Packing Structure of Antiparallel β-Sheet Polyalanine Region in a Sequential Model Peptide of Nephila clavipes Dragline Silk Studied Using ¹³C Solid-State NMR and MD Simulation

Abstract

Packing structures of polyalanine regions, which are considered to be the reason for the extremely high strength of spider dragline silks, were studied using a series of sequential peptides: (Glu) 4 GlyGlyLeuGlyGlyGlnGlyAlaGly(Ala) n GlyGlyAlaGly-GlnGlyGlyTyrGlyGly(Glu) 4 (n = 3−8) using 13 C solid-state NMR spectroscopy. The conformations of (Ala) n in the freeze-dried peptides changed gradually with increasing n from random coils to α-helices with partial antiparallel β-sheet (AP-β) structures. Conversely, all the insolubilized peptides, n = 6−8 after low-pH treatment and n = 4−8 after formic acid/methanol treatment, formed AP-β structures with significant amounts of staggered packing arrangements. These results are different from previously obtained results for pure alanine oligopeptides, that is, AP-β (Ala) n formed rectangular packing for less than n = 6 but staggered packings for n ≥ 7. The 13 C-labeled peptides were also used to confirm the staggered packing arrangements from NMR dynamics. Furthermore, a MD simulation supported the observed results.

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

“…In addition, the staggered packing arrangement was preserved even after the annealing and replica exchange molecular dynamics (REMD) simulations, suggesting that staggered packing is a reasonably stable structure [63]. The 13 C CP/MAS NMR spectra of a series of AP-β sheet poly-A samples were reported by Lee and Ramamoorthy [98] and Wildman et al [99].…”

“…The underlined sequence, a, is the selected sequence to study the conformations of amino acid residues in the Gly-rich region sandwiched by poly-A sequence on both sides reported previously [46,60,62]. The underlined sequence, b, is selected to study the conformation and packing structure of poly-A sequence sandwiched by partial Gly-rich sequences [63].…”

“…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]…”

“…On the other hand, molecular dynamics (MD) simulation is very effective to understand the local conformation and dynamics of Nephila clavipes (N. clavipes) dragline silks theoretically and to verify the experimental results [56,58,62,63,65]. MD simulation can play a critical role in connecting experimental restraints with potentially plausible molecular structures.…”

“…Therefore, it has been also used to understand the structure and structural changes of silk fibroins including silk fiber formation mechanism and the chain dynamics [73][74][75][76][77][78][79][80][81][82][83][84][85]. Especially, combination of solid-state NMR and MD simulations was very strong analytical tools to understand the local conformation and dynamics of the spider dragline silk in atomic resolution [56,58,62,63,65].…”