Structure of Model Peptides Based on Nephila clavipes Dragline Silk Spidroin (MaSp1) Studied by ¹³C Cross Polarization/Magic Angle Spinning NMR

To obtain detailed structural information for spider dragline spidroin (MaSp1), we prepared three versions of the consensus peptide GGLGGQGAGAAAAAAGGAGQGGYGGLGSQGAGR labeled with ¹³C at six different sites. The ¹³C CP/MAS NMR spectra were observed after treating the peptides with different reagents known to alter silk protein conformations. The conformation-dependent ¹³C NMR chemical shifts and peak deconvolution were used to determine the local structure and the fractional compositions of the conformations, respectively. After trifluoroacetic acid (solvent)/diethyl ether (coagulant) treatment, the N-terminal region of poly-Ala (PLA) sequence, Ala⁸ and Ala¹⁰, adopted predominantly the α-helix with a substantial amount of β-sheet. The central region, Ala¹⁵, Ala¹⁸, and Leu²⁶, and C-terminal region, Ala³¹, of the peptide were dominated by either 3₁-helix or α-helix. There was no indication of β-sheet, although peak broadening indicates that the torsion angle distribution is relatively large. After 9 M LiBr/dialysis treatment, three kinds of conformation, β-sheet, random coil, and 3₁-helix, appeared, in almost equal amounts of β-sheet and random coil conformations for Ala⁸ and Ala¹⁰ residues and distorted 3₁-helix at the central region of the peptide. In contrast, after formic acid/methanol and 8 M urea/acetonitrile treatments, all of the local structure tends to β-sheet, although small amounts of random coil are also observed. The peak pattern of the Ala C_β carbon after 8 M urea/acetonitrile treatment is similar to the corresponding patterns of silk fiber from Bombyx mori and Samia cynthia ricini. We also synthesized a longer ¹³C-labeled peptide containing two PLA blocks and three Gly-rich blocks. After 8 M urea/acetonitrile treatment, the conformation pattern was closely similar to that of the shorter peptide.