Structural Characterization of Silk-Based Water-Soluble Peptides (Glu)_n(Ala-Gly-Ser-Gly-Ala-Gly)₄ (n = 4−8) as a Mimic of Bombyx mori Silk Fibroin by ¹³C Solid-State NMR

Abstract

Peptides with a combination of hydrophilic and hydrophobic sequences mimicking the primary structure of Bombyx mori silk fibroin were synthesized and studied in the solid state by NMR using 13C selective labeling coupled with 13C conformation-dependent chemical shifts and 2D solid-state spin-diffusion NMR. The hydrophilic sequence was poly(l-glutamic acid) (E) n , and the hydrophobic one was the consensus sequence of the crystalline fraction of B. mori silk fibroin, (AGSGAG)4. The balance of hydrophilic and hydrophobic characters of the peptide was controlled by changing the relative length, n, of (E) n from 4 to 8. When n = 4 and 5, the structure of the hydrophobic sequence is basically Silk I (the structure of B. mori silk fibroin before spinning in the solid state), and the polyglutamate sequences are random coil. On the other hand, when n = 6−8, the structure of the polyglutamate sequence changes gradually from random coil to β-sheet, and the hydrophobic sequence adopts a mixture of β-sheet and random coil/distorted β-turn forms, although the fraction of the latter form decreases gradually by increasing the number n from 6 to 8. Molecular dynamics and molecular mechanics calculations were also performed to examine the stability of the aggregated domains of the peptides in the solid state. The conformational change of (E)4(AGSGAG)4 was monitored in the solid state by decreasing the pH of the aqueous solution during the sample preparation.

Regeneration of the femoral epicondyle on calcium-binding silk scaffolds developed using transgenic silk fibroin produced by transgenic silkworm

“…The peak assignments have already been reported [29]. The chemical shifts change depending on the secondary structures of the protein in the solid state and, therefore, these will be a marker of the structure.…”

“…These finding focus on poly-L-glutamic acid as the HAP nuclei-forming sites in proteins. In previous studies we have mimicked the self-assembly system in native bone [28,29]. A new strategy for bone repair was advanced by using the sequence [(AGSGAG) 4 E 8 AS] 4 , which combines the excellent mechanical properties of the B. mori silk fibroin repetitive domain (AGSGAG) n and the Ca binding properties of the poly-glutamic acid domain.…”

Insights into the production and characterization of electrospun fibers from regenerated silk fibroin

“…Analyses by CP-MAS 13 C NMR have also been conducted on B. morí silk fibroin, as well as related model peptides [46,[64][65][66][67][68][69], in order to characterize the secondary structure of SF in the worm gland before spinning (Silk I) and in the spun solid fiber (Silk II). We used degummed SF fibers (D) and lyophilized regenerated SF powder (L) as a reference for Silk II and Silk I structures, respectively (Fig.…”

Characterization of a Ca binding-amphipathic silk-like protein and peptide with the sequence (Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4with potential for bone repair

“…In our previous paper, 13 we reported the structural characterization of a family of silk-based water-soluble peptides, (Glu) n (Ala-Gly-Ser-Gly-Ala-Gly) 4 [(E) n (AGSGAG) 4 ] where n ¼ 4-8, using 13 C solid-state NMR. In these peptides, the sequence (AGSGAG) 4 is markedly hydrophobic, while hydrophilic (E) n not found in native silks is used to make the peptides water soluble when n is large enough.…”

“…In these peptides, the sequence (AGSGAG) 4 is markedly hydrophobic, while hydrophilic (E) n not found in native silks is used to make the peptides water soluble when n is large enough. The effect on the local conformation of changing the balance of hydrophilic and hydrophobic domains by varying the number n in the (E) n in these peptides was studied using 13 C solid state NMR. When n ¼ 4 and 5, the conformation of the hydrophobic sequence was essentially silk I (the structure of B. mori silk fibroin in the silk gland before it is spun into a fiber) while the sequence (E) n was random coil.…”