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. 相似文献
Spider dragline silk is a composite biopolymer that harbors extraordinary mechanical characteristics, and consists of a hierarchically arranged protein core surrounded by outer “skin” layers. However, the contribution of the successive fiber layers on material properties has not been well defined. Here, the influence of the different components on the physicochemical and mechanical properties of dragline is investigated. The crystal structure and the mechanical properties are not changed significantly after the removal of skin constituents, indicating that the core region of dragline silk fibers determines the structural and mechanical properties. Furthermore, the outer layers have little influence on supercontraction, suggesting they do not function as protection against the penetration of water molecules. On the other hand, the outer layers offer some protection against protease digestion. The present study provides insight into how the function and structure of silk fibers are modulated and facilitates the design of silk‐inspired functional materials. 相似文献
Selective replacement of the amorphous peptide domain of a spider silk with poly(ethylene glycol) gave N. clavipes silk-inspired polymers having similar solid-state structures and very good mechanical properties. The tendency of poly(alanine) having appropriate chain length to form beta-sheets and the facility with which the beta-sheets self-assemble have been retained in the polymers. Solid-state (13)C NMR, solid-state FTIR, X-ray diffraction, and AFM studies showed that the polymers formed predominantly antiparallel beta-sheets that self-assembled into discrete nanostructures. The longer the peptide segment was, the greater was the tendency to self-assemble into antiparallel beta-sheet aggregates. AFM revealed that the morphology of the polymers was a microphase-separated architecture that contained irregularly shaped 100-200 nm poly(alanine) nanodomains interspersed within the PEG phase. The results suggest that the poly(alanine) domain influences the solid-state properties of spider silk through beta-sheet self-assembly into temporary cross-links. The results further demonstrate that by selectively replacing certain segments of a naturally occurring biopolymer with a judiciously selected nonnative segment while, at the same time, retaining other segments known to be critical for the essential properties of the native biopolymer, a synthetic polymer with similar properties and function can be obtained. 相似文献
Spider dragline silk as a protein fiber can be pictured as the oriented organization of protein nanocrystals along the long axis with their spacing filled by amorphous protein domains. We used the surface of the spider dragline silk as a biological template to nucleate bone mineral hydroxylapatite (HAP) site-specifically from a HAP-supersaturated solution. HAP crystals were found to be nucleated on the surface of silks with their c axis preferentially oriented at an average angle of 72.9 degrees with respect to the long axis of the silks. The preferred orientation is nearly identical among the different mineralized silks that we studied. Other materials such as Au and CdS could be nucleated on the silks but did not show any preferred orientation. We believe that the oriented nucleation of HAP is directly related to the structures of silks and HAP. The mineralized silks will combine the good mechanical properties of the spider silks and the biocompatibility of HAP and may be assembled into ideal biomaterials as bone implants. 相似文献
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. 相似文献
Contrary to expectation, we demonstrate that spider dragline silk spun experimentally under water displays greater stiffness and higher resilience compared to silk spun "naturally" into air. We suggest that this consequence of extended wet-spinning is due to increased molecular orientation resulting from extension of the mobile phase. 相似文献
Spider dragline silk is a biopolymer with excellent mechanical properties. The development of recombinant spider silk protein (RSP)-based materials with these properties is desirable. Formic acid (FA) is a spinning solvent for regenerated Bombyx mori silk fiber with excellent mechanical properties. To use FA as a spinning solvent for RSP with the sequence of major ampullate spider silk protein from Araneus diadematus, we determined the conformation of RSP in FA using solution NMR to determine the role of FA as a spinning solvent. We assigned 1H, 13C, and 15N chemical shifts to 32-residue repetitive sequences, including polyAla and Gly-rich regions of RSP. Chemical shift evaluation revealed that RSP is in mainly random coil conformation with partially type II β-turn structure in the Gly-Pro-Gly-X motifs of the Gly-rich region in FA, which was confirmed by the 15N NOE data. In addition, formylation at the Ser OH groups occurred in FA. Furthermore, we evaluated the conformation of the as-cast film of RSP dissolved in FA using solid-state NMR and found that β-sheet structure was predominantly formed. 相似文献
Solid-state nuclear magnetic resonance (NMR) has been extensively used to elucidate spider silk protein structure and dynamics. In many of these studies, site-specific isotope enrichment is critical for designing particular NMR methods for silk structure determination. The commonly used isotope analysis techniques, isotope-ratio mass spectroscopy and liquid/gas chromatography-mass spectroscopy, are typically not capable of providing the site-specific isotope information for many systems because an appropriate sample derivatization method is not available. In contrast, NMR does not require any sample derivatization or separation prior to analysis. In this article, conventional liquid-state 1H NMR was implemented to evaluate incorporation of 13C/15N-labeled amino acids in hydrolyzed spider dragline silk. To determine site-specific 13C and 15N isotope enrichments, an analysis method was developed to fit the 1H–13C and 1H–15N J-splitting (JCH and JNH) 1H NMR peak patterns of hydrolyzed silk fiber. This is demonstrated for Nephila clavipes spiders, where [U–13C3,15N]-Ala and [1-13C,15N]-Gly were dissolved in their water supplies. Overall, contents for Ala and Gly isotopomers are extracted for these silk samples. The current methodology can be applied to many fields where site-specific tracking of isotopes is of interest. 相似文献
An asymmetric β-sheet structure of spider silk is said to induce optical second harmonic generation. In this paper, using an in-house nonscanning type femtosecond pulse laser second harmonic generation microscope, we characterized the behavior of the β-sheet of spider silk under an applied external force. The orientation of the β-sheets was more unidirectional when the silk was extended. One of the origins of the high mechanical strength of the dragline is suggested to be the physical arrangement of its β-sheets. 相似文献
Nephila clavipes dragline silk microstructure has been investigated by scanning transmission X-ray microscopy (STXM), a technique that allows quantitative mapping of the level of orientation of the peptide groups at high spatial resolution (<50 nm). Maps of the orientation parameter P2 have been derived for spider silk for the first time. Dragline silk presents a very fine microstructure in which small, highly oriented domains (average area of 1800 nm2, thus clearly bigger than individual beta-sheet crystallites) are dispersed in a dominant, moderately oriented matrix with several small unoriented domains. Our results also highlight the orientation of the noncrystalline fraction in silk, which has been underestimated in numerous structural models. No evidence of either a regular lamellar structure or any periodicity along the fiber was observed at this spatial resolution. The surface of fresh spider silk sections consists of a approximately 30-120 nm thick layer of highly oriented protein chains, which was found to vary with the reeling speed, where web building (0.5 cm/s) and lifeline (10 cm/s) spinning speeds were investigated. While the average level of orientation of the protein chains is unaffected by the spinning speed, STXM measurements clearly highlight microstructure differences. The slowpull fiber contains a larger fraction of highly oriented domains, while the protein chains are more homogeneously oriented in the fastpull fiber. In comparison, cocoon silk from the silkworm Bombyx mori presents a narrower orientation distribution. The strength-extensibility combination found in spider dragline silk is associated with its broad orientation distribution of highly interdigitated and unoriented domains. 相似文献
Genetically engineered spider silk‐like block copolymers were studied to determine the influence of polyalanine domain size on secondary structure. The role of polyalanine block distribution on β‐sheet formation was explored using FT‐IR and WAXS. The number of polyalanine blocks had a direct effect on the formation of crystalline β‐sheets, reflected in the change in crystallinity index as the blocks of polyalanines increased. WAXS analysis confirmed the crystalline nature of the sample with the largest number of polyalanine blocks. This approach provides a platform for further exploration of the role of specific amino acid chemistries in regulating the assembly of β‐sheet secondary structures, leading to options to regulate material properties through manipulation of this key component in spider silks.
Proton spectroscopy in solid-state NMR on catalytic materials offers new opportunities in structural characterization, in particular of reaction products of catalytic reactions such as hydrogenation reactions. Unfortunately, the 1H NMR line widths in magic-angle spinning solid-state spectra are often broadened by an incomplete averaging of 1H-1H dipolar couplings. We herein discuss two model compounds, namely the H2-splitting products of two phosphane-borane Frustrated Lewis Pairs (FLPs), to study potentials and limitations of proton solid-state NMR experiments employing magic-angle spinning frequencies larger than 100 kHz at a static magnetic field strength of 20.0 T. The 1H lines are homogeneously broadened as illustrated by spin-echo decay experiments. We study two structurally similar materials which however show significant differences in 1H line widths which we explain by differences in their 1H-1H dipolar networks. We discuss the benefit of fast MAS experiments up to 110 kHz to detect the resonances of the H+/H− pair in the hydrogenation products of FLPs. 相似文献
Dynamic mechanical and dielectric properties of amorphous regenerated films of silk fibroin were studied as a function of temperature. A mechanical loss tangent peak at about 175°C may be due to the segmental motion of the main chains in the amorphous silk fibroin film. The dynamic modulus of the amorphous silk fibroin increased at 185°C due to the crystallization of the silk fibroin. Dielectric loss tangent peaks were observed at about ?40°C and 175°C at 1 kHz. The former is ascribed to the local motion of the amorphous silk fibroin with absorbed water, while the latter seems to originate from the segmental motion of the main chains and the crystallization of silk fibroin. 相似文献
(13)C CPMAS NMR has been investigated in application to protein samples with a variety of deuteration patterns. Samples were prepared with protons in either all hydrogen positions, only in the exchangeable sites, or in the exchangeable sites plus select methyl groups. CP dynamics, T(1) relaxation times, and (13)C line widths have been compared. Using ubiquitin as a model system, reasonable (1)H-(13)C CP transfer is observed for the extensively deuterated samples. In the absence of deuterium decoupling, the (13)C line widths observed for the deuterated samples are identical to those observed for the perprotio samples with a MAS rate of 20 kHz. Extensive deuteration has little effect on the T(1) of the exchangeable protons. On the basis of these observations, it is clear that there are no substantive compromises accompanying the use of extensive deuteration in the design of (1)H, (15)N, or (13)C solid-state NMR methods. 相似文献
A molecular model is proposed for describing the unusual mechanical properties of spider dragline. We find that the high stretch and strength of the semi-crystalline fiber are due to the small size of the crystallites which create inside the amorphous region a thin interphase with modulus higher than in the bulk. 相似文献
Two kinds of amorphous aluminum hydroxides, a sample precipitated from admixing AlCl3 and NaOH aqueous solutions and the commercial product, were measured by 27Al and 1H solid-state NMR spectroscopy. Pentahedral and tetrahedral coordinations, as well as octahedral coordination of oxygen atoms for aluminum, are observed in 27Al magic angle spinning (MAS) spectra of both amorphous samples. In contrast, octahedral coordination is only observed in gibbsite, bayerite, and boehmite. According to 1H MAS-NMR spectra under conditions of high spinning rate (35 kHz) and high field (14.09 T), free waters and OH groups coupled with aluminum for amorphous samples are observed at approximately 5 and approximately 4.5 ppm, respectively, the latter peak being broader. This is consistent with the differential spectra between spin echo and transfer of populations in double resonance. We conclude that the subunits of AlO4, AlO5, and AlO6 in amorphous aluminum hydroxides are bound through hydrogen bonds with a wide distribution of bonding strength. 相似文献