title: NMR analysis of the fibronectin cell-adhesive sequence, Arg-Gly-Asp, in a recombinant silk-like protein and a model peptide.
abstract: It is well established that by introducing the cell-adhesive sequence Arg-Gly-Asp (RGD) from fibronectin into Bombyx mori silk fibroin by covalent coupling or bioengineering techniques, excellent biomaterials have been developed with the modified silk fibroin. However, there is no report about the structure and dynamics of the RGD moiety in the silk fibroin. To clarify the origin of such a high cell adhesion character and to design new recombinant silk protein with higher cell adhesion ability, it is necessary to characterize the structure and dynamics of the RGD moiety introduced into silk fibroin. In this study, the structure and dynamics of the RGD moiety in a recombinant silk-like protein, SLPF(10), consisting of the repeated silk fibroin sequence (AGSGAG)(3) and the sequence ASTGRGDSPA including the RGD moiety, were studied using solution NMR. The (1)H, (15)N, and (13)C chemical shifts indicate that the RGD moiety, as well as the silk fibroin sequence, takes a random coil form with high mobility in aqueous solution. Next, a (13)C solid-state NMR study was performed on a (13)C selectively labeled model peptide, AGSGAG[3-(13)C]A(7)GSGAGAGSGGT[2-(13)C]G(19)R[1-(13)C]G(21)DSPAGGGAGAGSGAG. After formic acid treatment, an increase in the β-sheet fraction for the AGSGAG sequence and peak narrowing of the residues around the RGD moiety were observed in the dry state. The latter indicates a decrease in the chemical shift distribution although the RGD moiety is still in random coil. A decrease in the peak intensities of the RGD moiety in the swollen state after immersing it in distilled water was observed, indicating high mobility of the RGD sequence in the peptide in the swollen state. Thus, the random coil state of the RGD moiety in the recombinant silk-like protein is maintained in aqueous solution and also in both dry and swollen state. This is similar to the case of the RGD moiety in fibronectin. The presence of the linker ASTG at the N-terminus and SPAGG at the C-terminus seems important to maintain the random coil form and the flexible state of the RGD sequence in order to permit access for binding to various integrins.
authors.affiliation: Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. asakura@cc.tuat.ac.jp
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Asakura
authors.forename: Hirohito
authors.initials: H
authors.lastname: Nishi
authors.forename: Aya
authors.initials: A
authors.lastname: Nagano
authors.forename: Ai
authors.initials: A
authors.lastname: Yoshida
authors.forename: Yasumoto
authors.initials: Y
authors.lastname: Nakazawa
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Kamiya
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D005353:Fibronectins / Q000737:chemistry*; D055767:Immobilized Proteins; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D047011:Silk / Q000737:chemistry*
chemical_list: D005353:Fibronectins; D055767:Immobilized Proteins; D010455:Peptides; D047011:Silk