Michael B. Hinman

Synthetic spider silk: a modular fiber

Spiders make their webs and perform a wide range of tasks with up to seven different types of silk fiber. These different fibers allow a comparison of structure with function, because each silk has distinct mechanical properties and is composed of peptide modules that confer those properties. By using genetic engineering to mix the modules in specific proportions, proteins with defined strength and elasticity can be designed, which have many potential medical and engineering uses.

Inducing β-Sheets Formation in Synthetic Spider Silk Fibers by Aqueous Post-Spin Stretching

As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce man-made fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spidroin 1 (MaSp1) consensus repeat sequence were expressed and spun into fibers. Mechanical test results showed that fiber spun from the higher molecular weight protein had better overall mechanical properties (70 KD versus 46 KD), whereas postspin stretch treatment in water helped increase fiber tensile strength significantly. Carbon-13 solid-state NMR studies of those fibers further revealed that the postspin stretch in water promoted protein molecule rearrangement and the formation of β-sheets in the polyalanine region of the silk. The rearrangement correlated with improved fiber mechanical properties and indicated that postspin stretch is key to helping the spider silk proteins in the fiber form correct secondary structures, leading to better quality fibers.

Nephila clavipes Flagelliform silk-like GGX motifs contribute to extensibility and spacer motifs contribute to strength in synthetic spider silk fibers.

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.

Distinct contributions of model MaSp1 and MaSp2 like peptides to the mechanical properties of synthetic major ampullate silk fibers as revealed in silico

All characterized major ampullate silks from orb-web weaving spiders are composites of primarily two different proteins: MaSp1 and MaSp2. The conserved association of MaSp1 and MaSp2 in these spider species, the highly conserved amino acid motifs, and variable ratios of MaSp1 to MaSp2 demonstrate the importance of both MaSp1 and MaSp2 to the strength and elasticity of the fiber. Computer simulated mechanical tests predicted differing roles for MaSp1 and MaSp2 in the mechanical properties of the fibers. Recombinant MaSp1 and MaSp2 proteins were blended and spun into fibers mimicking the computer-simulated conditions. Mechanical testing verified the differing roles of MaSp1 and MaSp2.

Modular Spider Silk Fibers: Defining New Modules and Optimizing Fiber Properties

Orb-web weaving spiders use multiple silk fibers to accomplish different tasks, combining repetitive peptide modules to produce different properties in each fiber. Each fiber is the product of a distinct gland, but is subject to a common spinning paradigm to produce an insoluble fiber from an aqueous-soluble protein dope. We start by presenting the cloning of the last of the six silks used by Nephila clavipes, the piriform silk spidroin. This piriform fiber presents a unique set of protein modules, which are used to attach other silk fibers to surfaces and to each other. Fiber spinning studies using major ampullate, minor ampullate, and flagelliform modules responsible for distinct secondary structures and therefore fiber properties will be presented. The properties of various synthetic fibers such as the initial (Young’s) modulus, tensile strength at break, strain at break, and toughness will be presented for a N. clavipes flagelliform/major ampullate hybrid synthetic fiber series, and an Argiope aurantia flagelliform/major ampullate hybrid synthetic fiber. Then, an N. clavipes major ampullate protein 1 synthetic fiber will be compared to itself in terms of how the fiber reacts to a post-spin draw in terms of properties and secondary structure. Finally, two flagelliform/major ampullate hybrid fibers made from slightly different elastic modules will be compared to show how minor changes in a single peptide module can change artificial spinning parameters substantially. Post-spin draw regimens on each fiber will demonstrate the importance of such procedures in optimizing fiber properties to take advantage of the modular protein sequences. Secondary structure studies at different stages of spinning will demonstrate the recruitment of secondary structures that greatly influence fiber properties.