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Spider silk proteome provides insight into the structural characterization of Nephila clavipes flagelliform spidroin.

ABSTRACT: The capture spiral of web from N. clavipes spider consists of a single type of spidroin - the flagelliform silk protein, a natural material representing a combination of strength and high elasticity. Flagelliform spider silk is the most extensible silk fibre produced by orb weaver spiders and the structure of this remarkable material is still largely unknown. In the present study we used a proteomic approach to elucidate the complete sequence and the post-translational modifications of flagelliform silk proteins. The long sequence of flagelliform silk protein presents 45 hydroxylated proline residues, which may contribute to explain the mechanoelastic property of these fibres, since they are located in the GPGGX motif. The 3D-structure of the protein was modelled considering the three domains together, i.e., the N- and C-terminal non-repetitive domains, and the central repetitive domain. In the resulting molecular model there is a predominance of random structures in the solid fibres of the silk protein. The N-terminal domain is composed of three ?-helices and the C-terminal domain is composed of one small helical section. Proteomic data reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based spider polymers.

SUBMITTER: Dos Santos-Pinto JRA

PROVIDER: S-EPMC6168590 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Spider silk proteome provides insight into the structural characterization of Nephila clavipes flagelliform spidroin.

Dos Santos-Pinto José Roberto Aparecido JRA Arcuri Helen Andrade HA Esteves Franciele Grego FG Palma Mario Sergio MS Lubec Gert G

Scientific reports 20181002 1

The capture spiral of web from N. clavipes spider consists of a single type of spidroin - the flagelliform silk protein, a natural material representing a combination of strength and high elasticity. Flagelliform spider silk is the most extensible silk fibre produced by orb weaver spiders and the structure of this remarkable material is still largely unknown. In the present study we used a proteomic approach to elucidate the complete sequence and the post-translational modifications of flagellif ...[more]

PMID: 30279551

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Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

Project description:BACKGROUND: Spidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers. Due to the highly variable nature of their repetitive sequences, spidroin evolutionary relationships have principally been determined from their non-repetitive carboxy (C)-terminal domains, though they offer limited character data. The few known spidroin amino (N)-terminal domains have been difficult to obtain, but potentially contain critical phylogenetic information for reconstructing the diversification of spider silks. Here we used silk gland expression data (ESTs) from highly divergent species to evaluate the functional significance and phylogenetic utility of spidroin N-terminal domains. RESULTS: We report 11 additional spidroin N-termini found by sequencing approximately 1,900 silk gland cDNAs from nine spider species that shared a common ancestor > 240 million years ago. In contrast to their hyper-variable repetitive regions, spidroin N-terminal domains have retained striking similarities in sequence identity, predicted secondary structure, and hydrophobicity. Through separate and combined phylogenetic analyses of N-terminal domains and their corresponding C-termini, we find that combined analysis produces the most resolved trees and that N-termini contribute more support and less conflict than the C-termini. These analyses show that paralogs largely group by silk gland type, except for the major ampullate spidroins. Moreover, spidroin structural motifs associated with superior tensile strength arose early in the history of this gene family, whereas a motif conferring greater extensibility convergently evolved in two distantly related paralogs. CONCLUSIONS: A non-repetitive N-terminal domain appears to be a universal attribute of spidroin proteins, likely retained from the origin of spider silk production. Since this time, spidroin N-termini have maintained several features, consistent with this domain playing a key role in silk assembly. Phylogenetic analyses of the conserved N- and C-terminal domains illustrate dramatic radiation of the spidroin gene family, involving extensive duplications, shifts in expression patterns and extreme diversification of repetitive structural sequences that endow spider silks with an unparalleled range of mechanical properties.

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Spider silk proteome provides insight into the structural characterization of Nephila clavipes flagelliform spidroin.

Publications

Spider silk proteome provides insight into the structural characterization of Nephila clavipes flagelliform spidroin.

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