Structural Characterization of Black Widow Spider Dragline Silk Proteins CRP1 and CRP4.

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.

KScons: a Bayesian approach for protein residue contact prediction using the knob-socket model of protein tertiary structure.

MOTIVATION: By simplifying the many-bodied complexity of residue packing into patterns of simple pairwise secondary structure interactions between a single knob residue with a three-residue socket, the knob-socket construct allows a more direct incorporation of structural information into the prediction of residue contacts. By modeling the preferences between the amino acid composition of a socket and knob, we undertake an investigation of the knob-socket construct's ability to improve the prediction of residue contacts. The statistical model considers three priors and two posterior estimations to better understand how the input data affects predictions. This produces six implementations of KScons that are tested on three sets: PSICOV, CASP10 and CASP11. We compare against the current leading contact prediction methods. RESULTS: The results demonstrate the usefulness as well as the limits of knob-socket based structural modeling of protein contacts. The construct is able to extract good predictions from known structural homologs, while its performance degrades when no homologs exist. Among our six implementations, KScons MST-MP (which uses the multiple structure alignment prior and marginal posterior incorporating structural homolog information) performs the best in all three prediction sets. An analysis of recall and precision finds that KScons MST-MP improves accuracy not only by improving identification of true positives, but also by decreasing the number of false positives. Over the CASP10 and CASP11 sets, KScons MST-MP performs better than the leading methods using only evolutionary coupling data, but not quite as well as the supervised learning methods of MetaPSICOV and CoinDCA-NN that incorporate a large set of structural features. CONTACT: qiwei.li@rice.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Dragline silk: a fiber assembled with low-molecular-weight cysteine-rich proteins.

Dragline silk has been proposed to contain two main protein constituents, MaSp1 and MaSp2. However, the mechanical properties of synthetic spider silks spun from recombinant MaSp1 and MaSp2 proteins have yet to approach natural fibers, implying the natural spinning dope is missing critical factors. Here we report the discovery of novel molecular constituents within the spinning dope that are extruded into dragline silk. Protein studies of the liquid spinning dope from the major ampullate gland, coupled with the analysis of dragline silk fibers using mass spectrometry, demonstrate the presence of a new family of low-molecular-weight cysteine-rich proteins (CRPs) that colocalize with the MA fibroins. Expression of the CRP family members is linked to dragline silk production, specifically MaSp1 and MaSp2 mRNA synthesis. Biochemical data support that CRP molecules are secreted into the spinning dope and assembled into macromolecular complexes via disulfide bond linkages. Sequence analysis supports that CRP molecules share similarities to members that belong to the cystine slipknot superfamily, suggesting that these factors may have evolved to increase fiber toughness by serving as molecular hubs that dissipate large amounts of energy under stress. Collectively, our findings provide molecular details about the components of dragline silk, providing new insight that will advance materials development of synthetic spider silk for industrial applications.