Spider silks are renowned for their remarkable mechanical properties. Due to combined high strength and extensibility, spider silk becomes the toughest biomaterial in nature and is superior to most man-made materials [1]. An individual female orb-weaving spider can spin a diverse suite of silk types from seven morphologically differentiated types of silk glands, and each silk type has unique material properties and specific tasks [2,3]. Among these silk types, dragline silk produced from major ampullate glands has received most attention for its extremely high strength and toughness, and can be used for bridgelines and web radii [4,5]. Dragline silk is primarily comprised of at least two major ampullate silk proteins (MaSp1 and MaSp2) that are co-expressed in major ampullate glands [6], [7], [8], [9]. These two spider silk proteins (spidroins) have a similar molecular structure, an enormous repetitive region flanked by relatively short and conserved amino- (N-) and carboxyl- (C-) terminal regions, which are similar to other spidroin types [10,11].

The primary structure of spidroins, especially the central repetitive sequence, largely determines the mechanical properties of spider silk fibers [12,13]. The repetitive regions of MaSp1 and MaSp2 proteins contain short and simple repeated sequence motifs [10]. For MaSp1, the repetitive region is dominated by two repeat motifs, poly-alanine (poly-A) and GGX (X usually represents A, Q or Y) [10]. The ubiquitous poly-A motifs are believed to form hydrophobic crystalline domains containing β-sheet secondary structures that contribute to high strength of the silk fiber [14,15]. However, the hydrophilic GGX motifs form 31-helical structures that are responsible for elasticity [16,17]. In addition to the two motif types as above, MaSp2 also contains a large proportion of GPG motifs that likely form β-turns providing extensibility for dragline silks [10,18,19]. Recently, two new silk proteins were found in major ampullate gland, which were named as MaSp3 and MaSp4, respectively, based on their distinct repetitive sequences [20,21]. These two proteins both lack the poly-A motifs typical of MaSp1 and MaSp2, which could result in distinct mechanical properties from MaSp1 or MaSp2. Moreover, the MaSp4 repetitive region contains abundant GPGPQ motifs that rarely appear in MaSps, which is believed to increase dragline silk extensibility [21]. Prior to the present study, however, only fragmentary MaSp4 sequence was available, being an impediment to investigating its structural and mechanical property as well as biological function [21].

Here, we present the full-length MaSp4 gene sequence from the orb-weaving spider, Aranues ventricosus. We described the organization, composition, and predicted hydrophobicity of MaSp4 and determined its phylogenetic placement in the spidroin gene family. To analyze the features of this new type of major ampullate spidroins, We designed the mini-MaSp4 (NRCM4) for recombinant production in E. coli and characterized the secondary structure and thermal stability of NRCM4 in solution. We also prepared the NRCM4 fibers by using wet-spinning and analyzed their structures and mechanical properties.