²H-Labeling of Silk Fibroin Fibers and Their Structural Characterization by Solid-State ²H NMR

Three kinds of 2 H-labeled Bombyx mori silk fibroin samples (with [2,2- 2 H 2 ]Gly, [3,3,3- 2 H 3 ]Ala, or [2,3,5,6- 2 H 4 ]Tyr) were obtained by oral administration of either the labeled amino acid or 2 H 2 O to 5th instar larvae. The administration of 2 H 2 O alone yielded a high degree of selective deuteration at the alanine methyl group, since the incorporation of 2 H 2 O 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 2 H-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 Cα− 2 H bond vectors in glycine and of Cα−Cβ 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- 2 H 2 ]Gly- and [3,3,3- 2 H 3 ]Ala-labeled silk, respectively. These values are identical with those obtained from the 2 H-NMR powder patterns of the unaligned samples, within experimental error. From the angular dependence of the quadrupole splittings, it was thus calculated that the Cα− 2 H bonds of glycine as well as the Cα−Cβ 2 H 3 bond of alanine make an angle of approximately 90° relative to the fiber axis. These steric constraints were then used to evaluate the torsion angles, φ and ψ, for the glycine and alanine residues within the protein backbone. These data, determined independently by solid-state 2 H NMR, thus verified and narrowed down the allowed region in the Ramachandran ( φ, ψ) map obtained from previous solid-state 13 C- and 15 N-NMR studies.