Structure of Characteristic Sequences in Nephila clavipes Dragline Silk (MaSp1) Studied with 13C Solid State NMR

Abstract

The dragline silk of the golden weaver Nephila clavipes is composed of two proteins, designated spidroin 1 (MaSp1) and spidroin 2 (MaSp2), and has been the focus of numerous recent studies because they are among the strongest natural protein fibers. 1-14The dominant MaSpl protein can be described as a block-copolymer consisting of polyalanine poly(Ala) runs and glycine-rich sections containing relatively high concentrations of amino acids with bulky, hydrophilic side chains. Several solid state NMR studies [3][4][5][6][7][8][9][10][11][12][13] have been applied to clarify the structure for native spider silk fibers. The poly(Ala) region in dragline silk fiber took -sheet structure, which was derived from the 13 C chemical shifts of Ala residues clearly. 4-8However, the structure of the glycine-rich region is not clear because of heterogeneity of the sequences. Thus, it seems difficult to study the precise structure of the native spider silk fiber using solid state NMR and should consider the large structural distributions, especially in the glycine-rich region. 5-9The structural analysis using NMR for the model peptides seems very effective because it is possible to prepare several model peptides with well-defined sequences by reference of the primary structure of MaSp1. However, there are no such researches with appropriate model peptides for the spider silk. In our previous papers, 15-17 the model peptides selected from the primary structure of the silk fibroins from Bombyx mori (B. mori) and Samia cynthia ricini (S. c. ricini) were effectively used to obtain the structural information using several solid state NMR methods. Especially, the primary structure of the silk fibroin from S. c. ricini is the repeated sequences which consist of alternative appearance of poly(Ala) region and glycinerich region, which is similar to the primary structure of the MaSpl protein of spider silk. [17][18][19] In this paper, 49 mer peptide with the sequence, GGLGGQGAGAAAAAAGGAGQGGYGGLGSQG-AGRGGQGAAAAAAGGAGQG selected from the primary structure of the MaSp1 protein of from Nephila clavipes dragline silk and the 13 C isotopelabeled model peptide with the same sequence were synthesized. The structure was studied with 13 C CP/ MAS NMR, especially, the conformation-dependent 13 C chemical shifts. The methods of the three kinds of treatments before NMR observation were changed in order to reproduce the structure before and after spinning of the spider silk. Namely, (1) the peptide was dissolved in trifruoroacetic acid and then dried (TFA treatment), (2) the peptide was dissolved in 9 M LiBr and then dialyzed against water (Dialysis treatment) and (3) the peptide was dissolved in 8 M urea and then precipitated in acetonitrile, and dried (AN treatment). EXPERIMENTALThe following peptides, 1-3, were synthesized by the solid-phase method. Peptide 1; YGGLGSQGAGRG Peptide 2; GGLGGQGAGAAAAAAGGAGQGGYG-GLGSQGAGRGGQGAAAAAAGGAGQG Peptide 3; GGLGGQGAGAAA[3-13 C CP/MAS NMR spectra were acquired on a Chemagnetics CMX-400 spectrometer operating at ...

Secondary Structure Adopted by the Gly-Gly-X Repetitive Regions of Dragline Spider Silk

“…The spacer was also poor in α-helices, with less than 1% of the spacer residues in α-helical conformation. The low abundance of α-helices and β-sheets in the spacer region is in agreement with the NMR results reported above and in the literature for the Gly-Gly-X region [22,23,24,25,26,27,28,29]. …”

“…Based on NMR chemical shifts, it has previously been reported and also indicated in this study that the spacer region is rich in 3 1 -helices with (φ, ψ) values near (−90°, 150°) [22,23,24,25,26,27,28,29]. We indeed saw a large population of secondary structure elements with these dihedral angles in the simulations, but these were classified as β-turns in our MD analysis.…”

“…The results for Ala and Gly are similar to previously reported solid-state NMR studies where components for β-sheet and 3 1 -helix were observed with nearly identical chemical shifts [29]. For the other 13 C labeled X amino acids including Gln and Tyr, the chemical shifts indicate that these amino acids are present in non-β-sheet conformations; however, the shifts do not match α-helical structures and shifts similar to those observed for Ala in a model 3 1 -helix for the most part (see Table 1) [22,32,33,34,35]. Specifically, the observed shifts for Cα and CO sites shift to the β-sheet side of random coil (to lower ppm) and the Cβ sites are close to random coil.…”

“…Previous NMR studies have shown that poly(Gly-Pro-Gly-X-X) found in MaSp2 forms type II β-turn structures [21]. Additionally, solid-state NMR has provided evidence that poly(Gly-Gly-X) found in MaSp1 forms 3 1 -helical structures similar to polyglycine II [22,23,24,25,26,27,28,29]. The poly(Gly-Gly-X) sequence is also found in minor ampullate and flagelliform (capture spiral) silk.…”

Microbially Synthesized Polymeric Amyloid Fiber Promotes β-Nanocrystal Formation and Displays Gigapascal Tensile Strength

“…Spidroin MaSp1 from Nephila clavipes is one of the most extensively characterized dragline spider silk proteins − and has been widely used for recombinant production. ,− The glycine-rich sequence of MaSp1 has a repetitive pattern of (GGX) n . In natural dragline silk fiber, this glycine-rich sequence serves as a flexible region connecting the polyalanine sequences that form a β-sheet in silk fiber. ,− Here, we mimic the design of natural silk protein and use this flexible glycine-rich sequence from MaSp1 to connect 16 β-strand-forming amyloid peptides ,, (Figure S1, Tables S2 and S3), generating a 16-mer polymeric amyloid protein ( i .…”