Lamellar structure of poly(Ala-Gly) or (AG)n in the solid was examined using 13C solid-state NMR and statistical mechanical approaches. Two doubly labeled versions, [1-13C]Gly14[1-13C]Ala15- and [1-13C]Gly18[1-13C]Ala19 of (AG)15 were examined by two-dimensional (2D) 13C spin diffusion NMR in the solid state. In addition five doubly labeled [15N,13C]-versions of the same peptide, (AG) 15 and 15 versions labeled [3-13C] in each of the successive Ala residues were utilized for REDOR and 13C CP/MAS NMR measurements, respectively. The observed spin diffusion NMR spectra were consistent with a structure containing a combination of distorted beta-turns with a large distribution of the torsion angles and antiparallel beta-sheets. The relative proportion of the distorted beta-turn form was evaluated by examination of 13C CP/MAS NMR spectra of [3-13C]Ala-(AG)15. In addition, REDOR determinations showed five kinds of atomic distances between doubly labeled 13C and 15N nuclei which were also interpreted in terms of a combination of beta-sheets and beta-turns. Our statistical mechanical analysis is in excellent agreement with our Ala Cbeta 13C CP/MAS NMR data strongly suggesting that (AG)15 has a lamellar structure.

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.

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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.