Thus, separate neurotoxic mechanisms could exist following the puzzling intermediates generated in the different cell compartments. These results not only reveal an important regulation of lipid membrane on PrP behavior but may also provide clues for designing stage-specific and prion-targeted therapy.
https://doi.org/10.1016/j.bbamem.2018.12.001
Jyotsana Lal
This article is dedicated to the memory of Loic Auvray
The results on Winsor phases, droplet and bicontinous microemulsions phases with polymer-grafted lipids studied by Small Angle Neutron Scattering (SANS) are reported below, together with the contrast variation techniques used to characterize the average curvature in the system. We have clearly shown that polymer-grafted lipids change the interaction between microemulsion droplets --it need not be just repulsive but could also be attractive. They induce structural changes or bring about complete phase changes as observed visually in the Winsor phases when added in sufficient amounts. In the bicontinous microemulsion phases, the polymer-grafted lipids decrease the persistence length, hence the bending rigidity, increase the apparent average thickness of the film, and cause a complex deformation of the film which brings about a negative curvature change at a semi-local scale. Contrary to the naive prediction that the polymer-grafted lipids should increase membrane rigidity our experiments show a decrease. This is a subtle effect caused by perhaps an indirect coupling between film curvature and concentration fluctuations.
Raphael Dos Santos Morais, Olivier Delalande, Javier Perez, Dominique Mias-Lucquin, Melanie Lagarrigue, Anne Martel, Anne-Elisabeth Molza, Angelique Cheron, Celine Raguenes-Nicol, Thomas Chenuel, Arnaud Bondon, Marie-Sousai Appavou, Elisabeth Le Rumeur, Sophie Combet, and Jean-Francois Hubert
Scaffolding proteins play important roles in supporting the plasma membrane (sarcolemma) of muscle cells. Among them, dystrophin strengthens the sarcolemma through protein-lipid interactions, and its absence due to gene mutations leads to the severe Duchenne muscular dystrophy. Most of the dystrophin protein consists of a central domain made of 24 spectrin-like coiled-coil repeats (R). Using small angle neutron scattering (SANS) and the contrast variation technique, we specifically probed the structure of the three first consecutive repeats 1–3 (R1–3), a part of dystrophin known to physiologically interact with membrane lipids. R1–3 free in solution was compared to its structure adopted in the presence of phospholipid-based bicelles. SANS data for the protein/lipid complexes were obtained with contrast-matched bicelles under various phospholipid compositions to probe the role of electrostatic interactions. When bound to anionic bicelles, large modifications of the protein threedimensional structure were detected, as revealed by a significant increase of the protein gyration radius from 42 5 1 to 60 5 4 A . R1–3/anionic bicelle complexes were further analyzed by coarse-grained molecular dynamics simulations. From these studies, we report an all-atom model of R1–3 that highlights the opening of the R1 coiled-coil repeat when bound to the membrane lipids. This model is totally in agreement with SANS and click chemistry/mass spectrometry data. We conclude that the sarcolemma membrane anchoring that occurs during the contraction/elongation process of muscles could be ensured by this coiled-coil opening. Therefore, understanding these structural changes may help in the design of rationalized shortened dystrophins for gene therapy. Finally, our strategy opens up new possibilities for structure determination of peripheral and integral membrane proteins not compatible with different high-resolution structural methods.
Dos Santos Morais R et al. Biophys J 115: 1231–1239, 2018.
https://doi.org/10.1016/j.bpj.2018.07.039
Maelenn Chevreuil, Didier Law-Hine, Jingzhi Chen, Stéphane Bressanelli, Sophie Combet, Doru Constantin, Jéril Degrouard, Johannes Möller, Mehdi Zeghal and Guillaume Tresset
The survival of viruses partly relies on their ability to self-assemble inside host cells. Although coarse-grained simulations have identified different pathways leading to assembled virions from their components, experimental evidence is severely lacking. Here, we use timeresolved small-angle X-ray scattering to uncover the nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging their full RNA genome. We reveal the formation of amorphous complexes via an en masse pathway and their relaxation into virions via a synchronous pathway. The binding energy of capsid subunits on the genome is moderate (~7kBT0, with kB the Boltzmann constant and T0 = 298 K, the room temperature), while the energy barrier separating the complexes and the virions is high (~ 20kBT0). A synthetic polyelectrolyte can lower this barrier so that filled capsids are formed in conditions where virions cannot build up. We propose a representation of the dynamics on a free energy landscape.
Unravelling a mechanism of action for a cecropin a‑melittin hybrid antimicrobial peptide: the induced formation of multilamellar lipid stacks
T. Silva, B. Claro, B. F. B. Silva, N. Vale, P. Gomes, M.-S. Gomes, S. S. Funari, J. Teixeira, D. Uhríková, and M. Bastos, Langmuir, 2018, 34 (5), pp 2158–2170.
An understanding of the mechanism of action of antimicrobial peptides is fundamental to the development of new and more active antibiotics. In the present work, we use a wide range of techniques (SANS, SAXD, DSC, ITC, CD, and confocal and electron microscopy) in order to fully characterize the interaction of a cecropin A-melittin hybrid antimicrobial peptide, CA(1-7)M(2-9), of known antimicrobial activity, with a bacterial model membrane of POPE/POPG in an effort to unravel its mechanism of action.
We found that CA(1-7)M(2-9) disrupts the vesicles, inducing membrane condensation and forming an onionlike structure of multilamellar stacks, held together by the intercalated peptides. SANS and SAXD revealed changes induced by the peptide in the lipid bilayer thickness and the bilayer stiffening in a tightly packed liquid-crystalline lamellar phase. The analysis of the observed abrupt changes in the repeat distance upon the phase transition to the gel state suggests the formation of an Lγ phase. To the extent of our knowledge, this is the first time that the Lγ phase is identified as part of the mechanism of action of antimicrobial peptides. The energetics of interaction depends on temperature, and ITC results indicate that CA(1-7)M(2-9) interacts with the outer leaflet. This further supports the idea of a surface interaction that leads to membrane condensation and not to pore formation.
As a result, we propose that this peptide exerts its antimicrobial action against bacteria through extensive membrane disruption that leads to cell death.
Structural characterization of nanoparticles formed by fluorinated poly(2-oxazoline)-based polyphiles,
Anna Riabtseva, Leonid I. Kaberov, Laurence Noirez, Vasyl Ryukhtin, Corinne Nardin, Bart Verbraeken, Richard Hoogenboom, Petr Stepanek, Sergey K. Filippov, European Polymer Journal 99 (2018) 518.
Abstract :
We report on the self-assembly behavior of poly(2-methyl-2-oxazoline)–block–poly(2-octyl-2-oxazoline) comprising different terminal perfluoroalkyl fragments in aqueous solutions. As reported previously [Kaberov et al. (2017)] such polyphiles can form a plethora of nanostructures depending of the composition and on the way of preparation. Here we report, for the first time, detailed information on the internal structure of the nanoparticles resulting from the self-assembly of these copolymers. Small-angle neutron and X-ray scattering (SANS/SAXS) experiments unambiguously prove the existence of polymersomes, wormlike micelles and their aggregates in aqueous solution. It is shown that increasing content of fluorine in the poly(2-oxazoline) copolymers results in a morphological transition from bilayered or multi-layered vesicles to wormlike micelles for solutions prepared by direct dissolution.
In contrast, nanoparticles prepared by dialysis of a polymer solution in a non-selective organic solvent against water are characterized by SAXS method. The internal structure of the nanoparticles could be assessed by fitting of the scattering data, revealing complex core-double shell architecture of spherical symmetry. Additionally, long range ordering is identified for all studied nanoparticles due to the crystallization of the poly(2-octyl-2-oxazoline) segments inside the nanoparticles.
Publication by Springer Eds of the book ( ISBN 978-1-4939-7634-8):
"Bacterial regulatory RNA"
Methods and Protocols © 2018
Editors: Veronique Arluison, , Claudio Valverde
Collection : Methods in Molecular Biology
Including the contribution:
"Techniques to analyse sRNA protein cofactor self-assembly in vitro"
D. Partouche, A. Malabirade, T. Bizien, M. Velez, S. Trepout, S. Marco, V. Militello, C. Sandt, F. Wien and V. Arluison (2018) Methods Mol Biol (2018).
© 2018
Bacterial Regulatory RNA
Methods and Protocols
Editors: Arluison, Veronique, Valverde, Claudio (Eds.)
Raphael Dos Santos Morais, Olivier Delalande, Javier Pérez, Liza Mouret, Arnaud Bondon, Anne Martel, Marie-Sousai Appavou, Elisabeth Le Rumeur, Jean-François Hubert, and Sophie Combet
Obtaining structural information on integral or peripheral membrane proteins is currently arduous due to the difficulty of their solubilization, purification, and crystallization (for X-ray crystallography (XRC) application). To overcome this challenge, bicelles are known to be a versatile tool for high-resolution structure determination, especially when using solution and/or solid state nuclear magnetic resonance (NMR) and, to a lesser extent, XRC. For proteins not compatible with these high-resolution methods, small-angle X-ray and neutron scattering (SAXS and SANS, respectively) are powerful alternatives to obtain structural information directly in solution. In particular, the SANS-based approach is a unique technique to obtain low-resolution structures of proteins in interactions with partners by contrast-matching the signal coming from the latter. In the present study, isotropic bicelles are used as a membrane mimic model for SANS-based structural studies of bound peripheral membrane proteins. We emphasize that the SANS signal coming from the deuterated isotropic bicelles can be contrast-matched in 100% D2O-based buffer, allowing us to separately and specifically focus on the signal coming from the protein in interaction with membrane lipids. We applied this method to the DYS-R11–15 protein, a fragment of the central domain of human dystrophin known to interact with lipids, and we were able to recover the signal from the protein alone. This approach gives rise to new perspectives to determine the solution structure of peripheral membrane proteins interacting with lipid membranes and might be extended to integral membrane proteins.
Stehane Longeville et Laura-Roxana Stingaciu
Translational diffusion of macromolecules in cell is generally assumed to be anomalous due high macromolecular crowding of the milieu. Red blood cells are a special case of cells filled quasi exclusively (95% of the dry weight of the cell) with an almost spherical protein: hemoglobin. Hemoglobin diffusion has since a long time been recognized as facilitating the rate of oxygen diffusion through a solution. We address in this paper the question on how hemoglobin diffusion in the red blood cells can help the oxygen capture at the cell level and hence to improve oxygen transport. We report a measurement by neutron spin echo spectroscopy of the diffusion of hemoglobin in solutions with increasing protein concentration. We show that hemoglobin diffusion in solution can be described as Brownian motion up to physiological concentration and that hemoglobin diffusion in the red blood cells and in solutions at similar concentration are the same. Finally, using a simple model and the concentration dependence of the diffusion of the protein reported here, we show that hemoglobin concentration observed in human red blood cells (≃≃330 g.L−1) corresponds to an optimum for oxygen transport for individuals under strong activity.
Following UV absorption, many biomolecular systems are endowed with mechanisms of excited-states deactivation that ensure their photochemical stability. These (ultra)fast processes indeed provide an efficient way to dissipate the electronic energy into vibration, avoiding then structural damages which could affect their biological function. In order to determine and document the basic physical phenomena controlling the lifetime of excited states and to highlight the link between electronic dynamic and structure, we have developed a synergetic theory-experience approach combining Quantum chemistry and Spectroscopies laser. The theoretical challenge is double: i) identify in these complex systems, the critical motions responsible of the electronic relaxation and ii) treat in an accurate and balanced way the different excited states. This challenge is addressed thanks the implementation of an innovative computational multi-step and multi-level strategy. Understanding the fate of the energy absorbed is of fundamental importance and relevant to potential applications in many fields such as photochemistry, biology or material sciences.
Motions of the carbonyl group of the Photoactive Yellow Protein (PYP) chromophore have been investigated, for the first time, by femtosecond time-resolved circular dichroism (tr-CD) spectroscopy, in the near-ultraviolet spectral region. The quantitative analysis of the tr-CD signals shows that, upon excitation, the carbonyl group undergoes a fast (≪0.8 ps) and unidirectional flipping motion in the excited state with an angle of ca. 17−53°. For the subset of proteins that do not enter the photocycle, tr-CD spectroscopy provides strong evidence that the carbonyl group moves back to its initial position, leading to the formation of a nonreactive ground-state intermediate of trans conformation. The initial ground state is then restored within ca. 3 ps.
Many complex molecular systems absorb light in the UV spectral range, including those of paramount biological importance, like DNA bases or proteins. The excited states created by UV absorption are endowed with mechanisms of deactivation which are of major importance for the photochemical stability of these species. The majority of these processes are ultrafast and provide a rapid and efficient way to dissipate the electronic energy into vibration, thus avoiding photochemical reactions.
In proteins, for instance, absorption in the near UV range is mainly due to the aromatic side chain of the tryptophan, tyrosine and phenylalanine amino acids and both spectroscopic and dynamic properties of these chromophores generally depend on their immediate environment, i.e., on the local conformation of the protein. In this context, synergetic theoretical/experimental studies of gas phase model peptides as proteins building blocks should lead to better understanding the photophysical phenomena involved in the relaxation mechanisms of proteins. Such an approach has been developed by a collaboration between the SBM team of LFP (CEA-CNRS URA2453), a theoretical team of the Ruđer Bošković Institut (Zagreb, Croatia) and two experimentalists of CLUPS (Paris Sud University, Orsay) in order to characterize the excited states of the stable conformers of a model peptide and establish the nonradiative relaxation mechanisms.1
Scientists at the "Laboratoire de chimie et biologie des métaux" (CEA-CNRS-Université J. Fourier, CEA-Grenoble), "Laboratoire de chimie des surfaces et interfaces" (CEA-Saclay) and a team at the Laboratoire d'innovation pour les technologies des énergies nouvelles et les nanomatériaux" (CEA Grenoble) have combined nanoscience and bio-inspired chemistry to develop, for the 1st time, a paltinum free material capable of catalyzing both production of hydrogen and its use in a fuel cell.
This result, important in view of a more competitive hydrogen economy is being published in the journal Science.
