X-ray nanodiffraction probes nanofibrillar assembly in spider silk

Major ampullate silk, which forms the radial threads and frame of orb-webs, combines high strength and toughness – attributes that have inspired extensive efforts to reproduce its properties via artificial spinning of recombinant silk proteins [1]. These mechanical properties are thought to arise from a hierarchical structure in which the two ~300 kDa core spidroin proteins, MaSp1 and MaSp2, assemble into nanofibrillar morphologies. However, the precise spatial organization of these nanofibrils, and its relationship to mechanical function, remains incompletely understood.

To address this, scanning X-ray nanodiffraction was used to map nanofibrillar organization in radial threads of Argiope bruennichi (Figure 1a). Beamline ID13, equipped with a focused ~ 180 nm X-ray beam, enabled scanning of the small-diameter (~ 5 µm) fibres at high spatial resolution (Figure 1b). Scans were conducted in 500 nm steps, allowing detailed analysis of the weak scattering signals characteristic of these radiation-sensitive biological fibres.
 

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Fig. 1: a) Argiope bruennichi spider in the hub of its orb-web, showing load-bearing radial threads composed of paired dragline fibres (inset: zoomed view), as well as sticky capture spiral and web-decorating silk (stabilimentum). b) Representative X-ray nanodiffraction pattern from a single radial thread. c) Optical image of a bent radial fiber. The two constituent fibres are superimposed due to bending. The overlaid density map from a nanodiffraction mesh scan highlights the intensity distribution of the (020)/(210) Bragg reflections, characteristic of the poly(L-alanine) crystalline domains. 

This approach builds on previous nanodiffraction studies of silks, including the detection of meso-scale (> 50 nm) nanofibrillar bundles in the silk of the bagworm moth [2], where thin sectioning was required to reduce surface protein interference. To avoid potential artefacts from sample preparation, the present study examined mechanically bent silk fibres (Figure 1c), a configuration known to promote nanofibrillar self-assembly at interfaces.

Nanodiffraction revealed the formation of meso-scale nanofibrillar bundles at both the outer and inner interfaces of the radial silk fibre core (Figure 2). This dual-interface bundling morphology suggests a structural strategy to limit defect propagation, potentially enhancing fracture toughness. These observations are consistent with previous spidroin-specific imaging studies, which showed an inhomogeneous radial distribution of MaSp1 and MaSp2 proteins [3], and support a two core-shell model for silk structure. In particular, the presence of crystalline nanofibril assembly at the inner core interface implies direct molecular interactions between MaSp1 and MaSp2 (Figure 2b).

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Fig. 2: a) Schematic cross-section of a radial fibre, indicating outer and inner core–surface interfaces. The model illustrates a proposed hexagonal packing of nanofibrils at the inner interface, composed of a central cylindrical nanofibril (~ 4 nm radius, red) surrounded by six peripheral fibrils (blue), forming a 7-mer cluster. Central and peripheral fibrils are tentatively assigned to MaSp2 and MaSp1, respectively. b) Experimental scattering pattern from the inner core interface (marked “x”), fitted with Gaussian profiles (blue lines). Solid blue arrows indicate peaks consistent with hexagonal lattice periodicities; open arrows denote additional interference features. The red curve represents a simulated SAXS pattern for a cylindrical nanofibrillar bundle with a radius of 33.5 nm. 

These structural insights provide experimental support for a molecular model predicting that mesoscale confinement of nanofibrils is essential for achieving optimal mechanical performance [4]. They also align with evolutionary hypotheses suggesting that the combination of MaSp1 and MaSp2 spidroins – rather than MaSp1 alone – confers enhanced strength, extensibility, and toughness [5].

The study demonstrates that scanning nanodiffraction can detect spatial variations in nanofibrillar assembly at submicron resolution without destructive sample preparation. This capability is particularly valuable for investigating the hierarchical architecture of complex biomaterials. The findings highlight the role of nanofibrillar morphology in determining spider silk performance and may guide the development of artificial high-performance silks based on recombinant spidroin proteins.

 
Principal publication and authors
Meso-Scale Nanofibrillar Organization in Spider’s Orb-Web Radial Fibers, C. Riekel & T.A. Grünewald, Adv. Funct. Mat. 35, 3, 2419631 (2024); https://doi.org/10.1002/adfm.202419631
 

References
[1] R. Fan et al., Adv. Funct. Mater. 35, 2410415 (2025).
[2] T. Yoshioka et al., Nano Lett. 23, 757-764 (2023).
[3] A. Sponner et al., Nat. Mater. 4, 772-775 (2005).
[4] T. Giesa et al., Nano Lett. 11, 5038-5046 (2011).
[5] T.A. Blackledge et al., Sci. Rep. 2, 782 (2012).