Solid-State NMR Analysis of (GA)₃S(AG)₃D(GA)₃S(AG)₃D(GA)₃S(AG)₃, a Peptide with a Lamellar Structure and a Calcium Binding Site, and Production of TS[(AG)₃D(GA)₃S]₁₆inEscherichia coli

Abstract

In an attempt to produce mineralized composite materials with potential use as biomaterials or scaffolds for tissue engineering, we designed silklike peptides based on Ala-Gly repeated sequences with a lamellar structure and Asp as a Ca binding site in the turn part as in Tirrell's work (for example: Macromolecules 1996, 29, 1540-1553). We further modified the design of the lamella structure by introducing a Ser residue between (GlyAla) 3 and (AlaGly) 3 sequences. At first, we synthesized three labeled versions of 41SDSDS, (GlyAla) 3 Ser-(AlaGly) 3 Asp(GlyAla) 3 Ser(AlaGly) 3 Asp(GlyAla) 3 Ser(AlaGly) 3 , with 13 C labeling in different positions to characterize the lamellar structure using 13 C CP/MAS and spin-diffusion solid-state NMR. The β-sheet fraction in Ala residues increased with increased distance from the Asp residue in the turn part. The introduced Ser residue took almost 100% β-sheet structure probably because it forms an extra hydrogen bond stabilizing the stem part of (AlaGly) n . Thus, position-selective and sensitive information useful to characterize the detailed lamella structure with heterogeneous local conformations, can be obtained by 13 C selective labeling of the peptide and determining 13 C conformation-dependent NMR chemical shifts. We then produced an analogous recombinant protein, 14DS16, ThrSer[(AlaGly) 3 Asp(GlyAla) 3 Ser] 16 in Escherichia coli as a possible biomaterial. Films of this protein treated with simulated body fluid were rapidly mineralized with hydroxyapatite.

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

“…For example, the peaks observed at 20 ppm and 17 ppm in Fig. 6a are assigned to the b-sheet and the random coil of the Ala residue respectively [24][25][26] and the fraction of random coil is slightly larger judging from the relative intensities. In addition, the relatively sharp peaks with a shoulder observed at 52 ppm and 30 ppm assigned to Glu Ca and Glu Cb carbons, respectively, indicate that the structure of the (E) 8 region is mainly b-sheet.…”

Analysis of the Structure of Bombyx mori Silk Fibroin by NMR

“…They proposed a γ-turn, but a β-turn was proposed in our study. Similar conclusions were reached by NMR studies of a (GA) n peptide containing regularly placed Ser, 147 and a study of the peptide (AGSGAG) 5 , supported by statistical mechanics. 137 Furthermore, it is noteworthy that almost all subdomains of fibroin start with multiple repeats of the sequence GAGAGS, followed later by Tyr that tend to occur every 6−12 residues.…”

“…They proposed a γ-turn, but a β-turn was proposed in our study. Similar conclusions were reached by NMR studies of a (GA) n peptide containing regularly placed Ser, and a study of the peptide (AGSGAG) 5 , supported by statistical mechanics …”

Elucidating silk structure using solid-state NMR

“…The silk-like peptides based on AG repeated sequences with a lamellar structure and Asp as a Ca binding site at the turn part were designed and produced in E. coli. 85 The lamellar structure formation was conrmed for the model peptide with different 13 C labeling positions by solid-state NMR. The surfaces of the recombinant silk protein with the lamellar structure are responsible for calcium binding and accelerated formation of hydroxyapatite.…”