Structural conversion of the spidroin C-terminal domain during assembly of spider silk fibers.

The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to ß-sheet transition upon fiber formation and highlight the helix No4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix No4 has the ability of pH-induced conversion into ß-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.

Insights into the material properties of dragline spider silk affecting Schwann cell migration.

Dragline silk of Trichonephila spiders has attracted attention in various applications. One of the most fascinating uses of dragline silk is in nerve regeneration as a luminal filling for nerve guidance conduits. In fact, conduits filled with spider silk can measure up to autologous nerve transplantation, but the reasons behind the success of silk fibers are not yet understood. In this study dragline fibers of Trichonephila edulis were sterilized with ethanol, UV radiation, and autoclaving and the resulting material properties were characterized with regard to the silk's suitability for nerve regeneration. Rat Schwann cells (rSCs) were seeded on these silks in vitro and their migration and proliferation were investigated as an indication for the fiber's ability to support the growth of nerves. It was found that rSCs migrate faster on ethanol treated fibers. To elucidate the reasons behind this behavior, the fiber's morphology, surface chemistry, secondary protein structure, crystallinity, and mechanical properties were studied. The results demonstrate that the synergy of dragline silk's stiffness and its composition has a crucial effect on the migration of rSCs. These findings pave the way towards understanding the response of SCs to silk fibers as well as the targeted production of synthetic alternatives for regenerative medicine applications.

Mesoscale Confinement in Bagworm Silk: A Hidden Structural Organization.

While silk fibers produced by silkworms and spiders are frequently described as a network of amorphous protein chains reinforced by crystalline ß-sheet nanodomains, the importance of higher-order, self-assembled structures has been recognized for advanced modeling of mechanical properties. General acceptance of hierarchical structural models is, however, currently limited by lack of experimental results. Indeed, X-ray scattering studies of spider's dragline-type fibers have been particularly limited by low crystallinities. Here we are reporting on probing the local structure of exceptionally crystalline bagworm silk fibers by X-ray nanobeam scattering. Probing the comparable thickness of cross sections with an X-ray nanobeam allows removing the strong scattering background from the outer sericin layer and reveals a hidden structural organization due to a radial gradient in diameters of mesoscale nanofibrillar bundles in the fibroin phase. Our results provide direct support for lateral interactions between nanofibrils.

Mesoscale Ordering of Phycocyanin Molecules in Langmuir-Blodgett Multilayers.

Phycocyanin molecules, which are part of light-harvesting complexes in cyanobacteria, can be assembled into mesoscale multilayer nanofilms by the Langmuir-Blodgett technique. Results obtained by quartz crystal microbalance and atomic force microscopy confirm the homogeneity and reproducibility of phycocyanin Langmuir-Blodgett multilayer deposition. We show by cryo-electron microdiffraction that amorphous phycocyanin Langmuir-Blodgett multilayers form, after annealing at 150 °C and cooling to room temperature, a layered nanofibrillar lattice with rotational disorder. Scanning X-ray nanodiffraction suggests that structural transformation is not homogeneous through the film but limited to patches of up to about 10 µm diameter.

Size-Exclusion Particle Separation Driven by Micro-Flows in a Quasi-Spherical Droplet: Modelling and Experimental Results.

Aqueous solution droplets are supported quasi contact-free by superhydrophobic surfaces. The convective flow in evaporating droplets allows the manipulation and control of biological molecules in solution. In previous works, super-hydrophobic drops on nano-patterned substrates have been used to analyze otherwise undetectable species in extremely low concentration ranges. Here, we used particle image velocimetry (PIV) for studying the flow field in water droplets containing polystyrene particles on a pillared silicon super-hydrophobic chip. The particles describe vortex-like motions around the droplet center as long as the evaporating droplet maintains a spherical shape. Simulations by a Finite Element Method (FEM) suggest that the recirculating flow is due to the temperature gradient along the droplet rim, generating a shear stress. Notably, the characteristics of the internal flow can be modulated by varying the intensity of the temperature gradient along the drop. We then used the flow-field determined by experiments and an approximate form of the Langevin equation to examine how particles are transported in the drop as a function of particle size. We found that larger particles with an average size of µ36 µm are preferentially transported toward the center of the substrate, differently from smaller particles with a 10-fold lower size that are distributed more uniformly in the drop. Results suggest that solutions of spherical particles on a super-hydrophobic chip can be used to separate soft matter and biological molecules based on their size, similarly to the working principle of a time-of-flight (ToF) mass analyzer, except that the separation takes place in a micro-sphere, with less space, less time, and less solution required for the separation compared to conventional ToF systems.

Mesoscale structures in amorphous silks from a spider's orb-web.

Of the 7-8 silk fibers making up an orb-web only the hierarchical structural organization of semicrystalline radial fibers -composed of major ampullate silk- has been studied in detail, given its fascinating mechanical features. While major ampullate silk's nanofibrillar morphology is well established, knowhow on mesoscale (> 50-100 nm) assembly and its contribution to mechanical performance is limited. Much less is known on the hierarchical structural organization of other, generally less crystalline fibers contributing to an orb-webs' function. Here we show by scanning X-ray micro&nanodiffraction that two fully amorphous, fine silk fibers from the center of an orb-web have different mesoscale features. One of the fibers has a fibrillar composite structure resembling stiff egg case silk. The other fiber has a skin-core structure based on a nanofibrillar ribbon wound around a disordered core. A fraction of nanofibrils appears to have assembled into mesoscale fibrils. This fiber becomes readily attached to the coat of major ampullate silk fibers. We observe that a detached fiber has ripped out the glycoprotein skin-layer containing polyglycine II nanocrystallites. The anchoring of the fiber in the coat suggests that it could serve for strengthening the tension and cohesion of major ampullate silk fibers.

Exploiting Sphingo- and Glycerophospholipid Impairment to Select Effective Drugs and Biomarkers for CMT1A.

In Charcot-Marie-Tooth type 1A (CMT1A), Schwann cells exhibit a preponderant transcriptional deficiency of genes involved in lipid biosynthesis. This perturbed lipid metabolism affects the peripheral nerve physiology and the structure of peripheral myelin. Nevertheless, the identification and functional characterization of the lipid species mainly responsible for CMT1A myelin impairment currently lack. This is critical in the pathogenesis of the neuropathy since lipids are many and complex molecules which play essential roles in the cell, including the structural components of cellular membranes, cell signaling, and membrane trafficking. Moreover, lipids themselves are able to modify gene transcription, thereby affecting the genotype-phenotype correlation of well-defined inherited diseases, including CMT1A. Here we report for the first time a comprehensive lipid profiling in experimental and human CMT1A, demonstrating a previously unknown specific alteration of sphingolipid (SP) and glycerophospholipid (GP) metabolism. Notably, SP, and GP changes even emerge in biological fluids of CMT1A rat and human patients, implying a systemic metabolic dysfunction for these specific lipid classes. Actually, SP and GP are not merely reduced; their expression is instead aberrant, contributing to the ultrastructural abnormalities that we detailed by X-ray diffraction in rat and human internode myelin. The modulation of SP and GP pathways in myelinating dorsal root ganglia cultures clearly sustains this issue. In fact, just selected molecules interacting with these pathways are able to modify the altered geometric parameters of CMT1A myelinated fibers. Overall, we propose to exploit the present SP and GP metabolism impairment to select effective drugs and validate a set of reliable biomarkers, which remain a challenge in CMT1A neuropathy.

Nanoscale Structural Features in Major Ampullate Spider Silk.

Spider major ampullate silk is often schematically represented as a two-phase material composed of crystalline nanodomains in an amorphous matrix. Here we are interested in revealing its more complex nanoscale organization by probing Argiope bruennichi dragline-type fibers using scanning X-ray nanodiffraction. This allows resolving transversal structural features such as an about 1 µm skin layer composed of around 100 nm diameter nanofibrils serving presumably as an elastic sheath. The core consists of a composite of several nm size crystalline nanodomains with poly(l-alanine) microstructure, embedded in a polypeptide network with short-range order. Stacks of nanodomains separated by less ordered nanosegments form nanofibrils with a periodic axial density modulation which is particularly sensitive to radiation damage. The precipitation of larger ß-type nanocrystallites in the outer core-shell is attributed to MaSp1 protein molecules.

Superhydrophobic Surfaces Boost Fibril Self-Assembly of Amyloid ß Peptides.

Amyloid ß (Aß) peptides are the main constituents of Alzheimer's amyloid plaques in the brain. Here we report how the unique microfluidic flows exerted by droplets sitting on superhydrophobic surfaces can influence the aggregation mechanisms of several Aß fragments by boosting their fibril self-assembly. Aß(25-35), Aß(1-40), and Aß(12-28) were dried both on flat hydrophilic surfaces (contact angle (CA) = 37.3°) and on nanostructured superhydrophobic ones (CA = 175.8°). By embedding nanoroughened surfaces on top of highly X-ray transparent Si3N4 membranes, it was possible to probe the solid residues by raster-scan synchrotron radiation X-ray microdiffraction (µXRD). As compared to residues obtained on flat Si3N4 membranes, a general enhancement of fibrillar material was detected for all Aß fragments dried on superhydrophobic surfaces, with a particular emphasis on the shorter ones. Indeed, both Aß(25-35) and Aß(12-28) showed a marked crystalline cross-ß phase with varying fiber textures. The homogeneous evaporation rate provided by these nanostructured supports, and the possibility to use transparent membranes, can open a wide range of in situ X-ray and spectroscopic characterizations of amyloidal peptides involved in neurodegenerative diseases and for the fabrication of amyloid-based nanodevices.

Methodological challenges of optical tweezers-based X-ray fluorescence imaging of biological model organisms at synchrotron facilities.

Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.

Toxicity of eosinophil MBP is repressed by intracellular crystallization and promoted by extracellular aggregation.

Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.

Directed Growth of Virus Nanofilaments on a Superhydrophobic Surface.

The evaporation of single droplets of colloidal tobacco mosaic virus (TMV) nanoparticles on a superhydrophobic surface with a hexagonal pillar-pattern results in the formation of coffee-ring type residues. We imaged surface features by optical, scanning electron, and atomic force microscopies. Bulk features were probed by raster-scan X-ray nanodiffraction. At ∼100 pg/µL nanoparticle concentration, the rim of the residue connects to neighboring pillars via fibrous extensions containing flow-aligned crystalline domains. At ∼1 pg/µL nanoparticle concentration, nanofilaments of ≥80 nm diameter and ∼20 µm length are formed, extending normal to the residue-rim across a range of pillars. X-ray scattering is dominated by the nanofilament form-factor but some evidence for crystallinity has been obtained. The observation of sheets composed of stacks of self-assembled nanoparticles deposited on pillars suggests that the nanofilaments are drawn from a structured droplet interface.

Thermal transformations of self-assembled gold glyconanoparticles probed by combined nanocalorimetry and X-ray nanobeam scattering.

Noble metal nanoparticles with ligand shells are of interest for applications in catalysis, thermo-plasmonics, and others, involving heating processes. To gain insight into the structure-formation processes in such systems, self-assembly of carbohydrate-functionalized gold nanoparticles during precipitation from solution and during further heating to ca. 340 °C was explored by in situ combination of nanobeam SAXS/WAXS and nanocalorimetry. Upon precipitation from solution, X-ray scattering reveals the appearance of small 2D domains of close-packed nanoparticles. During heating, increasing interpenetration of the nanoparticle soft shells in the domains is observed up to ca. 81 °C, followed by cluster formation at ca. 125 °C, which transform into crystalline gold nuclei at around 160 °C. Above ca. 200 °C, one observes the onset of coalescence and grain growth resulting in gold crystallites of average size of about 100 nm. The observed microstructural changes are in agreement with the in situ heat capacity measurements with nanocalorimetry.

A superhydrophobic chip based on SU-8 photoresist pillars suspended on a silicon nitride membrane.

We developed a new generation of superhydrophobic chips optimized for probing ultrasmall sample quantities by X-ray scattering and fluorescence techniques. The chips are based on thin Si3N4 membranes with a tailored pattern of SU-8 photoresist pillars. Indeed, aqueous solution droplets can be evaporated and concentrated at predefined positions using a non-periodic pillar pattern. We demonstrated quantitatively the deposition and aggregation of gold glyconanoparticles from the evaporation of a nanomolar droplet in a small spot by raster X-ray nanofluorescence. Further, raster nanocrystallography of biological objects such as rod-like tobacco mosaic virus nanoparticles reveals crystalline macro-domain formation composed of highly oriented nanorods.

Myelin organization in the nodal, paranodal, and juxtaparanodal regions revealed by scanning x-ray microdiffraction.

X-ray diffraction has provided extensive information about the arrangement of lipids and proteins in multilamellar myelin. This information has been limited to the abundant inter-nodal regions of the sheath because these regions dominate the scattering when x-ray beams of 100 µm diameter or more are used. Here, we used a 1 µm beam, raster-scanned across a single nerve fiber, to obtain detailed information about the molecular architecture in the nodal, paranodal, and juxtaparanodal regions. Orientation of the lamellar membrane stacks and membrane periodicity varied spatially. In the juxtaparanode-internode, 198-202 Å-period membrane arrays oriented normal to the nerve fiber axis predominated, whereas in the paranode-node, 205-208 Å-period arrays oriented along the fiber direction predominated. In parts of the sheath distal to the node, multiple sets of lamellar reflections were observed at angles to one another, suggesting that the myelin multilayers are deformed at the Schmidt-Lanterman incisures. The calculated electron density of myelin in the different regions exhibited membrane bilayer profiles with varied electron densities at the polar head groups, likely due to different amounts of major myelin proteins (P0 glycoprotein and myelin basic protein). Scattering from the center of the nerve fibers, where the x-rays are incident en face (perpendicular) to the membrane planes, provided information about the lateral distribution of protein. By underscoring the heterogeneity of membrane packing, microdiffraction analysis suggests a powerful new strategy for understanding the underlying molecular foundation of a broad spectrum of myelinopathies dependent on local specializations of myelin structure in both the PNS and CNS.

Virus particle assembly into crystalline domains enabled by the coffee ring effect.

Tobacco mosaic virus particles can be rapidly assembled into 3D-domains by capillary flow-driven alignment at the triple contact-line of an evaporating droplet. Virus particles of ∼150 Šdiameter can be resolved within individual domains at the outer rim of the "coffee-ring" type residue by atomic force microscopy. The crystalline domains can also be probed by X-ray microdiffraction techniques. Both techniques reveal that the rod-like virus particles are oriented parallel to the rim. We further demonstrate the feasibility of collection of hk0 reflection intensities in GISAXS geometry and show it allows calculating a low-resolution electron density projection along the rod axis.

Probing droplets on superhydrophobic surfaces by synchrotron radiation scattering techniques.

Droplets on artificially structured superhydrophobic surfaces represent quasi contact-free sample environments which can be probed by X-ray microbeams and nanobeams in the absence of obstructing walls. This review will discuss basic surface wettability concepts and introduce the technology of structuring surfaces. Quasi contact-free droplets are compared with contact-free droplets; processes related to deposition and evaporation on solid surfaces are discussed. Droplet coalescence based on the electrowetting effect allows the probing of short-time mixing and reaction processes. The review will show for several materials of biological interest that structural processes related to conformational changes, nucleation and assembly during droplet evaporation can be spatially and temporally resolved by raster-scan diffraction techniques. Orientational ordering of anisotropic materials deposited during solidification at pinning sites facilitates the interpretation of structural data.

Amyloid ß peptide conformational changes in the presence of a lipid membrane system.

Here we are presenting a comparative analysis of conformational changes of two amyloid ß peptides, Aß(25-35) and Aß(1-42), in the presence and absence of a phospholipid system, namely, POPC/POPS (1-palmitoyl-2-oleoylphospatidylcholine/palmitoyl-2-oleoylphospatidylserine), through Raman spectroscopy, synchrotron radiation micro Fourier-transform infrared spectroscopy, and micro X-ray diffraction. Ringlike samples were obtained from the evaporation of pure and mixed solutions of the proteins together with the POPC/POPS system on highly hydrophilic substrates. The results confirm the presence of a α-helical to ß-sheet transition from the internal rim of the ringlike samples to the external one in the pure Aß(25-35) residual, probably due to the convective flow inside the droplets sitting on highly hydrophilic substrates enhancing the local concentration of the peptide at the external edge of the dried drop. In contrast, the presence of POPC/POPS lipids in the peptide does not result in α-helical structures and introduces the presence of antiparallel ß-sheet material together with parallel ß-sheet structures and possible ß-turns. As a control, Aß(1-42) peptide was also tested and shows ß-sheet conformations independently from the presence of the lipid system. The µXRD analysis further confirmed these conclusions, showing how the absence of the phospholipid system induces in the Aß(25-35) a probable composite α/ß material while its coexistence with the peptide leads to a not oriented ß-sheet conformation. These results open interesting scenarios on the study of conformational changes of Aß peptides and could help, with further investigations, to better clarify the role of enzymes and alternative lipid systems involved in the amyloidosis process of Aß fragments.

Superhydrophobic surfaces allow probing of exosome self organization using X-ray scattering.

Drops of exosome dispersions from healthy epithelial colon cell line and colorectal cancer cells were dried on a superhydrophobic PMMA substrate. The residues were studied by small- and wide-angle X-ray scattering using both a synchrotron radiation micrometric beam and a high-flux table-top X-ray source. Structural differences between healthy and cancerous cells were detected in the lamellar lattices of the exosome macro-aggregates.

Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis.

The self-assembly of abnormally folded proteins into amyloid fibrils is a hallmark of many debilitating diseases, from Alzheimer's and Parkinson diseases to prion-related disorders and diabetes type II. However, the fundamental mechanism of amyloid aggregation remains poorly understood. Core sequences of four to seven amino acids within natural amyloid proteins that form toxic fibrils have been used to study amyloidogenesis. We recently reported a class of systematically designed ultrasmall peptides that self-assemble in water into cross-ß-type fibers. Here we compare the self-assembly of these peptides with natural core sequences. These include core segments from Alzheimer's amyloid-ß, human amylin, and calcitonin. We analyzed the self-assembly process using circular dichroism, electron microscopy, X-ray diffraction, rheology, and molecular dynamics simulations. We found that the designed aliphatic peptides exhibited a similar self-assembly mechanism to several natural sequences, with formation of α-helical intermediates being a common feature. Interestingly, the self-assembly of a second core sequence from amyloid-ß, containing the diphenylalanine motif, was distinctly different from all other examined sequences. The diphenylalanine-containing sequence formed ß-sheet aggregates without going through the α-helical intermediate step, giving a unique fiber-diffraction pattern and simulation structure. Based on these results, we propose a simplified aliphatic model system to study amyloidosis. Our results provide vital insight into the nature of early intermediates formed and suggest that aromatic interactions are not as important in amyloid formation as previously postulated. This information is necessary for developing therapeutic drugs that inhibit and control amyloid formation.