Quantifying the fraction of alanine residues in an α-helical conformation in hornet silk using solid-state NMR

Here, we demonstrate the first successful isotope labeling of Ala carbons in hornet silk produced by the larvae of Vespa (Vespinae, Vespidae) mandarinia. This labeled hornet silk was examined by high-resolution 13C solid-state NMR, and it was found that the fraction of Ala residues in α-helical conformations compared with Ala residues in the overall conformation of hornet silk can be quantitatively determined from Ala Cα NMR peaks. The value for this α-helical Ala fraction is close to that of the fraction of Ala residues in coiled-coil structures estimated in the four major hornet silk proteins by coiled-coil prediction analysis. This result indicates that most of the Ala residues in α-helices occur in those α-helices with a coiled-coil structure, and that the number of Ala residues in α-helices without a coiled-coil structure is small. Moreover, coiled-coil prediction analysis indicated that the potential coiled-coil domains are located only in the central portion of the protein chains of the major hornet silk proteins. From these results, we confirmed that the α-helical conformation mostly forms in the central portion of the hornet silk chains, whereas the ends of the protein chains are nearly devoid of α-helical structure. We deduce that the ends of the protein chains would preferentially adopt a β-sheet conformation.