Determining Secondary Structure in Spider Dragline Silk by Carbon−Carbon Correlation Solid-State NMR Spectroscopy

Abstract

Two-dimensional (2D) (13)C-(13)C NMR correlation spectra were collected on (13)C-enriched dragline silk fibers produced from Nephila clavipes spiders. The 2D NMR spectra were acquired under fast magic-angle spinning (MAS) and dipolar-assisted rotational resonance (DARR) recoupling to enhance magnetization transfer between (13)C spins. Spectra obtained with short (150 ms) recoupling periods were utilized to extract distinct chemical shifts for all carbon resonances of each labeled amino acid in the silk spectra, resulting in a complete resonance assignment. The NMR results presented here permit extraction of the precise chemical shift of the carbonyl environment for each (13)C-labeled amino acid in spider silk for the first time. Spectra collected with longer recoupling periods (1 s) were implemented to detect intermolecular magnetization exchange between neighboring amino acids. This information is used to ascribe NMR resonances to the specific repetitive amino acid motifs prevalent in spider silk proteins. These results indicate that glycine and alanine are both present in two distinct structural environments: a disordered 3(1)-helical conformation and an ordered beta-sheet structure. The former can be ascribed to the Gly-Gly-Ala motif while the latter is assigned to the poly(Ala) and poly(Gly-Ala) domains.

Nanostructure and molecular mechanics of spider dragline silk protein assemblies

“…Primary protein structures ( polypeptide sequences) are created with the amino acid sequence of the N. clavipes MaSp1 and MaSp2 proteins, which constitute the majority of the silk's core (Holland et al 2008). The MaSp1 sequence is (in one-letter amino acid codes): GGAGQGGYGGLGSQGAGRGGLGGQGAG AAAAAAGGAGQGGYGGLGSQGAGRGGLGGQG AG.…”

“…Two distinct proteins are typically found in dragline silks with similar sequences across species (Gatesy et al 2001). One of the most studied silk from spiders, Nephila clavipes dragline silk, contains MaSp1 and MaSp2 proteins, with different repeat units and possibly distinct mechanical functions (Hayashi & Lewis 1998;Hayashi et al 1999;Brooks et al 2005;Holland et al 2008). MaSp1 contains glycine (Gly or G)-rich Gly -Gly -X (GGX) repeats with poly-alanine (Ala or A) and GA domains, where X typically stands for alanine, tyrosine, leucine or glutamine.…”

A Materiomics Approach to Spider Silk: Protein Molecules to Webs

“…In fact, this model first identified the secondary structure composition of the amorphous regions, showing a lack of alpha-helix conformations, but rather a disorderly mixture of structures resembling 3 1 helices and beta-turns, supporting a series of earlier experimental investigations. 26,[28][29][30] Silk's extraordinary properties on the macroscopic scale ultimately stem from the balance of strength and extensibility at the molecular scale. To assess the function of different protein domains, molecular-level deformation mechanisms of the protein composite were examined.…”

Studying natural structural protein fibers by solid‐state nuclear magnetic resonance

“…To increase the spin diffusion transfer in the fiber and avoid loss of signal due to relaxation, Marcotte et al (64) have used short mixing times (few millisseconds) and rotary resonance conditions (66). Also known as DARR ( 13 C-1 H dipolar-assisted rotational resonance), this recoupling method increases magnetization exchange during a mixing period and was also employed by Holland et al (67) on N. clavipes dragline silk. It should be noted that it is important to perform a series of experiments with different mixing times to obtain intra-fiber correlations.…”

“…It also revealed significant disorder within silk's crystalline and noncrystalline domains. Recently, Holland et al (67) used another approach in which they have extracted slices of 2D PDSD spectra to assign the chemical shifts of several residues (Ala, Gly, Gln, and Ser) in 13 C-enriched dragline silk from N. clavipes, and ascribed the b-sheet structures and 3 1 -helical domains to specific amino acid motifs.…”