Hfq C-terminal region forms a ß-rich amyloid-like motif without perturbing the N-terminal Sm-like structure.

Hfq is a pleitropic actor that serves as stress response and virulence factor in the bacterial cell. To execute its multiple functions, Hfq assembles into symmetric torus-shaped hexamers. Extending outward from the hexameric core, Hfq presents a C-terminal region, described as intrinsically disordered in solution. Many aspects of the role and the structure of this region remain unclear. For instance, in its truncated form it can promote amyloid-like filament assembly. Here, we show that a minimal 11-residue motif at the C-terminal end of Hfq assembles into filaments with amyloid characteristics. Our data suggest that the full-length Hfq in its filamentous state contains a similar molecular fingerprint than that of the short ß-strand peptide, and that the Sm-core structure is not affected by filament formation. Hfq proteins might thus co-exist in two forms in vivo, either as isolated, soluble hexamers or as self-assembled hexamers through amyloid-reminiscent interactions, modulating Hfq cellular functions.

Membrane plasticity induced by myo-inositol derived archaeal lipids: chemical synthesis and biophysical characterization.

Archaeal membrane lipids have specific structures that allow Archaea to withstand extreme conditions of temperature and pressure. In order to understand the molecular parameters that govern such resistance, the synthesis of 1,2-di-O-phytanyl-sn-glycero-3-phosphoinositol (DoPhPI), an archaeal lipid derived from myo-inositol, is reported. Benzyl protected myo-inositol was first prepared and then transformed to phosphodiester derivatives using a phosphoramidite based-coupling reaction with archaeol. Aqueous dispersions of DoPhPI alone or mixed with DoPhPC can be extruded and form small unilamellar vesicles, as detected by DLS. Neutron, SAXS, and solid-state NMR demonstrated that the water dispersions could form a lamellar phase at room temperature that then evolves into cubic and hexagonal phases with increasing temperature. Phytanyl chains were also found to impart remarkable and nearly constant dynamics to the bilayer over wide temperature ranges. All these new properties of archaeal lipids are proposed as providers of plasticity and thus means for the archaeal membrane to resist extreme conditions.

Structural polymorphism of the low-complexity C-terminal domain of TDP-43 amyloid aggregates revealed by solid-state NMR.

Aberrant aggregation of the transactive response DNA-binding protein (TDP-43) is associated with several lethal neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Cytoplasmic neuronal inclusions of TDP-43 are enriched in various fragments of the low-complexity C-terminal domain and are associated with different neurotoxicity. Here we dissect the structural basis of TDP-43 polymorphism using magic-angle spinning solid-state NMR spectroscopy in combination with electron microscopy and Fourier-transform infrared spectroscopy. We demonstrate that various low-complexity C-terminal fragments, namely TDP-13 (TDP-43300-414), TDP-11 (TDP-43300-399), and TDP-10 (TDP-43314-414), adopt distinct polymorphic structures in their amyloid fibrillar state. Our work demonstrates that the removal of less than 10% of the low-complexity sequence at N- and C-termini generates amyloid fibrils with comparable macroscopic features but different local structural arrangement. It highlights that the assembly mechanism of TDP-43, in addition to the aggregation of the hydrophobic region, is also driven by complex interactions involving low-complexity aggregation-prone segments that are a potential source of structural polymorphism.

Switchable Lipids: From Conformational Switch to Macroscopic Changes in Lipid Vesicles.

It has been shown that the use of conformationally pH-switchable lipids can drastically enhance the cytosolic drug delivery of lipid vesicles. Understanding the process by which the pH-switchable lipids disturb the lipid assembly of nanoparticles and trigger the cargo release is crucial to optimize the rational design of pH-switchable lipids. Here, we gather morphological observations (FF-SEM, Cryo-TEM, AFM, confocal microscopy), physicochemical characterization (DLS, ELS), as well as phase behavior studies (DSC, 2H NMR, Langmuir isotherm, and MAS NMR) to propose a mechanism of pH-triggered membrane destabilization. We demonstrate that the switchable lipids are homogeneously incorporated with other co-lipids (DSPC, cholesterol, and DSPE-PEG2000) and promote a liquid-ordered phase insensitive to temperature variation. Upon acidification, the protonation of the switchable lipids triggers a conformational switch altering the self-assembly properties of lipid nanoparticles. These modifications do not lead to a phase separation of the lipid membrane; however, they cause fluctuations and local defects, which result in morphological changes of the lipid vesicles. These changes are proposed to affect the permeability of vesicle membrane, triggering the release of the cargo encapsulated in the lipid vesicles (LVs). Our results confirm that pH-triggered release does not require major morphological changes, but can result from small defects affecting the lipid membrane permeability.

Dynamic Sorting of Mobile and Rigid Molecules in Biomembranes by Magic-Angle Spinning 13C NMR.

Understanding the membrane dynamics of complex systems is essential to follow their function. As molecules in membranes can be in a rigid or mobile state depending on external (temperature, pressure) or internal (pH, domains, etc.) conditions, we propose an in-depth examination of NMR methods to filter highly mobile molecular parts from others that are in more restricted environments. We have thus developed a quantitative magic-angle spinning (MAS) 13C NMR approach coupled with cross-polarization (CP) and/or Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) on rigid and fluid unlabeled model membranes. We demonstrate that INEPT can detect only very mobile lipid headgroups in gel (solid-ordered) phases; the remaining rigid parts are only detected with CP. A direct correlation is established between the normalized line intensity as obtained by CP and the C-H (C-D) order parameters measured by wide-line 2H NMR or extracted from molecular dynamics: ICP/IDPeq ≈ 5|SCH|, indicating that when the order is greater than 0.2-0.3 (maximum value of 0.5 for chain CH2), only rigid parts can be filtered and detected using CP techniques. In very fluid (liquid-disordered) membranes, where there are many more active motions, both INEPT and CP detect resonances, with, however, a clear propensity of each technique to detect mobile and restricted molecular parts, respectively. Interestingly, the 13C NMR chemical shift of lipid hydrocarbon chains can be used to monitor order-disorder phase transitions and calculate the fraction of chain defects (rotamers) and the part of the transition enthalpy due to bond rotations (6-7 kJ·mol-1 for dimyristolphosphatidylcholine, DMPC). Cholesterol-containing membranes (liquid-ordered phases) can be dynamically contrasted as the rigid-body sterol is mainly detected by the CP technique, with a contact time of 1 ms, and the phospholipid by INEPT. Our work opens up a straightforward, robust, and cost-effective route for the determination of membrane dynamics by taking advantage of well-resolved conventional 13C NMR experiments without the need of isotopic labeling.

The Rigid Core and Flexible Surface of Amyloid Fibrils Probed by Magic-Angle-Spinning NMR Spectroscopy of Aromatic Residues.

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein-protein interactions. We studied aromatic residues in the two structurally homologous cross-ß amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of "breathing motions" over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-ß architecture.

Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

Solid-state NMR molecular snapshots of Aspergillus fumigatus cell wall architecture during a conidial morphotype transition.

While establishing an invasive infection, the dormant conidia of Aspergillus fumigatus transit through swollen and germinating stages, to form hyphae. During this morphotype transition, the conidial cell wall undergoes dynamic remodeling, which poses challenges to the host immune system and antifungal drugs. However, such cell wall reorganization during conidial germination has not been studied so far. Here, we explored the molecular rearrangement of Aspergillus fumigatus cell wall polysaccharides during different stages of germination. We took advantage of magic-angle spinning NMR to investigate the cell wall polysaccharides, without employing any destructive method for sample preparation. The breaking of dormancy was associated with a significant change in the molar ratio between the major polysaccharides ß-1,3-glucan and α-1,3-glucan, while chitin remained equally abundant. The use of various polarization transfers allowed the detection of rigid and mobile polysaccharides; the appearance of mobile galactosaminogalactan was a molecular hallmark of germinating conidia. We also report for the first time highly abundant triglyceride lipids in the mobile matrix of conidial cell walls. Water to polysaccharides polarization transfers revealed an increased surface exposure of glucans during germination, while chitin remained embedded deeper in the cell wall, suggesting a molecular compensation mechanism to keep the cell wall rigidity. We complement the NMR analysis with confocal and atomic force microscopies to explore the role of melanin and RodA hydrophobin on the dormant conidial surface. Exemplified here using Aspergillus fumigatus as a model, our approach provides a powerful tool to decipher the molecular remodeling of fungal cell walls during their morphotype switching.

Bio-membranes: Picosecond to second dynamics and plasticity as deciphered by solid state NMR.

Since the first membrane models in the 1970s, the concept of biological membranes has evolved considerably. The membrane is now seen as a very complex mixture whose dynamic behavior is even more complex. Solid-state NMR is well suited for such studies as it can probe the movements of the membrane from picoseconds to seconds. Two NMR observables can be used: motionally averaged spectra and relaxation times. They bring information on order parameters, phase transitions, correlation times, activation energies and membrane elasticity. Spectra are used to determine the nature of the membrane phase. The order parameters can be measured directly from spectra that are dominated by quadrupolar, dipolar and chemical shielding magnetic interactions and allow describing the lipid membrane as being very rigid at the glycerol and chain level and very fluid at its center and surface. Correlation times and activation energies can be measured for intramolecular motions (pico to nanoseconds), molecular motions (nano to 100 ns) and collective modes of membrane deformation (microseconds). Sterols modulate membrane phases, order parameters, correlation times and membrane elasticity. In general terms, sterols tend to act to reduce the impact of environmental changes on molecular order and dynamics. They can be described as regulators of membrane dynamics by keeping them in a state of dynamics that changes very little when the temperature or other factors change. The presence of such large-scale membrane dynamics is proposed as a means of adapting to evolutionary constraints.

Cell-free synthesis of amyloid fibrils with infectious properties and amenable to sub-milligram magic-angle spinning NMR analysis.

Structural investigations of amyloid fibrils often rely on heterologous bacterial overexpression of the protein of interest. Due to their inherent hydrophobicity and tendency to aggregate as inclusion bodies, many amyloid proteins are challenging to express in bacterial systems. Cell-free protein expression is a promising alternative to classical bacterial expression to produce hydrophobic proteins and introduce NMR-active isotopes that can improve and speed up the NMR analysis. Here we implement the cell-free synthesis of the functional amyloid prion HET-s(218-289). We present an interesting case where HET-s(218-289) directly assembles into infectious fibril in the cell-free expression mixture without the requirement of denaturation procedures and purification. By introducing tailored 13C and 15N isotopes or CF3 and 13CH2F labels at strategic amino-acid positions, we demonstrate that cell-free synthesized amyloid fibrils are readily amenable to high-resolution magic-angle spinning NMR at sub-milligram quantity.

Unraveling Membrane Perturbations Caused by the Bacterial Riboregulator Hfq.

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.

Structures of Pathological and Functional Amyloids and Prions, a Solid-State NMR Perspective.

Infectious proteins or prions are a remarkable class of pathogens, where pathogenicity and infectious state correspond to conformational transition of a protein fold. The conformational change translates into the formation by the protein of insoluble amyloid aggregates, associated in humans with various neurodegenerative disorders and systemic protein-deposition diseases. The prion principle, however, is not limited to pathogenicity. While pathological amyloids (and prions) emerge from protein misfolding, a class of functional amyloids has been defined, consisting of amyloid-forming domains under natural selection and with diverse biological roles. Although of great importance, prion amyloid structures remain challenging for conventional structural biology techniques. Solid-state nuclear magnetic resonance (SSNMR) has been preferentially used to investigate these insoluble, morphologically heterogeneous aggregates with poor crystallinity. SSNMR methods have yielded a wealth of knowledge regarding the fundamentals of prion biology and have helped to solve the structures of several prion and prion-like fibrils. Here, we will review pathological and functional amyloid structures and will discuss some of the obtained structural models. We will finish the review with a perspective on integrative approaches combining solid-state NMR, electron paramagnetic resonance and cryo-electron microscopy, which can complement and extend our toolkit to structurally explore various facets of prion biology.

Bacteria associated with wood tissues of Esca-diseased grapevines: functional diversity and synergy with Fomitiporia mediterranea to degrade wood components.

Fungi are considered to cause grapevine trunk diseases such as esca that result in wood degradation. For instance, the basidiomycete Fomitiporia mediterranea (Fmed) is overabundant in white rot, a key type of wood-necrosis associated with esca. However, many bacteria colonize the grapevine wood too, including the white rot. In this study, we hypothesized that bacteria colonizing grapevine wood interact, possibly synergistically, with Fmed and enhance the fungal ability to degrade wood. We isolated 237 bacterial strains from esca-affected grapevine wood. Most of them belonged to the families Xanthomonadaceae and Pseudomonadaceae. Some bacterial strains that degrade grapevine-wood components such as cellulose and hemicellulose did not inhibit Fmed growth in vitro. We proved that the fungal ability to degrade wood can be strongly influenced by bacteria inhabiting the wood. This was shown with a cellulolytic and xylanolytic strain of the Paenibacillus genus, which displays synergistic interaction with Fmed by enhancing the degradation of wood structures. Genome analysis of this Paenibacillus strain revealed several gene clusters such as those involved in the expression of carbohydrate-active enzymes, xylose utilization and vitamin metabolism. In addition, certain other genetic characteristics of the strain allow it to thrive as an endophyte in grapevine and influence the wood degradation by Fmed. This suggests that there might exist a synergistic interaction between the fungus Fmed and the bacterial strain mentioned above, enhancing grapevine wood degradation. Further step would be to point out its occurrence in mature grapevines to promote esca disease development.

Bolaamphiphile-based supramolecular gels with drugs eliciting membrane effects.

Supramolecular chemistry has garnered important interest in recent years toward improving therapeutic efficacy via drug delivery approaches. Although self-assemblies have been deeply investigated, the design of novel drugs leveraging supramolecular chemistry is less known. In this contribution, we show that a Low Molecular Weight Gel (LMWG) can elicit cancer cell apoptosis. This biological effect results from the unique supramolecular properties of a bolaamphiphile-based gelator, which allow for strong interaction with the lipid membrane. This novel supramolecular-drug paradigm opens up new possibilities for therapeutic applications targeting membrane lipids.

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation.

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph-mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.

High-Temperature Behavior of Early Life Membrane Models.

Origin of life scenarios generally assume an onset of cell formation in terrestrial hot springs or in the deep oceans close to hot vents, where energy was available for non-enzymatic reactions. Membranes of the protocells had therefore to withstand extreme conditions different from what is found on the Earth surface today. We present here an exhaustive study of temperature stability up to 80 °C of vesicles formed by a mixture of short-chain fatty acids and alcohols, which are plausible candidates for membranes permitting the compartmentalization of protocells. We confirm that the presence of alcohol has a strong structuring and stabilizing impact on the lamellar structures. Moreover and most importantly, at a high temperature (> 60 °C), we observe a conformational transition in the vesicles, which results from vesicular fusion. Because all the most likely environments for the origin of life involve high temperatures, our results imply the need to take into account such a transition and its effect when studying the behavior of a protomembrane model.

Novel self-replicating α-synuclein polymorphs that escape ThT monitoring can spontaneously emerge and acutely spread in neurons.

The conformational strain diversity characterizing α-synuclein (α-syn) amyloid fibrils is thought to determine the different clinical presentations of neurodegenerative diseases underpinned by a synucleinopathy. Experimentally, various α-syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe thioflavin T (ThT). We report that, concurrent with classical ThT-positive products, fibrillization in saline also gives rise to polymorphs invisible to ThT (τ-). The generation of τ- fibril polymorphs is stochastic and can skew the apparent fibrillization kinetics revealed by ThT. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. They present a yet undescribed atomic organization and show an exacerbated propensity toward self-replication in cortical neurons, and in living mice, their injection into the substantia nigra pars compacta triggers a synucleinopathy that spreads toward the dorsal striatum, the nucleus accumbens, and the insular cortex.

Tandem NMR and Mass Spectrometry Analysis of Human Nuclear Membrane Lipids.

The human nuclear membrane is composed of a double bilayer, the inner membrane being linked to the protein lamina network and the outer nuclear membrane continuous with the endoplasmic reticulum. Nuclear membranes can form large invaginations inside the nucleus; their specific roles still remain unknown. Although much of the protein identification has been determined, their lipid composition remains largely undetermined. In order to understand the mechanical and dynamic properties of nuclear membranes we investigated their lipid composition by two quantitative methods, namely, 31P and 1H multidimensional NMR and mass spectrometry, using internal standards. We also developed a nondetergent nuclei extraction protocol allowing to produce milligram quantities of nuclear membrane lipids. We found that the nuclear membrane lipid extract is composed of a complex mixture of phospholipids with different phosphatidylcholine species present in large amounts. Negatively charged lipids, with elevated amounts of phosphatidylinositol (PI), were also present. Mass spectrometry confirmed the phospholipid composition and provided further information on acyl-chain length and unsaturation. Lipid chain lengths ranged between 30 and 38 carbon atoms (two chains summed up) with a high proportion of 34 carbon atom length for most species. PI lipids have high amounts of chain lengths with 36-38 carbons. Independent of the chain length unsaturations were highly elevated with one to two double bonds per lipid species.

The unprecedented membrane deformation of the human nuclear envelope, in a magnetic field, indicates formation of nuclear membrane invaginations.

Human nuclear membrane (hNM) invaginations are thought to be crucial in fusion, fission and remodeling of cells and present in many human diseases. There is however little knowledge, if any, about their lipid composition and dynamics. We therefore isolated nuclear envelope lipids from human kidney cells, analyzed their composition and determined the membrane dynamics after resuspension in buffer. The hNM lipid extract was composed of a complex mixture of phospholipids, with high amounts of phosphatidylcholines, phosphatidylinositols (PI) and cholesterol. hNM dynamics was determined by solid-state NMR and revealed that the lamellar gel-to-fluid phase transition occurs below 0 °C, reflecting the presence of elevated amounts of unsaturated fatty acid chains. Fluidity was higher than the plasma membrane, illustrating the dual action of Cholesterol (ordering) and PI lipids (disordering). The most striking result was the large magnetic field-induced membrane deformation allowing to determine the membrane bending elasticity, a property related to hydrodynamics of cells and organelles. Human Nuclear Lipid Membranes were at least two orders of magnitude more elastic than the classical plasma membrane suggesting a physical explanation for the formation of nuclear membrane invaginations.