Nephila clavipes Flagelliform Silk-Like GGX Motifs Contribute to Extensibility and Spacer Motifs Contribute to Strength in Synthetic Spider Silk Fibers

Abstract

Flagelliform spider silk is the most extensible silk fiber produced by orb weaver spiders, though not as strong as the dragline silk of the spider. The motifs found in the core of the Nephila clavipes flagelliform Flag protein are: GGX, spacer, and GPGGX. Flag does not contain the polyalanine motif known to provide the strength of dragline silk. To investigate the source of flagelliform fiber strength, four recombinant proteins were produced containing variations of the three core motifs of the Nephila clavipes flagelliform Flag protein that produces this type of fiber. The as-spun fibers were processed in 80% aqueous isopropanol using a standardized process for all four fiber types, which produced improved mechanical properties. Mechanical testing of the recombinant proteins determined that the GGX motif contributes extensibility and the spacer motif contributes strength to the recombinant fibers. Recombinant protein fibers containing the spacer motif were stronger than the proteins constructed without the spacer that contained only the GGX motif or the combination of the GGX and GPGGX motifs. The mechanical and structural X-ray diffraction analysis of the recombinant fibers provide data that suggests a functional role of the spacer motif that produces tensile strength though the spacer motif is not clearly defined structurally. These results indicate that the spacer is likely a primary contributor of strength with the GGX motif supplying mobility to the protein network of native N. clavipes flagelliform silk fibers.

The Nephila clavipes genome highlights the diversity of spider silk genes and their complex expression

“…In total, >32 million features are annotated on the N. clavipes draft genome (Supplementary Table 8), which was achieved using (i) our transcriptome from all isolates, (ii) results from two gene prediction algorithms, (iii) libraries of transposable elements and repeated motifs, and (iv) coding sequences from related species 18,19 (Supplementary Table 8). Using gene modeling, we conservatively predict 14,025 genes present in the N. clavipes genome; 2,023 gene models transcribe >1 alternative spliceoform, resulting in 3,937 additional transcripts for a total of 17,962 mRNAs in the final gene set (Supplementary Table 9).…”

“…The silk classes of a typical 'gluey silk' orb-weaver (Araneoidea) female include (i) major ampullate silk, which exhibits great tensile strength and is employed in draglines, bridgelines, and web radii 11,12 ; (ii) minor ampullate silk, used for inelastic temporary spirals during web building 11,12 ; (iii) cement-like piriform silk that bonds fibers together and to other substrates 13,14 ; (iv) strong, yet flexible aciniform silk used for prey wrapping and egg case insulation 15 ; (v) tubuliform and cylindriform silk that constitutes the tough outer layer of egg cases 16,17 ; (vi) flagelliform silk that exhibits unparalleled extensibility and is used in the capture spiral 18,19 ; and (vii) the viscous and sticky aggregate silk that aids in prey capture [20][21][22][23][24] . Many spider species produce just a subset of these silk classes, and some produce yet other silk types, including cribellate silk 25 .…”

Identification of Wet-Spinning and Post-Spin Stretching Methods Amenable to Recombinant Spider Aciniform Silk

“…Wet-spinning requires a viscous solution 9,42 ; however, there is a fine balance between sufficient viscosity and an overly viscous state that cannot be extruded through the spinner. After trial and error, it was found that 5% w/v W 3 being insufficiently viscous and 10% w/v W 3 being sufficiently viscous to allow for wet-spinning but difficult to routinely prepare and employ, an optimum dope composition of 8% w/v W 3 in 70% HFIP/30% H 2 O was established.…”

“…Most studies characterizing post-spin stretching of silk-based fibers have reported carrying out this step manually with the use of forceps or tweezers 9,11,12,42 , with some reports of using controlled post-spin stretching apparatuses (e.g. 16,44 ).…”

Crystal Structure of the Nephila clavipes Major Ampullate Spidroin 1A N-terminal Domain Reveals Plasticity at the Dimer Interface

“…Dragline silk, one of the strongest types of silk, is produced in the major ampullate gland and is used as the web frame and lifeline for the spider (5). The proteins that form the core of dragline silk are termed major ampullate spidroins (MaSp), 2 and they have three main domains: a long, flexible, highly repetitive central domain known as the repeat region flanked by smaller, globular, non-repetitive N-terminal and C-terminal domains (NTD and CTD, respectively) (6 -8).…”