Characterization of a Ca binding-amphipathic silk-like protein and peptide with the sequence (Glu)8(Ala-Gly-Ser-Gly-Ala-Gly)4 with 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.