Fig. 2

Structure, dynamics and assembly of native ex vivo L. hesperus major ampullate spidroins (MaSps). A Schematic organization of MaSps, the prevailing amino acid motifs of the repetitive core domain, and their corresponding secondary structure. n = 20–100. B ¹H/¹⁵N HSQC NMR spectrum of isotopically enriched MaSps illustrates the disordered, unstructured nature of the repetitive core regions, as indicated by the poor chemical shift dispersion ( < 0.5 ppm) in the ¹H dimension. Reprinted from [22] with permission from Elsevier. C NMR relaxation measurements show that the spidroins exhibit rapid subnanosecond backbone dynamics in the major ampullate gland. The NOE values are negative at 500 MHz (black) and moderately positive at 800 MHz (red), which is consistent with this interpretation. Reprinted from [22] with permission from Elsevier. D NMR diffusion measurements of spidroins within intact MA glands show restricted diffusion. The diffusion coefficient as a function of the interpulse delay (∆) shows exponential decay, indicating a diffusion restriction length scale of ~250–300 nm. This finding supports the silk protein micelle theory, which states that silk proteins are clustered in micelle-like preassemblies in the gland environment. Adapted from [23]. E ¹H/¹⁵N HSQC NMR spectrum of isotopically enriched MaSp incubated in 4 M urea to disrupt micelles showing chemical shift perturbations for residues that are associated with micelle stabilization. The largest perturbations are observed for the amino acid motifs GAA and AAG (circled), while poly-A shows small perturbations, and the changes in GGQ are negligible. Adapted with permission from [24]. Copyright 2021 American Chemical Society. F CryoEM tomography imaging reveals that silk protein micelles are best described as complex micellar nanoparticles composed of assembled subdomains with an open core occupied by solvent. Adapted from [23]. G Significant chemical shift perturbations in ¹³C occur for alanine Cα and Cβ sites in the GAA and AAG motifs, which terminate the poly-A runs that bridge the GGX regions. This is consistent with the disruption of hydrophobic interactions as micelles disassemble in the denaturant. Adapted with permission from [24]. Copyright 2021 American Chemical Society