H-2-Labeling of silk fibroin fibers and their structural characterization by solid-state H-2 NMR

Three kinds of H-2-labeled Bombyx mori silk fibroin samples (with [2,2-H-2(2)]Gly, [3,3,3-H-2(3)]Ala, or [2,3,5,6-H-2(4)]Tyr) were obtained by oral administration of either the labeled amino acid or (H2O)-H-2 to 5th instar larvae. The administration of (H2O)-H-2 alone yielded a high degree of selective deuteration at the alanine methyl group, since the incorporation of (H2O)-H-2 occurs between fumarate and malate in the tricarboxylic acid (TCA) cycle of the silk fibroin synthetic pathway. Uniaxially oriented silk fibers were prepared as samples for H-2-NMR spectroscopy. An analysis of the quadrupole echo line shape was carried out in order to determine the angle of the deuterium-labeled group relative to the fiber axis, i.e., of the C alpha-H-2 bond vectors in glycine and of C alpha-C beta(2)H(3) in alanine. With the fiber axis aligned parallel to the magnetic field, quadrupole splittings were obtained as 117.8 and 39.8 kHz for [2,2-H-2(2)]Gly- and [3,3,3-H-2(3)]Ala-labeled silk, respectively. These values are identical with those obtained from the 2H-NMR powder patterns of the unaligned samples, within experimental error. From the angular dependence of the quadrupole splittings, it was thus calculated that the C alpha-H-2 bonds of glycine as well as the C alpha-C beta(2)H(3) bond of alanine make an angle of approximately 90 degrees relative to the fiber axis. These steric constraints were then used to evaluate the torsion angles, phi and psi, for the glycine and alanine residues within the protein backbone. These data, determined independently by solid-state H-2 NMR, thus verified and narrowed down the allowed region in the Ramachandran (phi, psi) map obtained from previous solid-state C-13- and N-15-NMR studies.