Qiushi Mou

Mechanical and physical properties of recombinant spider silk films using organic and aqueous solvents.

Spider silk has exceptional mechanical and biocompatibility properties. The goal of this study was optimization of the mechanical properties of synthetic spider silk thin films made from synthetic forms of MaSp1 and MaSp2, which compose the dragline silk of Nephila clavipes. We increased the mechanical stress of MaSp1 and 2 films solubilized in both HFIP and water by adding glutaraldehyde and then stretching them in an alcohol based stretch bath. This resulted in stresses as high as 206 MPa and elongations up to 35%, which is 4× higher than the as-poured controls. Films were analyzed using NMR, XRD, and Raman, which showed that the secondary structure after solubilization and film formation in as-poured films is mainly a helical conformation. After the post-pour stretch in a methanol/water bath, the MaSp proteins in both the HFIP and water-based films formed aligned β-sheets similar to those in spider silk fibers.

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 β-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 Å × 23.2 Å × 17.3 Å. 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. (31)P 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 β-sheet structure. However, NMR results show that the rigid phosphorus environment is mostly recovered after the silk is re-treated with calcium. The (31)P spin-lattice (T1) 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 (31)P T1 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.

Determining hydrogen-bond interactions in spider silk with 1H–13C HETCOR fast MAS solid-state NMR and DFT proton chemical shift calculations

Two-dimensional (2D) (1)H-(13)C heteronuclear correlation (HETCOR) solid-state NMR spectra collected with fast magic angle spinning (MAS) are used in conjunction with density functional theory (DFT) proton chemical shift calculations to determine the hydrogen-bonding strength for ordered β-sheet and disordered 310-helical structures in spider dragline silk. The hydrogen-bond strength is determined to be identical for both structures in spider silk with a 1.83-1.84 Å NH···OC hydrogen-bond distance.

X-ray Intermolecular Structure Factor (XISF): Separation of intra- and intermolecular interactions from total X-ray scattering data

XISF is a MATLAB program developed to separate intermolecular structure factors from total X-ray scattering structure factors for molecular liquids and amorphous solids. The program is built on a trust-region-reflective optimization routine with the r.m.s. deviations of atoms physically constrained. XISF has been optimized for performance and can separate intermolecular structure factors of complex molecules.

Protein secondary structure of Green Lynx spider dragline silk investigated by solid-state NMR and X-ray diffraction

In this study, the secondary structure of the major ampullate silk from Peucetia viridans (Green Lynx) spiders is characterized by X-ray diffraction and solid-state NMR spectroscopy. From X-ray diffraction measurement, β-sheet nanocrystallites were observed and found to be highly oriented along the fiber axis, with an orientational order, fc≈0.98. The size of the nanocrystallites was determined to be on average 2.5nm×3.3nm×3.8nm. Besides a prominent nanocrystalline region, a partially oriented amorphous region was also observed with an fa≈0.89. Two-dimensional (13)C-(13)C through-space and through-bond solid-state NMR experiments were employed to elucidate structure details of P. viridans silk proteins. It reveals that β-sheet nanocrystallites constitutes 40.0±1.2% of the protein and are dominated by alanine-rich repetitive motifs. Furthermore, based upon the NMR data, 18±1% of alanine, 60±2% glycine and 54±2% serine are incorporated into helical conformations.