Gregory P. Holland

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.

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 evidence for elastin-like β-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).

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 β-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.

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.

Abundant ammonia in primitive asteroids and the case for a possible exobiology

Carbonaceous chondrites are asteroidal meteorites that contain abundant organic materials. Given that meteorites and comets have reached the Earth since it formed, it has been proposed that the exogenous influx from these bodies provided the organic inventories necessary for the emergence of life. The carbonaceous meteorites of the Renazzo-type family (CR) have recently revealed a composition that is particularly enriched in small soluble organic molecules, such as the amino acids glycine and alanine, which could support this possibility. We have now analyzed the insoluble and the largest organic component of the CR2 Grave Nunataks (GRA) 95229 meteorite and found it to be of more primitive composition than in other meteorites and to release abundant free ammonia upon hydrothermal treatment. The findings appear to trace CR2 meteorites’ origin to cosmochemical regimes where ammonia was pervasive, and we speculate that their delivery to the early Earth could have fostered prebiotic molecular evolution.

Inducing β-Sheets Formation in Synthetic Spider Silk Fibers by Aqueous Post-Spin Stretching

As a promising biomaterial with numerous potential applications, various types of synthetic spider silk fibers have been produced and studied in an effort to produce man-made fibers with mechanical and physical properties comparable to those of native spider silk. In this study, two recombinant proteins based on Nephila clavipes Major ampullate Spidroin 1 (MaSp1) consensus repeat sequence were expressed and spun into fibers. Mechanical test results showed that fiber spun from the higher molecular weight protein had better overall mechanical properties (70 KD versus 46 KD), whereas postspin stretch treatment in water helped increase fiber tensile strength significantly. Carbon-13 solid-state NMR studies of those fibers further revealed that the postspin stretch in water promoted protein molecule rearrangement and the formation of β-sheets in the polyalanine region of the silk. The rearrangement correlated with improved fiber mechanical properties and indicated that postspin stretch is key to helping the spider silk proteins in the fiber form correct secondary structures, leading to better quality fibers.

Combining flagelliform and dragline spider silk motifs to produce tunable synthetic biopolymer fibers.

The two Flag/MaSp 2 silk proteins produced recombinantly were based on the basic consensus repeat of the dragline silk spidroin 2 protein (MaSp 2) from the Nephila clavipes orb weaving spider. However, the proline‐containing pentapeptides juxtaposed to the polyalanine segments resembled those found in the flagelliform silk protein (Flag) composing the web spiral: (GPGGX1 GPGGX2)2 with X1/X2 = A/A or Y/S. Fibers were formed from protein films in aqueous solutions or extruded from resolubilized protein dopes in organic conditions when the Flag motif was (GPGGX1 GPGGX2)2 with X1/X2 = Y/S or A/A, respectively. Post‐fiber processing involved similar drawing ratios (2–2.5×) before or after water‐treatment. Structural (ssNMR and XRD) and morphological (SEM) changes in the fibers were compared to the mechanical properties of the fibers at each step. Nuclear magnetic resonance indicated that the fraction of β‐sheet nanocrystals in the polyalanine regions formed upon extrusion, increased during stretching, and was maximized after water‐treatment. X‐ray diffraction showed that nanocrystallite orientation parallel to the fiber axis increased the ultimate strength and initial stiffness of the fibers. Water furthered nanocrystal orientation and three‐dimensional growth while plasticizing the amorphous regions, thus producing tougher fibers due to increased extensibility. These fibers were highly hygroscopic and had similar internal network organization, thus similar range of mechanical properties that depended on their diameters. The overall structure of the consensus repeat of the silk‐like protein dictated the mechanical properties of the fibers while protein molecular weight limited these same properties. Subtle structural motif re‐design impacted protein self‐assembly mechanisms and requirements for fiber formation.

Silk structure studied with nuclear magnetic resonance.

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