| Solid-state Nuclear Magnetic Resonance (NMR) is well suited for studying the structure of insoluble, heterogeneous materials like spider silk fibers. In this study, various solid-state NMR techniques are used to investigate and characterize the higher-order structure of major ampullate silk fibers from three different species of spiders, Argiope aurantia, Latrodectus hesperus, and Nephila clavipes. Major ampullate silk from all three species is comprised of two proteins, major ampullate spidroin 1 and 2 (MaSp1/MaSp2). However, the ratio of MaSp1 to MaSp2 differs among species. Therefore, silk from specific species are used with contrasting ratios of MaSp1 to MaSp2 to probe the structure of one protein over the other. The known conformational dependence of both the 13C and 15 N isotropic chemical shift is used to characterize the secondary structure of both spider silk proteins. Through-space hetero-nuclear (15N-13 C) and homo-nuclear (13C-13C) experiments utilizing dipolar-transfer are used for the assignment of chemical shifts that lead to the characterization of the protein structure. Additionally, through-bond 13C-13C double-quantum (DQ) / single quantum (SQ) refocused INADEQUATE experiment has been successfully used for chemical shift assignment and protein characterization. In addition to characterizing silk protein structure, quantitative correlations have been made between the primary amino acid sequence of the proteins and the secondary protein structures, providing a quantitative framework for synthetic silk production. Additionally, the major ampullate gland that produces the MaSp1 and MaSp2 proteins was excised from the spider prior to silk fiber formation to interrogate the pre-folded state of the proteins. The protein structure in the gland was investigated in the aqueous state utilizing high resolution-magic angle spinning (HR-MAS) NMR. The gland was then dried and characterized utilizing the same ssNMR experiments used to characterize the fiber. Utilizing solid-state NMR to study the protein structure of spider silk, pre-spun silk proteins, and dried silk proteins has provided a comprehensive picture of spider silk protein secondary structure and has formed a basis for better understanding the proteins that will lead to advances in the formation of synthetic fibers. |
