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

Abstract

Bombyx mori silk fibroin with the main sequence (Ala-Gly-Ser-Gly-Ala-Gly)n is a promising scaffold for bone regeneration not only on account of its excellent mechanical property as a structural matrix, but also for its slow biodegradability with adequate control of hydroxyapatite (HAP) deposition. Seeking to develop a material that might stimulate bone regeneration, we prepared a recombinant calcium binding-amphipathic silk-like protein [(Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4]4 by expression in E. coli. We also prepared the homologous peptide (Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4 by solid phase synthesis. The poly-L-glutamic acid was introduced into both protein and peptide because this sequence is involved in HAP-nucleating domains of bone sialoprotein. The recombinant protein was shown to bind relatively large quantities of Ca2+ ions in solution by a spectrophotometric assay and in the solid state by X-ray photoelectron spectroscopy. Changes in the electronic structure and local conformation of the peptide resulting from Ca2+ binding were studied using 13C solution NMR, especially 13C chemical shifts. We obtained evidence that Ca2+ bound to the poly-L-glutamic acid domains but not to the predominantly hydrophobic (Ala-Gly-Ser-Gly-Ala-Gly)4 domains. A remarkable conformational change induced by adsorption of the synthetic peptide on the HAP surface was also demonstrated using 13C solid state NMR.