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

摘要

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_3Ser-(AlaGly)3Asp(GlyAla)3Ser(AlaGly)3Asp(GlyAla)3Ser(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)_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)3Asp(GlyAla)3Ser]_(16)in Escherichia coli as a possible biomaterial.Films of this protein treated with simulated body fluid were rapidly mineralized with hydroxyapatite.