Reversible Assembly of β-Sheet Nanocrystals within Caddisfly Silk

Nuclear magnetic resonance (NMR) and X-ray diffraction (XRD) experiments reveal the structural importance of divalent cation-phosphate complexes in the formation of beta-sheet nanocrystals from phosphorylated serine-rich regions within aquatic silk from caddisfly larvae of the species Hesperophyla consimilis. Wide angle XRD data on native caddisfly silk show that the silk contains a significant crystalline component with a repetitive orthorhombic unit cell aligned along the fiber axis with dimensions of 5.9 angstrom x 23.2 angstrom x 17.3 angstrom. These nanocrystalline domains depend on multivalent cations, which can be removed through chelation with ethylenediaminetetraacetic acid (EDTA). A comparison of wide angle X-ray diffraction data before and after EDTA treatment reveals that the integrated peak area of reflections corresponding to the nanocrystalline regions decreases by 15-25% while that of the amorphous background reflections increases by 20%, indicating a partial loss of crystallinity. P-31 solid-state NMR data on native caddisfly silk also show that the phosphorylated serine-rich motifs transform from a rigid environment to one that is highly mobile and water-solvated after treatment with EDTA. The removal of divalent cations through exchange and chelation has therefore caused a collapse of the beta-sheet structure. However, NMR results show that the rigid phosphorus environment is mostly recovered after the silk is re-treated with calcium. The P-31 spin-lattice (T-1) relaxation times were measured at 7.6 +/- 3.1 and 1 +/- 0.5 s for this calcium-recovered sample and the native silk sample, respectively. The shorter P-31 T-1 relaxation times measured for the native silk sample are attributed to the presence of paramagnetic iron that is stripped away during EDTA chelation treatment and replaced with diamagnetic calcium.