posted on 2024-02-12, 06:04authored byDongqing Wu, Anamaria Koscic, Sonja Schneider, Romeo C. A. Dubini, Diana C. Rodriguez Camargo, Sabine Schneider, Petra Rovó
Despite the considerable
interest in the recombinant production
of synthetic spider silk fibers that possess mechanical properties
similar to those of native spider silks, such as the cost-effectiveness,
tunability, and scalability realization, is still lacking. To address
this long-standing challenge, we have constructed an artificial spider
silk gene using Golden Gate assembly for the recombinant bacterial
production of dragline-mimicking silk, incorporating all the essential
components: the N-terminal domain, a 33-residue-long major-ampullate-spidroin-inspired
segment repeated 16 times, and the C-terminal domain (N16C). This
designed silk-like protein was successfully expressed in Escherichia coli, purified, and cast into films from
formic acid. We produced uniformly 13C–15N-labeled N16C films and employed solid-state magic-angle spinning
nuclear magnetic resonance (NMR) for characterization. Thus, we could
demonstrate that our bioengineered silk-like protein self-assembles
into a film where, when hydrated, the solvent-exposed layer of the
rigid, β-nanocrystalline polyalanine core undergoes a transition
to an α-helical structure, gaining mobility to the extent that
it fully dissolves in water and transforms into a highly dynamic random
coil. This hydration-induced behavior induces chain dynamics in the
glycine-rich amorphous soft segments on the microsecond time scale,
contributing to the elasticity of the solid material. Our findings
not only reveal the presence of structurally and dynamically distinct
segments within the film’s superstructure but also highlight
the complexity of the self-organization responsible for the exceptional
mechanical properties observed in proteins that mimic dragline silk.