Changes in the Local Structure of Nephila clavipes Dragline Silk Model Peptides upon Trifluoroacetic Acid, Low pH, Freeze-Drying, and Hydration Treatments Studied by ¹³C Solid-State NMR

Abstract

The conformational analysis of spider dragline silks is difficult because of the amorphous character of the silks. In this article, the fractions of several conformations were determined for three 47-mer peptides, (Glu) 4 (Ala) 6 GlyGly 12 Ala 13 Gly 14 GlnGlyGlyTyrGlyGlyLeuGlySerGlnGly 25 Ala 26 Gly 27 -ArgGlyGlyLeuGlyGlyGln-Gly 35 Ala 36 Gly 37 (Ala) 6 (Glu) 4 , with three underlined 13 C-labeled blocks using a 13 C CP/MAS NMR method. The conformations of the 13 C-labeled sites change significantly depending on the location of the labeled blocks when treated with trifluoroacetic acid, low pH, and freeze-drying. The conformations of Ala 36 and Gly 37 residues are strongly influenced by the specific conformation of the (Ala) 6 sequence at the C-terminal side, but those of other residues, Ala 13 and Gly 14 , and Ala 26 and Gly 27 , are basically not influenced by the conformations of (Ala) 6 . Through hydration of the β-sheet peptide, sharp peaks with random coil could be observed depending on the position of the residue, and this result could be interpreted via the change in the Ramachandran map obtained from the molecular dynamics simulation.

Structure and Dynamics of Spider Silk Studied with Solid-State Nuclear Magnetic Resonance and Molecular Dynamics Simulation

“…Instead of this, the use of synthetic peptides with isotopically labelings in specific positions provides a sequential model of the Gly-rich region permitting site-specific structural information. Therefore, the selectively stable-isotope labeled 47-mer peptides flanked by (Ala) 6 at both ends were synthesized as summarized in Table 1 [46,60,62]. The sequence was selected in Figure 3 as underlined sequence, a and poly-Glu blocks were attached at both N-and C-termini to make them water-soluble [94].…”

“…Table 1. 47-mer peptides as the sequential models of the Gly-rich region in Figure 3 as underlined sequence, a flanked by (Ala) 6 at both ends [46,60,62]. (I) The peptides, 2-7 were used for determinations of both the inter-nuclear distances of 15 N and 13 C labeled sites, and the fractions of the local conformations of 13 C labeled Gly residues.…”

“…The unique characteristic of the spider silks, supercontraction, which occurs in the process of hydration [8][9][10], has been the interest of many NMR researchers studying the spider dragline silk [27,30,31,34,35,37,43,45,[47][48][49]53,54,57,[59][60][61]. For example, Figure 10 shows the aliphatic region from fully relaxed 13 C directly detected MAS NMR spectrum of N. clavipes major ampullate silk in the hydrated state reported by Jenkins et al [48].…”

“…The structure and dynamics of N. clavipes dragline silks was also studied by using the selectively stable-isotope labeled 47-mer peptides flanked by (Ala) 6 at both ends listed in Table 1 (II) [60]. Figure 11 is the expanded regions (10−70 ppm) of the 13 C-labeled 47 mer peptides with three 13 37 , respectively in the dry and hydrated states [60].…”

“…Spiders can be fed on an isotopically labeled diet, allowing isotopic labeling of silk within the silk gland: either uniformly or by feeding amino acids labeled at individual positions [27][28][29][30][31][32][34][35][36][37]40,[42][43][44][45][47][48][49][50][53][54][55][56]59,61]. In addition, synthetic peptides with isotopically labelings in specific positions have proved very useful model systems permitting site-specific structural information [33,38,39,41,46,51,52,60,[62][63][64][65]. The conformation-dependent 13 C chemical shift in the 13 C cross polarization and magic angle spinning (CP/MAS) NMR spectra of silks and the model peptides coupled with selective 13 C labeling can be used effectively for determination of local structure at secondary structure level in an amino acid-specific manner [66][67][68][69][70]…”