Silk-Like Protein with Persistent Radicals Identified in Oyster Adhesive by Solid-State NMR

The cement produced by the Eastern oyster, Crassostrea virginica, may provide blueprints for waterproof biocompatible adhesives synthesized under benign conditions. The composition of this organic-inorganic composite, and of an organic extract, was characterized by ¹³C and ¹H solid-state NMR and also compared with C. virginica shell and its organic extract. Quantification of the organic fraction by ¹³C and ¹H NMR spectroscopy consistently showed 3 wt % organics in cement, which was higher than the 1.2 wt % in the shell. According to ¹³C NMR with spectral editing, the organic fraction of cement consisted of 73% protein, 25% polysaccharide, and 2% lipid. The organic acid-insoluble extract from the cement was mostly made up of protein remarkably rich in alanine and glycine. The unusual amino acid content matched the composition of silk-like proteins in the C. virginica or C. gigas genomes, including spidron-1-like and shelk2 previously found to be upregulated at the mantle edge. The corresponding extract from the shell contained 32% glycine and was also enriched in serine but not alanine, which was consistent with a previous wet-chemistry study. The ¹³C and ¹H NMR spin-lattice relaxation in the organic component of cement and the acid-insoluble extract was 4-40 times faster than in the shell and showed pronounced nonexponentiality, indicating a high concentration of persistent radicals in the organic components of cement, in agreement with a prior EPR study. The presence of radicals in the acid-insoluble cement fraction was confirmed by observation of a paramagnetic shift anisotropy. ¹³C NMR corroborated prior observations that the calcium carbonate in the shell and pseudonacre was mostly calcite, whereas cement had an enhanced aragonite fraction. Surprisingly, ¹H-¹³C NMR indicated that aragonite in cement was more distant from the organic fraction than was calcite. These results help advance our understanding of how oysters achieve adhesion within their wet environment.