Spiders store spidroins in their silk glands as high concentration aqueous solutions, spinning these dopes into fibres with outstanding mechanical properties. Aciniform (or wrapping) silk is the toughest spider silk and is devoid of the short amino acid sequence motifs characteristic of the other spidroins. Using solution-state NMR spectroscopy, we demonstrate that the 200 amino acid Argiope trifasciata AcSp1 repeat unit contrasts with previously characterized spidroins, adopting a globular 5-helix bundle flanked by intrinsically disordered N- and C-terminal tails. Split-intein-mediated segmental NMR-active isotope-enrichment allowed unambiguous demonstration of modular and malleable “beads-on-a-string” concatemeric behaviour. Concatemers form fibres upon manual drawing with silk-like morphology and mechanical properties, alongside secondary structuring and orientation consistent with native AcSp1 fibres. AcSp1 structural stability varies locally, with the fifth helix denaturing most readily. The structural transition of aciniform spidroin from a mostly α-helical dope to a mixed α-helix/β-sheet-containing fibre can be directly related to spidroin architecture and stability.

To better understand the effect of mechanical stress during the spinning of silk, the protein orientation and conformation of Bombyx mori regenerated silk fibroin (RSF) films have been studied as a function of deformation in a static mode or in real time by tensile-Raman experiments and polarization modulation infrared linear dichroism (PM-IRLD), respectively. The data show that either for step-by-step or continuous stretching, elongation induces the progressive formation of β-sheets that align along the drawing axis, in particular above a draw ratio of 2. The formation of β-sheets begins before their alignment during a continuous drawing. Unordered chains were, however, never found to be oriented, which explains the very low level of orientation of the amorphous phase of the natural fiber. Stress-perturbed unordered chains readily convert into β-sheets, the strain-induced transformation following a two-state process. The final level of orientation and β-sheet content are lower than those found in the native fiber, indicating that various parameters have to be optimized in order to implement a spinning process as efficient as the natural one. Finally, during the stress relaxation period in a step-by-step drawing, there is essentially no change of the content and orientation of the β-sheets, suggesting that only unordered structures tend to reorganize.

The major ampullate (MA) silk of spider is known to be composed of oriented beta-sheet nanocrystals dispersed within an amorphous matrix. The presence of an interphase has also been proposed, but it has not been reported for the fibroin of the silkworm Bombyx mori (B. mori). To obtain quantitative information regarding this third phase, the deuteration of B. mori silk and Nephila clavipes MA silk has been probed by attenuated total reflection infrared spectroscopy. The spectral decomposition of the amide II region has allowed determination of the level of orientation and content of the different secondary structures. The data reveal that, in addition to the amorphous domains, part of the beta-sheets is deuterated upon immersion in D2O for both silks. The D2O-inaccessible beta-sheets are associated with crystallites, while the interphase is composed of D2O-accessible ones. It is found that the former beta-sheets are slightly more oriented along the fiber axis than the latter ones, which suggests that the interphase beta-sheets are located at both ends of the crystals. The total beta-sheet content is similar for B. mori silk (50 + 4%) and MA silk (46 + 4%). However, 27 + 3% of the beta-sheets of MA silk are D2O-accessible compared to 8 +/- 3% for B. mori silk. These data suggest that around 5 amino acids for B. mori silk and 9 amino acids for MA silk would be involved in the interphase beta-sheets. The higher amount of interphase beta-sheets for MA silk is believed to contribute to its higher toughness.

The synthesis of four amphiphilic thieno[3,4-c]pyrrole-4,6-dione (TPD)-based alternating copolymers and their behavior at the air-water interface are reported. Homogeneous and stable monolayers of TPD-based copolymers were prepared. Brewster angle microscopy (BAM) was utilized to characterize the morphology and topography of these Langmuir films, UV-vis absorption spectroscopy as well as atomic force microscopy has revealed a regular transfer of some copolymers on glass substrates. It was possible to obtain homogeneous Langmuir-Blodgett films of up to 30 layers. Infrared dichroic measurements revealed an edge-on orientation. These Langmuir-Blodgett films made of conjugated polymers are therefore good candidates for organic field-effect transistors (OFETs).

In this study, we demonstrate the use of eco-friendly native spider silk as an efficient optical fiber in air, highly bent fibers, and physiological liquid. We also integrated the silk filament in a photonic chip made of polymer microstructures fabricated by UV lithography. The molding process is non-destructive for silk and leads to an efficient micro-optical coupling between silk and synthetic optical structures. These optical performances combined with the unique biocompatibility, bioresorbability, flexibility, and tensile strength of silk filaments pave the way for new applications in biological media and for original biophotonic purposes.

The kinetics of adsorption of solubilized spider major ampullate (MA) silk fibers at the air-water interface and the molecular structure and mechanical properties of the interfacial films formed have been studied using various physical techniques. The data show that Nephila clavipes MA proteins progressively adsorb at the interface and ultimately form a highly cohesive thin film. In situ infrared spectroscopy shows that as soon as they reach the interface the proteins predominantly form β sheets. The protein secondary structure does not change significantly as the film grows, and the amount of β sheet is the same as that of the natural fiber. This suggests that the final β-sheet content is mainly dictated by the primary structure and not by the underlying formation process. The measure of the shear elastic constant at low strain reveals a very strong, viscous, cohesive assembly. The β sheets seem to form cross-links dispersed within an intermolecular network, thus probably playing a major role in the film strength. More importantly, the molecular weight seems to be a crucial factor because interfacial films made from the natural proteins are ~7 times stronger and ~3 times more viscous than those obtained previously with shorter recombinant proteins. Brewster angle microscopy at the air-water interface and transmission electron microscopy of transferred films have revealed a homogeneous organization on the micrometer scale. The images suggest that the structural assembly at the air-water interface leads to the formation of macroscopically solid and highly cohesive networks. Overall, the results suggest that natural spider silk proteins, although sharing similarities with recombinant proteins, have the particular ability to self-assemble into ordered materials with exceptional mechanical properties.

Summary form only given. Silk fibers produced by nature are among the most interesting materials. Besides their high degree of biocompatibility, they are bioresorbable, ecologically friendly, and offer excellent mechanical properties. In particular, silks of spider and worm have aroused a huge interest as seen by the large literature on their structural organization [1, 2]. Thanks to material engineering possibility [3, 4], regenerated worm silk has been intensively studied and used in different fields such as electronics and photonics [5, 6]. On the other hand, native silks fibers are utilized in textile applications and in surgery procedures but have not yet deserved attention for photonic applications. However, the exceptional mechanical properties of natural silk mainly originate from an elaborated hierarchical organization which has not yet been reproduced in regenerated silk.We demonstrate the promising optical fiber behavior of pristine dragline silk from the spider Nephila clavipes, as well as its integration in a photonic chip made of synthetic polymer microstructures patterned by UV-lithography. The spinning process of the native fiber leads to a diameter of 5 microns, homogeneous over long distances. Since silk manipulation is straightforward, optical propagation in straight and loop configurations has been validated. In free space, attenuation coefficient (propagation losses) and coupling losses were estimated by the cut-back method on straight fiber at 10.5 dB/cm and 10 dB, respectively.In addition, the integration of the pristine silk fiber on a photonic chip and working optical hybrid devices based on synthetic polymer and natural silk fibers were demonstrated. Fig. 1a) is a scanning electron microscope of a chip composed of native spider silk embedded in SU8 microstructures (disk-reservoirs MD1 and MD2) designed by UV-lithography7. During light injection in a SU8 waveguide connected to MD1, light propagates to MD1 where light is confined at the edge- of the disk. Fig. 1b is a micro-beam profile of the chip during light injection and shows that light is confined along the silk fiber and at the edges of MD2. This confirms the optical coupling between synthetic polymer microstructures and silk fiber. These results highlight the potential role of this unique material for biophotonic applications either in free space or in integrated chip for which light propagation and/or sensing in biocompatible media is needed.

Propagation properties of pristine spider dragline silk are presented. The 5 μm-diameter fibers have been also integrated with polymer microstructures leading to successful optical coupling. These results pave the way for biophotonics applications.

Structural data relative to spider silk fibers such as the flagelliform (Flag) silk are fundamental to understand the origin of their outstanding mechanical properties. However, due to its small diameter and limited availability, experimental data relative to the structure of the Flag silk are almost nonexistent. Raman spectromicroscopy is one of the rare techniques that can provide structural information about this type of silk. We have thus used this technique to characterize Flag silk fibers spun by three orb-weaving spiders in their native state. The polarized spectra reveal that Flag fibers spun by Araneus diadematus and Argiope aurantia spiders unexpectedly contain beta-sheets and exhibit a certain level of molecular orientation. In contrast, the Flag silk from Nephila clavipes possesses very few beta-sheets and a low molecular orientation, the proteins being essentially disordered. These observations account for the higher strength and lower extensibility of Araneus diadematus and Argiope aurantia Flag fibers compared with Nephila clavipes. The analysis of the primary structure strongly suggests that the "spacer" motifs of the Flag silk are more prominent and have a higher beta-sheet propensity for spider species that spin fibers containing beta-sheets. These results nicely support the Raman data and provide the first hypothesis regarding the role played by these spacers in the structure of Flag silk. The structural data obtained provide a molecular basis for the tensile properties of these fibers showing that overall, considering the different types of spider silk, the beta-sheet appears to be a universal structural element used by nature to provide strength to fibers that exhibit dissimilar tensile properties.

Studies at the liquid-air interface: a powerful toolbox to study interactions between biomolecules

We have been studying the behavior of several globular food proteins at the air-solution interface and their capacity to stabilize aqueous foams. Our aim is to understand the relation of foaming ability to the properties of the adsorbed protein layer, and to understand the relation of the properties of the adsorbed protein layer to the structural and physicochemical characteristics of the adsorbed protein(s). We used a combination of techniques providing insight in the adsorption kinetics (ellipsometry, surface tension), in the interfacial shear rheology and in the molecular conformational rearrangements of adsorbed proteins (polarization-modulation infrared reflection-absorption spectroscopy, PM-IRRAS). We compared proteins with distinctive molecular features, native and chemically or physically modified forms of one protein, and studied the interfacial behavior of proteins in binary solutions. We found that depending on the physicochemical adsorption conditions, different proteins form qualitatively different interfacial layer, as regards monolayer or multilayer adsorption, or the interfacial shear rheology. Strikingly, very minor structural modifications prior to adsorption to the air-solution interface could lead to dramatic changes in the interfacial behavior, in parallel to dramatic changes in the foaming ability. We also showed that in the case of binary solutions of oppositely charged proteins, a clear co-adsorption occurs, leading to very high surface concentrations and to deeply modified interfacial film properties, which cannot be extrapolated from the addition of individual behaviors. In addition, preliminary results about the osmotic pressure of bulk, very highly concentrated protein solutions suggest that in interfacial studies, due to the protein crowding close to the interface, intermolecular interactions could be of prominent importance in the understanding of interfacial properties.

Adsorption of purified apo-ovotransferrin at the air-water interface was studied by ellipsometry, surface tension, polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS), and shear elastic constant measurements. No significant difference was observed between pH 6.5 and 8.0 as regards the final value of surface concentration and surface pressure. However at low concentration, a weak barrier to adsorption is evidenced at pH 6.5 and confirmed by PM-IRRAS measurements. At a pH where the protein net charge is negative (pH 8.0), the behavior of ovotransferrin at the air-water interface is more influenced by charge effects rather than bulk concentration effects. At this pH, the interface exhibits a low shear elastic constant and a spectral signature not usual for globular proteins.

Raman spectroscopy has long been proved to be a useful tool to study the conformation of protein‐based materials such as silk. Thanks to recent developments, linearly polarized Raman spectromicroscopy has appeared very efficient to characterize the molecular structure of native single silk fibers and spinning dopes because it can provide information relative to the protein secondary structure, molecular orientation, and amino acid composition. This review will describe recent advances in the study of the structure of silk by Raman spectromicroscopy. A particular emphasis is put on the spider dragline and silkworm cocoon threads, other fibers spun by orb‐weaving spiders, the spinning dope contained in their silk glands and the effect of mechanical deformation. Taken together, the results of the literature show that Raman spectromicroscopy is particularly efficient to investigate all aspects of silk structure and production. The data provided can lead to a better understanding of the structure of the silk dope, transformations occurring during the spinning process, and structure and mechanical properties of native fibers. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 322–336, 2012.

Monolayers on a Langmuir trough constitute a great biomimetic model to characterize protein-protein or protein-lipid interaction, where the physical state of the interfacial layer is completely controlled. We present here three studies performed on monolayers, with a wide panel of experimental (optical, spectroscopical, rheological) techniques. i) Surface properties and conformation of Nephila clavipes Spider recombinant silk proteins (MaSpI and MaSp2) was studied at the air-water interface: we show that the mechanism of assembly of both proteins is different, although both proteins share the same sequence pattern and a close hydrophobicity. They both exhibit a certain propensity to form b-sheets that may be important for the efficiency of the natural spinning process. ii) The dystrophin molecular organization and its anchoring in a lipidic environment depend on the rod fragment used and on the lipid nature. Moreover the interaction is guided by the lateral surface pressure. This lipid packing variation is essential to understand the role of the dystrophin during compression-extension cycle of the muscle membrane. iii) We evidence that non additive behavior of mixtures of food globular proteins leads to enhanced foaming properties or to self assembled objects.

Spiders that spin orb webs secrete seven types of silk. Although the spinning process of the dragline thread is beginning to be understood, the molecular events that occur in spiders' opisthosomal glands, which produce the other fibers, are unknown due to a lack of data regarding their initial and final structures. Taking advantage of the efficiency of Raman spectromicroscopy in investigating micrometer-sized biological samples, we have determined the secondary structure of proteins in the complete set of glands of the orb-weaving spider Nephila clavipes. The major and minor ampullate silks in the sac of their glands have identical secondary structures typical of natively unfolded proteins. Spidroins are converted into fibers containing highly oriented β-sheets. The capture spiral represents a distinct structural singleton. The proteins are highly disordered prior to spinning and undergo no molecular change or alignment upon spinning. The cylindrical, aciniform, and piriform proteins are folded in their initial state with a predominance of α-helices, but whereas the cylindrical gland forms a fiber similar to the major ampullate thread, the aciniform and piriform glands produce fibers dominated by moderately oriented β-sheets and α-helices. The conformation of the proteins before spinning is related to intrinsic characteristics of their primary structure. Proteins that are unfolded in the gland have repeat sequences composed of submotifs and display no sequence regions with aggregation propensity. By contrast, the folded proteins have neither submotifs nor aggregation-prone sequence regions. Taken together, the Raman data show a remarkable diversity of molecular transformations occurring upon spinning.

Bovine lactoferricin (LfcinB) is an antimicrobial peptide obtained from the pepsin cleavage of lactoferrin. The activity of LfcinB has been extensively studied on diverse pathogens, but its mechanism of action still has to be elucidated. Because of its nonspecificity, its mode of action is assumed to be related to interactions with membranes. In this study, the interaction of LfcinB with a negatively charged monolayer of dipalmitoylphophatidylglycerol has been investigated as a function of the surface pressure of the lipid film using in situ Brewster angle and polarization modulation infrared reflection absorption spectroscopy and on transferred monolayers by atomic force microscopy and polarized attenuated total reflection in infrared spectroscopy. The data show clearly that LfcinB forms stable films at the air-water interface. They also reveal that the interaction of LfcinB with the lipid monolayer is modulated by the surface pressure. At low surface pressure, LfcinB inserts within the lipid film with its long molecular axis oriented mainly parallel to the acyl chains, while at high surface pressure, LfcinB is adsorbed under the lipid film, the hairpin being preferentially aligned parallel to the plane of the interface. The threshold for which behavior changes is 20 mN/m. At this critical surface pressure. LfcinB interacts with the monolayer to form discoidal lipid-peptide assemblies. This structure may actually represent the mechanism of action of this peptide. The results obtained on monolayers are correlated by fluorescent probe release measurements of dye-containing vesicles made of lipids in different phases and support the important role of the lipid fluidity and packing on the activity of LfcinB.

Due to its unmatched hardness and chemical inertia, diamond offers many advantages over other materials for extreme conditions and routine analysis by attenuated total reflection (ATR) infrared spectroscopy. Its low refractive index can offer up to a 6-fold absorbance increase compared to germanium. Unfortunately, it also results for strong bands in spectral distortions compared to transmission experiments. The aim of this paper is to present a methodological approach to determine quantitatively the degree of the spectral distortions in ATR spectra. This approach requires the determination of the optical constants (refractive index and extinction coefficient) of the investigated sample. As a typical example, the optical constants of the fibroin protein of the silk worm Bombyx mori have been determined from the polarized ATR spectra obtained using both diamond and germanium internal reflection elements. The positions found for the amide I band by germanium and diamond ATR are respectively 6 and 17 cm(-1) lower than the true value dtermined from the k(nu) spectrum, which is calculated to be 1659 cm(-1). To determine quantitatively the effect of relevant parameters such as the film thickness and the protein concentration, various spectral simulations have also been performed. The use of a thinner film probed by light polarized in the plane of incidence and diluting the protein sample can help in obtaining ATR spectra that are closer to their transmittance counterparts. To extend this study to any system, the ATR distortion amplitude has been evaluated using spectral simulations performed for bands of various intensities and widths. From these simulations, a simple empirical relationship has been found to estimate the band shift from the experimental band height and width that could be of practical use for ATR users. This paper shows that the determination of optical constants provides an efficient way to recover the true spectrum shape and band frequencies of distorted ATR spectra.

Silk fibers harvested from the web, cocoon, and prey wrapping of the spider Nephila clavipes have been studied by polarized Raman spectromicroscopy. The technique is efficient to differentiate the various types of silk by probing monofilaments produced by the major ampullate (MA), minor ampullate (MI), cylindriform, flagelliform, and aciniform glands. The spectra show that the MA, MI, and cylindriform silks belong to the same structural class and are composed of highly oriented beta-sheets (35-37%) with other slightly oriented secondary structures. Spectral markers of particular motifs involved in the beta-sheets have been identified. The flagelliform silk represents a second, very peculiar structural class. It displays a heterogeneous disordered conformation without any preferential orientation. Such characteristics certainly play a role in the large extensibility of this silk. The aciniform silk represents a third class of silk dominated by moderately oriented beta-sheets (approximately 30%) and alpha-helices (approximately 24%). Such a structure seems important in explaining the high toughness of this silk.