Single Honeybee Silk Protein Mimics Properties of Multi-Protein Silk

Abstract

Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk.

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

“…High-resolution solid-state 13 C NMR spectra were recorded using an Avance 600 WB (Bruker, Karlsruhe, Germany), with a magnetic field of 14.1T. The spectrometers were operated at a 13 C NMR frequency of 150.94 MHz.…”

“…The samples were placed in a solid-state probe and spun at a magic-angle spinning (MAS) frequency of 10.0 kHz in a 4.0-mm-f zirconia rotor. For the 13 C 901 single pulse (direct polarization (DP)) experiments, the 901 pulse length for the 13 C measurements was 3.75 ms. In the cross-polarization (CP) experiments, a 1 H 901 pulse length of 3.5 ms and 1 H-13 C CP contact of 70 kHz was employed.…”

“…Actually, the larval saliva used as the labeling larvae bait in this study contains only 6% Ala. 22 Quantitative analysis of the fraction of Ala in an a-helical conformation in hornet silk In Figure 3, the expanded Ala C ¼ O, C a and C b regions of the 13 C CP-MAS spectra are shown for unlabeled and labeled cocoons with thin and thick lines, respectively. In the Ala C a peak region, the spectra of hornet cocoons labeled at two different labeling rates (two Figure 2 The 13 C CP-MAS NMR spectra of cocoons produced by hornet larvae (a) and DL-Ala-13 C 3 administered hornet larvae (b) of V. mandarinia, together with the spectra of DL-Ala-13 C 3 powder (c) and the cocoon in Figure 2B 13 C CP-MAS NMR spectra of hornet cocoons produced by larvae (solid thin lines) and DL-Ala-13 C 3 administered larvae (solid thick lines) of V. mandarinia. The Ala C a region of the spectra of hornet cocoons produced by V. simillima xanthoptera Cameron larva (dotted thin line) and another DL-Ala-13 C 3 administered V. mandarinia larva with different labeling rate (dotted thick line) is also shown.…”

“…This correspondence was due to the fact that no other peak overlapped with the Ala C a peaks, which is significant for the analysis of the fraction of Ala in an a-helical conformation in hornet silk. The abundance ratio of Ala residues in a-helix and b-sheet can be estimated from the intensity ratio of the Ala C a peaks in a-helix and b-sheet, if only the quantitative high-resolution 13 C NMR spectrum of hornet silk is obtained.…”

“…12 In addition, hornet silk contains coiled-coil structures, 10 which are a common feature of the silks of the social Hymenoptera (bees, ants and hornets). 3,13 The coiled-coil structure consists of several a-helices wound around each other. 14 The a-helices that form the coiled-coils are characterized by a 7-residue (heptad) repeat, conventionally denoted as (abcdefg) n .…”