Solid-State NMR Analysis of (GA) sub(3)S(AG) sub(3)D(GA) sub(3)S(AG) sub(3)D(GA) sub(3)S(AG) sub(3), a Peptide with a Lamellar Structure and a Calcium Binding Site, and Production of TS[(AG) sub(3)D(GA) sub(3)S] sub(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) sub(3) and (AlaGly) sub(3) sequences. At first, we synthesized three labeled versions of 41SDSDS, (GlyAla) sub(3)Ser(AlaGly) sub(3)Asp(GlyAla) sub(3)Ser(AlaGly) sub(3)Asp(GlyAla) sub( 3) Ser(AlaGly) sub(3), with super(13)C labeling in different positions to characterize the lamellar structure using super(13)C CP/MAS and spin-diffusion solid-state NMR. The beta2-sheet fraction in Ala residues increased with increased distance from the Asp residue in the turn part. The introduced Ser residue took almost 100% beta2-sheet structure probably because it forms an extra hydrogen bond stabilizing the stem part of (AlaGly) sub(nd). Thus, position-selective and sensitive information useful to characterize the detailed lamella structure with heterogeneous local conformations, can be obtained by super(13)C selective labeling of the peptide and determining super(13)C conformation-dependent NMR chemical shifts. We then produced an analogous recombinant protein, 14DS16, ThrSer[(AlaGly) sub(3)Asp(GlyAla) sub(3)Ser] sub(16) in Escherichia coli as a possible biomaterial. Films of this protein treated with simulated body fluid were rapidly mineralized with hydroxyapatite.