Two Paenibacillus spp. strains promote grapevine wood degradation by the fungus Fomitiporia mediterranea: from degradation experiments to genome analyses.

Ascomycetes, basidiomycetes and deuteromycetes can degrade wood, but less attention has been paid to basidiomycetes involved in Esca, a major Grapevine Trunk Disease. Using a wood sawdust microcosm system, we compared the wood degradation of three grapevine cultivars inoculated with Fomitiporia mediterranea M. Fisch, a basidiomycete responsible for white-rot development and involved in Esca disease. The grapevine cultivar Ugni blanc was more susceptible to wood degradation caused by F. mediterranea than the cultivars Cabernet Sauvignon and Merlot. Solid-state Nuclear Magnetic Resonance (NMR) spectroscopy showed that F. mediterranea preferentially degrades lignin and hemicellulose over cellulose (preferential, successive or sequential white-rot). In addition, co-inoculation of sawdust with two cellulolytic and xylanolytic bacterial strains of Paenibacillus (Nakamura) Ash (Paenibacillus sp. (S231-2) and P. amylolyticus (S293)), enhanced F. mediterranea ability to degrade Ugni blanc. The NMR data further showed that the increase in Ugni blanc sawdust degradation products was greater when bacteria and fungi were inoculated together. We also demonstrated that these two bacterial strains could degrade the wood components of Ugni blanc sawdust. Genome analysis of these bacterial strains revealed numerous genes predicted to be involved in cellulose, hemicellulose, and lignin degradation, as well as several other genes related to bacteria-fungi interactions and endophytism inside the plant. The occurrence of this type of bacteria-fungus interaction could explain, at least in part, why necrosis develops extensively in certain grapevine varieties such as Ugni blanc.

Integrating Mechanics and Bioactivity: A Detailed Assessment of Elasticity and Viscoelasticity at Different Scales in 2D Biofunctionalized PEGDA Hydrogels for Targeted Bone Regeneration.

Methods for promoting and controlling the differentiation of human mesenchymal stem cells (hMSCs) in vitro before in vivo transplantation are crucial for the advancement of tissue engineering and regenerative medicine. In this study, we developed poly(ethylene glycol) diacrylate (PEGDA) hydrogels with tunable mechanical properties, including elasticity and viscoelasticity, coupled with bioactivity achieved through the immobilization of a mixture of RGD and a mimetic peptide of the BMP-2 protein. Despite the key relevance of hydrogel mechanical properties for cell culture, a standard for its characterization has not been proposed, and comparisons between studies are challenging due to the different techniques employed. Here, a comprehensive approach was employed to characterize the elasticity and viscoelasticity of these hydrogels, integrating compression testing, rheology, and atomic force microscopy (AFM) microindentation. Distinct mechanical behaviors were observed across different PEGDA compositions, and some consistent trends across multiple techniques were identified. Using a photoactivated cross-linker, we controlled the functionalization density independently of the mechanical properties. X-ray photoelectrin spectroscopy and fluorescence microscopy were employed to evaluate the functionalization density of the materials before the culturing of hMSCs on them. The cells cultured on all functionalized hydrogels expressed an early osteoblast marker (Runx2) after 2 weeks, even in the absence of a differentiation-inducing medium compared to our controls. Additionally, after only 1 week of culture with osteogenic differentiation medium, cells showed accelerated differentiation, with clear morphological differences observed among cells in the different conditions. Notably, cells on stiff but stress-relaxing hydrogels exhibited an overexpression of the osteocyte marker E11. This suggests that the combination of the functionalization procedure with the mechanical properties of the hydrogel provides a potent approach to promoting the osteogenic differentiation of hMSCs.

Magic-angle spinning NMR spectral editing of polysaccharides in whole cells using the DREAM scheme.

Most bacterial, plant and fungal cells possess at their surface a protective layer called the cell wall, conferring strength, plasticity and rigidity to withstand the osmotic pressure. This molecular barrier is crucial for pathogenic microorganisms, as it protects the cell from the local environment and often constitutes the first structural component encountered in the host-pathogen interaction. In pathogenic molds and yeasts, the cell wall constitutes the main target for the development of clinically-relevant antifungal drugs. In the past decade, solid-state NMR has emerged as a powerful analytical technique to investigate the molecular organization of microbial cell walls in the context of intact cells. 13C NMR chemical shift is an exquisite source of information to identify the polysaccharides present in the cell wall, and two-dimensional 13C-13C correlation experiments provide an efficient tool to rapidly access the polysaccharide composition in whole cells. Here we investigate the use of the adiabatic DREAM (for dipolar recoupling enhancement through amplitude modulation) recoupling scheme to improve solid-state NMR analysis of polysaccharides in intact cells. We demonstrate the advantages of two-dimensional 13C-13C experiments using the DREAM recoupling scheme. We report the spectral editing of polysaccharide signals by varying the radio-frequency carrier position. We provide practical considerations for the implementation of DREAM experiments to characterize polysaccharides in whole cells. We demonstrate the approach on intact fungal cells of Neurospora crassa and Aspergillus fumigatus, a model and a pathogenic filamentous fungus, respectively. The approach could be envisioned to efficiently reduce the spectral crowding of more complex cell surfaces, such as cell wall and peptidoglycan in bacteria.

NMR resonance assignment of the cell death execution domain BELL2 from multicellular bacterial signalosomes.

Signalosomes are high-order protein machineries involved in complex mechanisms controlling regulated immune defense and cell death execution. The immune response is initiated by the recognition of exogeneous or endogenous signals, triggering the signalosome assembly process. The final step of signalosome fate often involves membrane-targeting and activation of pore-forming execution domains, leading to membrane disruption and ultimately cell death. Such cell death-inducing domains have been thoroughly characterized in plants, mammals and fungi, notably for the fungal cell death execution protein domain HeLo. However, little is known on the mechanisms of signalosome-based immune response in bacteria, and the conformation of cell death executors in bacterial signalosomes is still poorly characterized. We recently uncovered the existence of NLR signalosomes in various multicellular bacteria and used genome mining approaches to identify putative cell death executors in Streptomyces olivochromogenes. These proteins contain a C-terminal amyloid domain involved in signal transmission and a N-terminal domain, termed BELL for Bacteria analogous to fungal HeLL (HeLo-like), presumably responsible for membrane-targeting, pore-forming and cell death execution. In the present study, we report the high yield expression of S. olivochromogenes BELL2 and its characterization by solution NMR spectroscopy. BELL is folded in solution and we report backbone and sidechain assignments. We identified five α-helical secondary structure elements and a folded core much smaller than its fungal homolog HeLo. This study constitutes the first step toward the NMR investigation of the full-length protein assembly and its membrane targeting.

A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion.

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.

Analytical Framework to Understand the Origins of Methyl Side-Chain Dynamics in Protein Assemblies.

Side-chain motions play an important role in understanding protein structure, dynamics, protein-protein, and protein-ligand interactions. However, our understanding of protein side-chain dynamics is currently limited by the lack of analytical tools. Here, we present a novel analytical framework employing experimental nuclear magnetic resonance (NMR) relaxation measurements at atomic resolution combined with molecular dynamics (MD) simulation to characterize with a high level of detail the methyl side-chain dynamics in insoluble protein assemblies, using amyloid fibrils formed by the prion HET-s. We use MD simulation to interpret experimental results, where rotameric hops, including methyl group rotation and χ1/χ2 rotations, cannot be completely described with a single correlation time but rather sample a broad distribution of correlation times, resulting from continuously changing local structure in the fibril. Backbone motion similarly samples a broad range of correlation times, from ∼100 ps to µs, although resulting from mostly different dynamic processes; nonetheless, we find that the backbone is not fully decoupled from the side-chain motion, where changes in side-chain dynamics influence backbone motion and vice versa. While the complexity of side-chain motion in protein assemblies makes it very challenging to obtain perfect agreement between experiment and simulation, our analytical framework improves the interpretation of experimental dynamics measurements for complex protein assemblies.

Cryo-EM observation of the amyloid key structure of polymorphic TDP-43 amyloid fibrils.

The transactive response DNA-binding protein-43 (TDP-43) is a multi-facet protein involved in phase separation, RNA-binding, and alternative splicing. In the context of neurodegenerative diseases, abnormal aggregation of TDP-43 has been linked to amyotrophic lateral sclerosis and frontotemporal lobar degeneration through the aggregation of its C-terminal domain. Here, we report a cryo-electron microscopy (cryo-EM)-based structural characterization of TDP-43 fibrils obtained from the full-length protein. We find that the fibrils are polymorphic and contain three different amyloid structures. The structures differ in the number and relative orientation of the protofilaments, although they share a similar fold containing an amyloid key motif. The observed fibril structures differ from previously described conformations of TDP-43 fibrils and help to better understand the structural landscape of the amyloid fibril structures derived from this protein.

Molecular characterization of the N-terminal half of TasA during amyloid-like assembly and its contribution to Bacillus subtilis biofilm formation.

Biofilms are bacterial communities that result from a cell differentiation process leading to the secretion of an extracellular matrix (ECM) by part of the population. In Bacillus subtilis, the main protein component of the ECM is TasA, which forms a fiber-based scaffold that confers structure to the ECM. The N-terminal half of TasA is strongly conserved among Bacillus species and contains a protein domain, the rigid core (RcTasA), which is critical for the structural and functional properties of the recombinant protein. In this study, we demonstrate that recombinantly purified RcTasA in vitro retains biochemical properties previously observed for the entire protein. Further analysis of the RcTasA amino acid sequence revealed two aggregation-prone stretches and a region of imperfect amino acid repeats, which are known to contribute to functional amyloid assembly. Biochemical characterization of these stretches found in RcTasA revealed their amyloid-like capacity in vitro, contributing to the amyloid nature of RcTasA. Moreover, the study of the imperfect amino acid repeats revealed the critical role of residues D64, K68 and D69 in the structural function of TasA. Experiments with versions of TasA carrying the substitutions D64A and K68AD69A demonstrated a partial loss of function of the protein either in the assembly of the ECM or in the stability of the core and amyloid-like properties. Taken together, our findings allow us to better understand the polymerization process of TasA during biofilm formation and provide knowledge into the sequence determinants that promote the molecular behavior of protein filaments in bacteria.

Efficient 18.8 T MAS-DNP NMR reveals hidden side chains in amyloid fibrils.

Amyloid fibrils are large and insoluble protein assemblies composed of a rigid core associated with a cross-ß arrangement rich in ß-sheet structural elements. It has been widely observed in solid-state NMR experiments that semi-rigid protein segments or side chains do not yield easily observable NMR signals at room temperature. The reasons for the missing peaks may be due to the presence of unfavorable dynamics that interfere with NMR experiments, which result in very weak or unobservable NMR signals. Therefore, for amyloid fibrils, semi-rigid and dynamically disordered segments flanking the amyloid core are very challenging to study. Here, we show that high-field dynamic nuclear polarization (DNP), an NMR hyperpolarization technique typically performed at low temperatures, can circumvent this issue because (i) the low-temperature environment (~ 100 K) slows down the protein dynamics to escape unfavorable detection regime, (ii) DNP improves the overall NMR sensitivity including those of flexible side chains, and (iii) efficient cross-effect DNP biradicals (SNAPol-1) optimized for high-field DNP (≥ 18.8 T) are employed to offer high sensitivity and resolution suitable for biomolecular NMR applications. By combining these factors, we have successfully established an impressive enhancement factor of ε ~ 50 on amyloid fibrils using an 18.8 T/ 800 MHz magnet. We have compared the DNP efficiencies of M-TinyPol, NATriPol-3, and SNAPol-1 biradicals on amyloid fibrils. We found that SNAPol-1 (with ε ~ 50) outperformed the other two radicals. The MAS DNP experiments revealed signals of flexible side chains previously inaccessible at conventional room-temperature experiments. These results demonstrate the potential of MAS-DNP NMR as a valuable tool for structural investigations of amyloid fibrils, particularly for side chains and dynamically disordered segments otherwise hidden at room temperature.

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.

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.

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.

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.

The Pga59 cell wall protein is an amyloid forming protein involved in adhesion and biofilm establishment in the pathogenic yeast Candida albicans.

The human commensal fungus Candida albicans can attach to epithelia or indwelling medical devices and form biofilms, that are highly tolerant to antifungal drugs and can evade the immune response. The cell surface protein Pga59 has been shown to influence adhesion and biofilm formation. Here, we present evidence that Pga59 displays amyloid properties. Using electron microscopy, staining with an amyloid fibre-specific dye and X-ray diffraction experiments, we showed that the predicted amyloid-forming region of Pga59 is sufficient to build up an amyloid fibre in vitro and that recombinant Pga59 can also adopt a cross-ß amyloid fibre architecture. Further, mutations impairing Pga59 amyloid assembly led to diminished adhesion to substrates and reduced biofilm production. Immunogold labelling on amyloid structures extracted from C. albicans revealed that Pga59 is used by the fungal cell to assemble amyloids within the cell wall in response to adhesion. Altogether, our results suggest that Pga59 amyloid properties are used by the fungal cell to mediate cell-substrate interactions and biofilm formation.

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.

Evidence for [2Fe-2S]2+ and Linear [3Fe-4S]1+ Clusters in a Unique Family of Glycine/Cysteine-Rich Fe-S Proteins from Megavirinae Giant Viruses.

We have discovered a protein with an amino acid composition exceptionally rich in glycine and cysteine residues in the giant virus mimivirus. This small 6 kDa protein is among the most abundant proteins in the icosahedral 0.75 µm viral particles; it has no predicted function but is probably essential for infection. The aerobically purified red-brownish protein overproduced inEscherichia coli contained both iron and inorganic sulfide. UV/vis, EPR, and Mössbauer studies revealed that the viral protein, coined GciS, accommodated two distinct Fe-S clusters: a diamagnetic S = 0 [2Fe-2S]2+ cluster and a paramagnetic S = 5/2 linear [3Fe-4S]1+ cluster, a geometry rarely stabilized in native proteins. Orthologs of mimivirus GciS were identified within all clades of Megavirinae, a Mimiviridae subfamily infecting Acanthamoeba, including the distantly related tupanviruses, and displayed the same spectroscopic features. Thus, these glycine/cysteine-rich proteins form a new family of viral Fe-S proteins sharing unique Fe-S cluster binding properties.

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.

Structural determinants of REMORIN nanodomain formation in anionic membranes.

Remorins are a family of multigenic plasma membrane phosphoproteins involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nanosized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains; however, the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here, we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated phosphatidylcholine, phosphatidylinositol phosphates, and sterol, we investigate the clustering of remorins to the membrane and monitor the formation of nanosized membrane domains. REM1.3 promoted membrane nanodomain organization on the exposed external leaflet of both spherical lipid vesicles and flat supported lipid bilayers. Our results reveal that REM1.3 drives a mechanism allowing lipid reorganization, leading to the formation of remorin-enriched nanodomains. Phosphorylation of the N-terminal IDD by the calcium protein kinase CPK3 influences this clustering and can lead to the formation of smaller and more disperse domains. Our work reveals the phosphate-dependent involvement of the N-terminal IDD in the remorin-membrane interaction process by driving structural rearrangements at lipid-water interfaces.

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.