Supporting Information

Protein Secondary Structure of Green Lynx Spider Dragline Silk Investigated by Solid-state NMR and X-ray DiffractionDian Xu, Xiangyan Shi, Forrest Thompson, Warner S. Weber, Qiushi Mou and Jeffery L. Yarger*

*Email: jyarger@gmail.comDepartment of Chemistry and Biochemistry, Magnetic Resonance Research Center, Arizona State University, Tempe, AZ 85287-1604, United States

Supplemental Figures and Tables

NMR Amino Acid Analysis. The NMR amino acid analysis was done using previously described method.1 2 mg of natural abundance GL dragline silk was soaked in 6 M HCl solution in a 25 ml glass tube for hydrolysis. Following degassing and flame sealing, the glass tube was then heated in oven at 108 °C for 2 days. The remaining solution was rotary-evaporated to remove all water and HCl, then the sample was dissolved in 700 l 99.9% D2O from Cambridge Isotopes for 1H NMR experiment. 1D solution-state 1H NMR experiment of the sample was collected at 298 K on the Varian VNMRS 500 MHz spectrometer equipped with a HCN triple resonance probe with z-axis gradients. A sweep width of 8012.8 Hz with 16 k points was collected and 16 transients were averaged with 10 s recycle delay for the 1H spectrum. The data was processed with a 0.2 Hz Lorentzian apodization and 2 times of zero filling. The 1D 1H NMR spectrum was assigned with the aid of a 2D 1H-1H COrrelation SpectroscopY (COSY) NMR spectrum. And the peaks for each amino acid were integrated using Vnmrj 4.0 and the peak integrals were used to calculate the molar percentages.

Table S1. Amino acid composition (molar percentage) of Peucetia viridans spider major ampullate silk from fitting the 1H NMR spectrum of hydrolyzed silk. Glx represents glutamine and glutamate; Asx represents aspartate and aspartic acid.

Amino acid Gly Ala Glx Asx Ser Pro TyrPercentage(%) 38.5±0.6 27.7±0.2 8.8±0.2 5.0±0.2 4.0±0.2 3.0±0.2 2.7±0.2Amino acid Val Leu Thr Ile Phe ArgPercentage(%) 2.6±0.2 2.5±0.2 1.9±0.1 1.9±0.1 1.2±0.1 0.3±0.1

Supporting Information

Figure S1. SEM image of the Peucetia viridans spider minor ampullate silk. The diameter of the minor silk (~ 200 nm) is much smaller compared to the major silk (~ 1 m).

Supporting Information

Figure S2. 1H solution-state NMR spectrum of hydrolyzed Peucetia viridans spider major ampullate silk. (A) Full spectrum; (B) aromatic region; (C) backbone region and (D) side chain region.

Figure S3 2D 13C-13C through-bond double quantum/single quantum (DQ/SQ) refocused INADEQUATE NMR spectra of U-[13C, 15N]-L-proline labeled Peucetia viridans spider wet major ampullate silk with 1 s (left) and 3 s (right) recycle delays.

Supporting Information

Figure S4 The log-log plot of the small-angle X-ray scattering (SAXS) structure factors on the Green Lynx (Peucetia viridans) silk and several other species. The SAXS structure factor shows perfect power law relation (linear relation in log-log plot) in the q range of 0.16 to 0.6 nm-1, indicating that the nano-crystals in Green Lynx is mass fractal with self-similar hierarchical spacial arrangement. The mass fractal morphology and self-similar property begin to break down on the length scale of 40 nm.

References(1) Shi, X. Y.; Holland, G. P.; Yarger, J. L. Anal Biochem 2013, 440, 150-157.