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.

Spider silk is one of nature's most remarkable biomaterials due to extraordinary strength and toughness not found in today's synthetic materials. Of the seven types of silk, wrapping silk (AcSp1) is the most extensible of the types of silks and has no sequence similarity to the other types. Here we report the chemical shifts for the AcSp1 199 amino acid protein repeat unit and its anticipated secondary structure based on secondary chemical shifts.

Biomolecular NMR spectroscopy frequently employs estimates of protein secondary structure using secondary chemical shift (Deltadelta) values, measured as the difference between experimental and random coil chemical shifts (RCCS). Most published random coil data have been determined in aqueous conditions, reasonable for non-membrane proteins, but potentially less relevant for membrane proteins. Two new RCCS sets are presented here, determined in dimethyl sulfoxide (DMSO) and chloroform:methanol:water (4:4:1 by volume) at 298 K. A web-based program, CS-CHEMeleon, has been implemented to determine the accuracy of secondary structure assessment by calculating and comparing Deltadelta values for various RCCS datasets. Using CS-CHEMeleon, Deltadelta predicted versus experimentally determined secondary structures were compared for large datasets of membrane and non-membrane proteins as a function of RCCS dataset, Deltadelta threshold, nucleus, localized parameter averaging and secondary structure type. Optimized Deltadelta thresholds are presented both for published and for the DMSO and chloroform:methanol:water derived RCCS tables. Despite obvious RCCS variations between datasets, prediction of secondary structure was consistently similar. Strikingly, predictive accuracy seems to be most dependent upon the type of secondary structure, with helices being the most accurately predicted by Deltadelta values using five different RCCS tables. We suggest caution when using Deltadelta-based restraints in structure calculations as the underlying dataset may be biased. Comparative assessment of multiple RCCS datasets should be performed, and resulting Deltadelta-based restraints weighted appropriately relative to other experimental restraints.

Conclusion:This type of analysis can be used to non-invasively study changes in immune cell recruitment in individual mice over time, potentially allowing improved application and combination of immunotherapies.3

a b s t r a c tArtificial spider silk proteins may form fibers with exceptional strength and elasticity. Wrapping silk, or aciniform silk, is the toughest of the spider silks, and has a very different protein composition than other spider silks. Here, we present the characterization of an aciniform protein (AcSp1) subunit named W 1 , consisting of one AcSp1 199 residue repeat unit from Argiope trifasciata. The structural integrity of recombinant W 1 is demonstrated in a variety of buffer conditions and time points. Furthermore, we show that W 1 has a high thermal stability with reversible denaturation at $71°C and forms self-assembled nanoparticle in near-physiological conditions. W 1 therefore represents a highly stable and structurally robust module for protein-based nanoparticle formation.