Conformational study of silklike peptides modified by the addition of the calcium-binding sequence from the shell nacreous matrix protein MSI60 using 13C CP/MAS NMR spectroscopy.
The calcium-binding site of the pearl oyster (Pinctada fucata) nacreous layer matrix protein MSI60 was introduced between different Ala-Gly repeating regions derived from the primary sequences of several silk fibroins. Several different organic solvents whose effect on the repetitive domains of silk peptides is well-understood were used to modify the secondary structure of the flanking Ala-Gly repeating regions. The local conformations of the flanking Ala-Gly repeating regions as well as the calcium-binding motif, MSI60, were determined by 13C CP/MAS NMR spectroscopy. The secondary structures of the polyalanine, poly(Ala), domains were modified by the solvent treatments in a predictable fashion, suggesting that only the solvent treatment and not the conformation of the MSI60 domain affected the conformation of poly(Ala) regions. Ala-Gly domains behaved differently, taking random coil conformation regardless of the choice of solvent, indicating that their secondary structure is affected by the central MSI60 domain. The conformation of the MSI60 domain is not altered by the solvent treatments, suggesting that it may retain its ability to bind calcium ions. This was confirmed using a calcium-binding assay. The assay further showed that the calcium-binding capability of MSI60 in the synthetic peptides was most effective when the flanking domain was in the beta-sheet structure.
Asakura
Tetsuo
T
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. asakura@cc.tuat.ac.jp
Hamada
Megumi
M
Ha
Sung-Won
SW
Knight
David P
DP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002247:Carbon Isotopes; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods* / Q000592:standards; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D049911:Pinctada / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012015:Reference Standards; D012680:Sensitivity and Specificity
D002135:Calcium-Binding Proteins; D002247:Carbon Isotopes; D016326:Extracellular Matrix Proteins; D010455:Peptides; D002118:Calcium