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]16 in Escherichia coli

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 beta-sheet fraction in Ala residues increased with increased distance from the Asp residue in the turn part. The introduced Ser residue took almost 100% beta-sheet structure probably because it forms an extra hydrogen bond stabilizing the stem part of (AlaGly). 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)3Ser]16 in Escherichia coli as a possible biomaterial. Films of this protein treated with simulated body fluid were rapidly mineralized with hydroxyapatite.