Characterizing the secondary protein structure of black widow dragline silk using solid-state NMR and X-ray diffraction.

This study provides a detailed secondary structural characterization of major ampullate dragline silk from Latrodectus hesperus (black widow) spiders. X-ray diffraction results show that the structure of black widow major ampullate silk fibers is comprised of stacked ß-sheet nanocrystallites oriented parallel to the fiber axis and an amorphous region with oriented (anisotropic) and isotropic components. The combination of two-dimensional (2D) (13)C-(13)C through-space and through-bond solid-state NMR experiments provide chemical shifts that are used to determine detailed information about the amino acid motif secondary structure in black widow spider dragline silk. Individual amino acids are incorporated into different repetitive motifs that make up the majority of this protein-based biopolymer. From the solid-state NMR measurements, we assign distinct secondary conformations to each repetitive amino acid motif and, hence, to the amino acids that make up the motifs. Specifically, alanine is incorporated in ß-sheet (poly(Alan) and poly(Gly-Ala)), 3(1)-helix (poly(Gly-Gly-Xaa), and α-helix (poly(Gln-Gln-Ala-Tyr)) components. Glycine is determined to be in ß-sheet (poly(Gly-Ala)) and 3(1)-helical (poly(Gly-Gly-X(aa))) regions, while serine is present in ß-sheet (poly(Gly-Ala-Ser)), 3(1)-helix (poly(Gly-Gly-Ser)), and ß-turn (poly(Gly-Pro-Ser)) structures. These various motif-specific secondary structural elements are quantitatively correlated to the primary amino acid sequence of major ampullate spidroin 1 and 2 (MaSp1 and MaSp2) and are shown to form a self-consistent model for black widow dragline silk.

Reproducing natural spider silks' copolymer behavior in synthetic silk mimics.

Dragline silk from orb-weaving spiders is a copolymer of two large proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2). The ratio of these proteins is known to have a large variation across different species of orb-weaving spiders. NMR results from gland material of two different species of spiders, N. clavipes and A. aurantia , indicates that MaSp1 proteins are more easily formed into ß-sheet nanostructures, while MaSp2 proteins form random coil and helical structures. To test if this behavior of natural silk proteins could be reproduced by recombinantly produced spider silk mimic protein, recombinant MaSp1/MaSp2 mixed fibers as well as chimeric silk fibers from MaSp1 and MaSp2 sequences in a single protein were produced based on the variable ratio and conserved motifs of MaSp1 and MaSp2 in native silk fiber. Mechanical properties, solid-state NMR, and XRD results of tested synthetic fibers indicate the differing roles of MaSp1 and MaSp2 in the fiber and verify the importance of postspin stretching treatment in helping the fiber to form the proper spatial structure.

X-ray diffraction study of nanocrystalline and amorphous structure within major and minor ampullate dragline spider silks.

Synchrotron X-ray micro-diffraction experiments were carried out on Nephila clavipes (NC) and Argiope aurantia (AA) major (MA) and minor ampullate (MiA) fibers that make up dragline spider silk. The diffraction patterns show a semi-crystalline structure with ß-poly(l-alanine) nanocrystallites embedded in a partially oriented amorphous matrix. A superlattice reflection 'S' diffraction ring is observed, which corresponds to a crystalline component larger in size and is poorly oriented, when compared to the ß-poly(l-alanine) nanocrystallites that are commonly observed in dragline spider silks. Crystallite size, crystallinity and orientation about the fiber axis have been determined from the wide-angle X-ray diffraction (WAXD) patterns. In both NC and AA, the MiA silks are found to be more highly crystalline, when compared with the corresponding MA silks. Detailed analysis on the amorphous matrix shows considerable differences in the degree of order of the oriented amorphous component between the different silks studied and may play a crucial role in determining the mechanical properties of the silks.

Structure and dynamics of aromatic residues in spider silk: 2D carbon correlation NMR of dragline fibers.

The structure of aromatic residues in spider silk has remained relatively uncharacterized. This is primarily due to the relatively low abundance of aromatic residues in dragline spider silk. NMR characterization has been further hindered by the inability to (13)C-isotopically enrich these amino acids in the silk effectively. In the present contribution, it is shown that feeding spiders an aqueous solution of [U-(13)C/(15)N]-l-phenylalanine (13)C-enriches Tyr to a level of 20-30% in dragline spider silk. This enables the collection of 2D through-bond (13)C double quantum/single quantum (DQ/SQ) correlation spectra with the refocused INADEQUATE solid-state NMR pulse sequence. These 2D spectra provide the complete unambiguous assignment of the Tyr resonances in dragline silks from two spider species: N. clavipes and A. aurantia. Additionally, weak resonances are detected for Phe in A. aurantia dragline silk that is present at levels <1%. The conformation dependence of the C(alpha), C(beta), and carbonyl chemical shifts show that Tyr and Phe present in the GGX and GPGXX motifs in the spider silk proteins MaSp1 and MaSp2 are in disordered helical structures and not incorporated in the beta-sheet domains.

Quantitative Correlation between the protein primary sequences and secondary structures in spider dragline silks.

Synthetic spider silk holds great potential for use in various applications spanning medical uses to ultra lightweight armor; however, producing synthetic fibers with mechanical properties comparable to natural spider silk has eluded the scientific community. Natural dragline spider silks are commonly made from proteins that contain highly repetitive amino acid motifs, adopting an array of secondary structures. Before further advances can be made in the production of synthetic fibers based on spider silk proteins, it is imperative to know the percentage of each amino acid in the protein that forms a specific secondary structure. Linking these percentages to the primary amino acid sequence of the protein will establish a structural foundation for synthetic silk. In this study, nuclear magnetic resonance (NMR) techniques are used to quantify the percentage of Ala, Gly, and Ser that form both beta-sheet and helical secondary structures. The fraction of these three amino acids and their secondary structure are quantitatively correlated to the primary amino acid sequence for the proteins that comprise major and minor ampullate silk from the Nephila clavipes spider providing a blueprint for synthetic spider silks.

Solid-state NMR comparison of various spiders' dragline silk fiber.

Major ampullate (dragline) spider silk is a coveted biopolymer due to its combination of strength and extensibility. The dragline silk of different spiders have distinct mechanical properties that can be qualitatively correlated to the protein sequence. This study uses amino acid analysis and carbon-13 solid-state NMR to compare the molecular composition, structure, and dynamics of major ampullate dragline silk of four orb-web spider species ( Nephila clavipes , Araneus gemmoides , Argiope aurantia , and Argiope argentata ) and one cobweb species ( Latrodectus hesperus ). The mobility of the protein backbone and amino acid side chains in water exposed silk fibers is shown to correlate to the proline content. This implies that regions of major ampullate spidroin 2 protein, which is the only dragline silk protein with any significant proline content, become significantly hydrated in dragline spider silk.

Determining secondary structure in spider dragline silk by carbon-carbon correlation solid-state NMR spectroscopy.

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.

Solid-state NMR investigation of major and minor ampullate spider silk in the native and hydrated states.

Silks spun from the major (Ma) and minor (Mi) ampullate glands by the spider Nephila clavipes respond to water differently. Specifically, Ma silk supercontracts (shrinks 40-50% in length) while Mi silk does not contract at all when hydrated with water. In the present study, 1H --> 13C cross polarization magic angle spinning (CP-MAS), 13C MAS NMR collected with dipolar decoupling, and two-dimensional wide-line separation spectra are presented on Mi silk in its native and hydrated state and comparisons are made to Ma silk. This combination of NMR data demonstrates that water plasticizes Mi and Ma silk similarly, with an increase in chain dynamics observed in regions containing Gly, Glu, Ser, Tyr, Leu, and a fraction of Ala when the Mi silk is hydrated. Resonances that correspond to the poly(Ala) and poly(Gly Ala) motifs of Ma and Mi silk are predominately rigid indicating that water does not penetrate these beta-sheet domains.

Quantifying the fraction of glycine and alanine in beta-sheet and helical conformations in spider dragline silk using solid-state NMR.

Solid-state two-dimensional refocused INADEQUATE MAS NMR experiments resolve distinct helical and beta-sheet conformational environments for both alanine and glycine in Nephila clavipes dragline silk fibers; the fraction of alanine and glycine in beta-sheet structures is determined to be 82% +/- 4% and 28% +/- 5%, respectively.

Identification of Multiple Diffusion Rates in Mixed Solvent Anion Exchange Membranes Using High Resolution MAS NMR.

High resolution magic angle spinning (HRMAS) 1H NMR in combination with 2D exchange NOESY and pulsed field gradient (PFG) NMR diffusion experiments have been used to characterize 1 N methanol swollen polymer anion exchange membranes (AEM) presently being developed for alkaline fuel cells. Standard static 1H NMR experiments on these materials have proven unsuccessful due to severe signal broadening. New experimental methods for increased resolution are needed to determine distinct solvent environments and transport properties. Using HRMAS NMR, resonances from water and methanol in both a free (bulk-like) environment and membrane-associated environment within the AEM were observed. 1H HRMAS PFG NMR experiments identified different molecular diffusion environments in the solvent, while 1H 2D NOESY exchange NMR experiments confirmed spatial contacts between membrane-associated species and the membrane. These results demonstrate that 1H HRMAS is an ideal technique for the characterization of individual environments and diffusion rates in polymer membranes with mixed solvent systems.

Proton-detected heteronuclear single quantum correlation NMR spectroscopy in rigid solids with ultra-fast MAS.

In this article, we show the potential for utilizing proton-detected heteronuclear single quantum correlation (HSQC) NMR in rigid solids under ultra-fast magic angle spinning (MAS) conditions. The indirect detection of carbon-13 from coupled neighboring hydrogen nuclei provides a sensitivity enhancement of 3- to 4-fold in crystalline amino acids over direct-detected versions. Furthermore, the sensitivity enhancement is shown to be significantly larger for disordered solids that display inhomogeneously broadened carbon-13 spectra. Latrodectus hesperus (Black Widow) dragline silk is given as an example where the sample is mass-limited and the sensitivity enhancement for the proton-detected experiment is 8- to 13-fold. The ultra-fast MAS proton-detected HSQC solid-state NMR technique has the added advantage that no proton homonuclear decoupling is applied during the experiment. Further, well-resolved, indirectly observed carbon-13 spectra can be obtained in some cases without heteronuclear proton decoupling.

Solid-state NMR evidence for elastin-like beta-turn structure in spider dragline silk.

Two-dimensional homo- and heteronuclear solid-state MAS NMR experiments on (13)C/(15)N-proline labeled Argiope aurantia dragline silk provide evidence for an elastin-like beta-turn structure for the repetitive Gly-Pro-Gly-X-X motif prevalent in major ampullate spidroin 2 (MaSp2).