Spider dragline silk represents a biomaterial with outstanding mechanical properties, possessing high-tensile strength and toughness. In black widows at least eight different proteins have been identified as constituents of dragline silk. These represent major ampullate spidroins MaSp1, MaSp2, MaSp’, and several low-molecular weight cysteine-rich protein (CRP) family members, including CRP1, CRP2, and CRP4. Molecular modeling predicts that CRPs contain a cystine slipknot motif, but experimental evidence to support this assertion remains to be reported. To advance scientific knowledge regarding CRP function, we recombinantly expressed and purified CRP1 and CRP4 from bacteria and investigated their secondary structure using circular dichroism (CD) under different chemical and physical conditions. We demonstrate by far-UV CD spectroscopy that these proteins contain similar secondary structure, having substantial amounts of random coil conformation, followed by lower levels of beta sheet, alpha helical and beta turn structures. CRPs are thermally and pH stable; however, treatment with reagents that disrupt disulfide bonds impact their structural conformations. Cross-linking mass spectrometry (XL-MS) data also support computational models of CRP1. Taken together, the chemical and thermal stability of CRPs, the cross-linking data, coupled with the structural sensitivity to reducing agents, are experimentally consistent with the supposition CRPs are cystine slipknot proteins.

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for forcedependent accessibility of structure that makes use of a wireless Mini-Radio Centrifuge Force Microscope (MR.CFM) to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules were used to determine how the triple helix responds to force. As the primary load-and tension-bearing protein in vertebrates, the physical properties of collagen are of significant biomedical interest. How collagen's triple helix responds to applied force is controversial, with different studies inferring incompatible outcomes: overwinding, unwinding, shearing, or maintaining its zero-force structure. Because proteolytic cleavage requires a locally unwound triple helix, our experiments reveal how local collagen structure changes in response to applied force. Our first results show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix. The generality of this result will be discussed in the context of collagen's sequence heterogeneity.

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