Structural Diversity of Native Major Ampullate, Minor Ampullate, Cylindriform, and Flagelliform Silk Proteins in Solution

Abstract

The foundations of silk spinning, the structure, storage, and activation of silk proteins, remain highly debated. By combining solution small-angle neutron and X-ray scattering (SANS and SAXS) alongside circular dichroism (CD), we reveal a shape anisotropy of the four principal native spider silk feedstocks from Nephila edulis . We show that these proteins behave in solution like elongated semiflexible polymers with locally rigid sections. We demonstrated that minor ampullate and cylindriform proteins adopt a monomeric conformation, while major ampullate and flagelliform proteins have a preference for dimerization. From an evolutionary perspective, we propose that such dimerization arose to help the processing of disordered silk proteins. Collectively, our results provide insights into the molecular-scale processing of silk, uncovering a degree of evolutionary convergence in protein structures and chemistry that supports the macroscale micellar/pseudo liquid crystalline spinning mechanisms proposed by the community.

Molecular Dynamics of Synthetic Flagelliform Silk Fiber Assembly

“…A recent study has shown that native spider silk proteins also presented specific secondary structure profiles in solution. [ 42 ] According with the authors, Flag proteins were partially folded and presented β‐turn conformational structures. Their results suggest that spider silk proteins in general behave in solution like elongated semiflexible polymers with locally rigid sections, agreeing with the multiple conformational structure found for rNcFlag2222.…”

“…Other aspects such as pH, ionic conditions, fiber spinning speed, and the presence of His tag may also have influenced the spinning process and the success of fiber assembly. [ 5,42,53,54 ]…”

“…rNcFlag2222 is composed by the elastic repetitive motifs (GPGGX/GGX) and the spacer region present in native N. cuentata Flag silk. Similar to natural spider silks, rNcFlag2222 behaved in solution like elongated semiflexible polymers with rigid sections, [ 42 ] characterized mostly by the presence of helical structures, β‐turn, and β‐sheet structural conformations.…”

“…Other aspects such as pH, ionic conditions, fiber spinning speed, and the presence of His tag may also have influenced the spinning process and the success of fiber assembly. [5,42,53,54] According to Raman spectromicroscopy and WAXS structural analyses, the synthetic rNcFlag2222 spun fiber is a mixture of secondary structures, mainly composed of helices (α-helices/PPII helices), β-turns, and stacked β-sheets. These data concur with the CD spectra obtained for rNcFlag2222 diluted in different solvents.…”

Seeking Solvation: Exploring the Role of Protein Hydration in Silk Gelation

“…Based on various nuclear magnetic resonance (NMR) methods, Asakura and co-workers [28,[46][47][48] suggested that 'the highly concentrated silk solution contained in the middle silk gland has residues in energetically favored conformations close to average random coil values, but forms a hydrogen-bonded network that keeps it in a repeated type II β-turn structure'. Other studies have also demonstrated predominantly random coil, helical or β-turn conformations in various native, recombinant or redissolved spidroin (spider silk protein) solutions [49][50][51][52][53][54][55][56][57].…”