Natural silk proteins

Despite the diversity of silk proteins from different organisms and silk types, these proteins contain common patterns. Generally, silkworm silk protein (fibroin) and spider silk protein (spidroin) comprise a highly repetitive core domain that contains alternating crystalline and amorphous regions strongly influencing the mechanical properties of the spun fibers.^[11,93,94] Alanine-rich stretches, such as An or (GA)n, build up β-sheets, which are stacked into crystallites and are responsible for the high strength of the silk fiber. In contrast, glycine-rich amino acid motifs, such as (GGX)n (X = tyrosine, glutamine, leucine), fold into 3₁-helices, β-turns and β-spirals (Figure 3).^[95,96] These glycine-rich stretches serve as an amorphous matrix for the crystallites and thus provide elasticity and flexibility to the fiber. Non-repetitive and highly conserved amino- and carboxy-terminal domains flank the repetitive core domain.^[22,28,97] and play an important role during protein storage at high concentrations and in triggering protein assembly (see chapter 1.4).^[98-104]

1.2.1. Fibroin

As mentioned above, fibroin consist of 3 different components: heavy chain, light chain and a glycoprotein called P25. Whereas H- and L-chain are linked together by a disulfide bond at the carboxy-terminus of both proteins,[24,105,106] P25 is non-covalently linked through hydrophobic interactions.^[107] P25 is assumed to act as a chaperone and aids during the transport and secretion of the highly insoluble fibroin H-chain,^[24,107] which is considered to determine the mechanical properties of the silk fibers. H-chain mainly consists of non-polar and hydrophobic glycine (45.9 %) and alanine (30.3 %) residues.^[108,109] The much smaller fibroin L-chain on the other hand exhibits a more hydrophilic nature, due to a lower content of alanine (14 %) and glycine (9 %) residues.^[110]

Two crystalline polymorphs are usually distinguished for fibroin: Silk I and Silk II.^[111-114]

Whereas Silk I refers to the dissolved, metastable form during storage in the silk glands, Silk II relates to the solid fibroin detected in spun silk fibers.^[115] While the detailed structure of Silk I is not fully understood and was described as lacking secondary structure,

INTRODUCTION

or being partially disordered,^[115-117] Silk II closely resembles the structure of spider silk spidroins. Similar to spider silk, the crystalline domains of silkworm fibers are also based on antiparallel β-sheets and the amorphous regions are made up of β-turns and –loops.

However, in contrast to spidroins, the anti-parallel β-sheets of fibroin do not consist of poly-alanine stretches, but of (GX)n-repeats, where X predominately represents alanine, serine, tyrosine, valine or threonine residues.^[108,118] Even though the crystalline domains constitute a higher total volume (40-50 %) of silkworm silk fibers compared to spider silk fibers (30-40 %), these domains are to a greater extent aligned in parallel to the fiber axis in spider silk fibers, emphasizing the influence of the alignment on the mechanical properties of the fiber.

1.2.2. Spidroin

One repeat of the repetitive core domain comprises 40-200 amino acids and theses amino acid motifs are repeated up to 100 times. Figure 3 shows an overview of structural motifs found in the different silk types of A. diadematus and N. clavipes.

Figure 3: Structural motifs of various spider silk proteins from A. diadematus and N. clavipes. X: predominantly tyrosine, leucine, glutamine, alanine, and serine. aa = amino acid (taken from ^[14] by courtesy of the publisher Elsevier).

These glycine-rich stretches serve as an amorphous matrix for the crystallites and thus provide elasticity and flexibility to the fiber. Contrary to the large repetitive core domain,

INTRODUCTION

the terminal domains only consist of 100-150 amino acids and they are folded into α-helical secondary structures, which are arranged in five helix bundles.[83,119-121] Apart from enabling spidroin storage at high concentrations, the terminal domains play an important role in triggering spidroin assembly (see chapter 1.4.2).^[98-104]

MI silk comprises two spidroins, MiSp1 and MiSp2, which have a molecular weight of approx. 250 kDa. Even though MI silk has similar mechanical properties as MA silk, their composition differs greatly. MI spidroins of N. clavipes contain almost no proline residues and their glutamic acid content is significantly reduced.^[122] Similar to MA spidroins, the repetitive region of MI spidroins fold into crystalline and amorphous structures. Even though MI silk possesses a high tensile strength, NMR studies showed that only a small fraction of alanine residues take part in its β-sheet crystals. In contrast to MA silk, the crystallites in MI silk contain a significant amount of glycine, since the length of repeated alanine residues is as low as three and these regions are always flanked by GA-blocks.

Whereas MiSp1 mainly consists of alternating GGXGGY (X = glutamine or alanine) and (GA)_y(A)_z (y = 3-6 and z = 2-5) motifs, the repeat unit of MiSp2 comprises alternating (GGX)_n (X = tyrosine, glutamine, alanine; n = 1-3) and GAGA motifs.^[123] The repetitive regions of the spidroin, which include the glycine-alanine crystalline β-sheet stacks, separated by amorphous α-helical GGX domains, alternate with 137 amino acid-long non-repetitive serine-rich spacer regions. In MA silk, the additional hydrophobic interactions of the polyalanine-blocks (An) may account for the high tensile strength.^[123] Due to its high glycine content, the strength of MI silk cannot solely be due to its β-sheet structures, and Dicko et al.^[66] assumed that cross-linking combined with specific matrix properties different to those of MA spidroins have an impact on the high strength of MI silk.

FTIR-measurements during stretching of MA and MI silk fibers showed significant differences in the structural behavior of the two silks. While the β-sheets in MA silk remained mostly unchanged, the disordered regions decreased and coiled structures became visible.^[123] Conversely, in MI silk no conformational changes of the amorphous structures were visible, and only the changes of the β-sheet crystals were observed prior to breaking of the fiber. It is assumed that the GGX and spacer regions in MI silk cannot reversibly withstand the same axial tension as the β-turns in MA silk can.^[123] The minor ampullate spidroin from A. diadematus ADF1 is similar to the MiSps of N. clavipes. This 174 amino acid-long protein also comprises two repeating domains, namely (GA)y(A)z and GGYGQGY. However, compared to MiSps, tyrosines and glutamines in ADF1 are not as highly conserved, and the length of each repeat varies.^[123] The gene sequence of the

INTRODUCTION

carboxy-terminal domain of ADF1 on the other hand has a high sequence conservation with the N. clavipes MiSp gene and it even consists of a possible non-repetitive spacer region at the 5’ end of the gene, which is different to that of MiSps, but one stretch is identical coding for 13 amino acids.^[123]

In contrast to MA and MI silk, flagelliform silk is mainly composed of one 500 kDa protein, which contains more proline and valine and less alanine residues than the other two silk types.^[122] Contrary to other silk proteins, X-ray diffraction measurements showed no crystalline fraction in flagelliform silk, which is attributed to the lack of β-sheet-building structures such as polyalanine or (GA)n-sequences.^[124] Flagelliform proteins of N. clavipes mostly contain (GGX)n and (GPGGX)2 motifs (X = serine or tyrosine), which build 31-helices and β-turn spirals, and are responsible for the high elasticity and flexibility of this silk.[68,73,125] More than 40 adjacent linked β-turns form spring-like spirals, presumably adding to the extraordinary extensibility (> 200 %) of the fiber.^[126]

Major and minor ampullate spidroins, as well as flagelliform spidroin, all consist of one or more of four amino acid motifs, A_n, (GA)_n, (GGX)_n and GPGX_n, in different compositions and arrangements.^[127] Furthermore, these types of silks are all involved in prey capture, and their functions are dependent on their outstanding mechanical properties. At the same time, the accessory role of pyriform, tubuliform and aciniform silks is also reflected in the composition of the respective protein. Since they hardly contain any of the typical amino acid motifs found in MaSps, MiSps and flag,^[40,89] these motifs appear not to be crucial for silks not involved in prey capture.^[89] Nevertheless, pyriform, aciniform and tubuliform proteins all contain highly-conserved carboxy-terminal domains.

The protein component of the small diameter fibers found in attachment disc silk is called pyriform spidroin (PySp). PySp1 (pyriform spidroin of L. hesperus), apart from other spidroins, does not contain conventional subrepeat modules and it lacks glycine and proline residues within its repeat units.^[81] Additionally, instead of long poly-alanine stretches, PySp1 only contains 3 consecutive alanine residues in a regular pattern.^[81] Other aspects, setting pyriform spidroins apart from the spider silk protein family, are their high degree of polar and charged amino acid residues, as well as a high glutamine content.^[81,82]

These features are suspected to have evolved due to PySps distinctive feature of being spun into an aqueous matrix of the attachment discs.^[81] Additionally, high glutamine content may aid protein aggregation which is necessary for a rapid solidification of the attachment discs.^[82,128]

INTRODUCTION

Tubuliform spidroin (TuSp) is the major component of tubuliform silk, which the spider uses to build the eggcase. Similar to PySps, the serine-rich and glycine-poor TuSps does not contain any of the typical amino acid motifs found in MaSps, MiSps and flag.^[40,89]. Instead, TuSp comprises a series of new amino acid motifs such as Sn, (SA)n, (SQ)n and GX (X = glutamine, asparagine, isoleucine, leucine, alanine, valine, tyrosine, phenylalanine and aspartic acid).^[89] The high content of large-side-chain amino acid likely accounts for twisted crystalline structures found during transmission electron microscopy (TEM) studies,^[129] which may explain the lower stiffness of tubuliform silk fibers compared to minor ampullate silk fibers.^[89,130]

Similar to TuSps, poly-alanine and glycine-alanine stretches are also not present in AcSp1, the spidroin constituting aciniform silk fibers of the banded garden spider Argiope trifasciata.^[62] In contrast to other spidroins, AcSp1 consists of over 200 amino acid long, complex repeats, which are virtually identical to each other.^[62,131] Even the most common subrepeat, poly-serine, only accounts for 8.5 % of the repeat unit, and the amino acid motif TGPSG only occurs twice in one AcSp1 repeat unit.^[62] Despite a similar alternation between hydrophobic and hydrophilic regions in AcSp1 compared to MaSps, the amino acid composition of AcSp1 is much more evenly distributed than in MaSps.^[131]