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Hydroxyapatite (HAP) exhibits a highly oriented hierarchical structure in biological hard tissues. The formation and selective crystalline orientation of HAP is a process that involves functional biomineralization proteins abundant in acidic residues. To obtain insights into the process of HAP mineralization and acidic residue binding, synthesized HAP with specific lattice planes including (001), (100), and (011) are structurally characterized following the adsorption of aspartic acid (Asp). The adsorption affinity of Asp on HAP surfaces is evaluated quantitatively and demonstrates a high dependency on the HAP morphological form. Among the synthesized HAP nanoparticles (NPs), Asp exhibits the strongest adsorption affinity to short HAP nanorods, which are composed of (100) and (011) lattice planes, followed by nanosheets with a preferential expression of the (001) facet, to which Asp displays a similar but slightly lower binding affinity. HAP nanowires, with the (100) lattice plane preferentially developed, show significantly lower affinity to Asp and evidence of multilayer adsorption compared to the previous two types of HAP NPs. A combination of solid-state NMR (SSNMR) techniques including 13 C and 15 N CP-MAS, relaxation measurements and 13 C- 31 P Rotational Echo DOuble Resonance (REDOR) is utilized to characterize the molecular structure and dynamics of Asp-HAP bionano interfaces with 13 C- and 15 N-enriched Asp. REDOR is used to determine 13 C- 31 P internuclear distances, providing insight into the Asp binding geometry where stronger 13 C- 31 P dipolar couplings correlate with binding affinity determined from Langmuir isotherms. The carboxyl sites are identified as the primary binding groups, facilitated by their interaction with surface calcium sites. The Asp chelation conformations revealed by SSNMR are further refined with molecular dynamics (MD) simulation where specific models strongly agree between the SSNMR and MD models for the various surfaces.

Selective one-dimensional 13 C- 13 C spin-diffusion solid-state nuclear magnetic resonance (SSNMR) provides evidence for CH/π ring packing interactions between Pro and Tyr residues in 13 C-enriched Latrodectus hesperus dragline silk. The secondary structure of Pro-containing motifs in dragline spider silks consistently points to an elastin-like type II β-turn conformation based on 13 C chemical shift analysis. 13 C- 13 C spin diffusion measurements as a function of mixing times allow for the measurement of spatial proximity between the Pro and Tyr rings to be ∼0.5-1 nm, supporting strong Pro-Tyr ring interactions that likely occur through a CH/π mechanism. These results are supported by molecular dynamics (MD) simulations and analysis and reveals new insights into the secondary structure and Pro-Tyr ring stacking interactions for one of nature's toughest biomaterials.

Copper phosphide (Cu 3- x P) nanocrystals are promising materials for nanoplasmonics due to their substoichiometric composition, enabling the generation and stabilization of excess delocalized holes and leading to localized surface plasmon resonance (LSPR) absorption in the near-IR. We present three Cu-coupled redox chemistries that allow postsynthetic modulation of the delocalized hole concentrations and corresponding LSPR absorption in colloidal Cu 3- x P nanocrystals. Changes in the structural, optical, and compositional properties are evaluated by powder X-ray diffraction, electronic absorption spectroscopy, 31 P magic-angle spinning solid-state nuclear magnetic resonance spectroscopy, and elemental analysis. The redox chemistries presented herein can be used to access nanocrystals with LSPR energies of 660-890 meV, a larger range than has been possible through synthetic tuning alone. In addition to utilizing previously reported redox chemistries used for copper chalcogenide nanocrystals, we show that the largest structural and LSPR modulation is achieved using a divalent metal halide and trioctylphosphine. Specifically, nanocrystals treated with zinc iodide and trioctylphosphine have the smallest unit-cell volume (295.2 Å 3 ) reported for P 6 3 cm Cu 3- x P, indicating more Cu vacancies than have been previously observed. Overall, these redox chemistries present valuable insight into controlling the optical and structural properties of Cu 3- x P.

Producing recombinant spider silk fibers that exhibit mechanical properties approaching native spider silk is highly dependent on the constitution of the spinning dope. Previously published work has shown that recombinant spider silk fibers spun from dopes with phosphate-induced pre-assembly (biomimetic dopes) display a toughness approaching native spider silks far exceeding the mechanical properties of fibers spun from dopes without pre-assembly (classical dopes). Dynamic light scattering experiments comparing the two dopes reveal that biomimetic dope displays a systematic increase in assembly size over time, while light microscopy indicates liquid-liquid-phase separation (LLPS) as evidenced by the formation of micron-scale liquid droplets. Solution nuclear magnetic resonance (NMR) shows that the structural state in classical and biomimetic dopes displays a general random coil conformation in both cases; however, some subtle but distinct differences are observed, including a more ordered state for the biomimetic dope and small chemical shift perturbations indicating differences in hydrogen bonding of the protein in the different dopes with notable changes occurring for Tyr residues. Solid-state NMR demonstrates that the final wet-spun fibers from the two dopes display no structural differences of the poly(Ala) stretches, but biomimetic fibers display a significant difference in Tyr ring packing in non-β-sheet, disordered helical domains that can be traced back to differences in dope preparations. It is concluded that phosphate pre-orders the recombinant silk protein in biomimetic dopes resulting in LLPS and fibers that exhibit vastly improved toughness that could be due to aromatic ring packing differences in non-β-sheet domains that contain Tyr.

Functionalizing silica nanoparticles with a lipid bilayer shell is a common first step in fabricating drug delivery and biosensing devices that are further decorated with other biomolecules for a range of nanoscience applications and therapeutics. Although the molecular structure and dynamics of lipid bilayers have been thoroughly investigated on larger 100 nm-1 μm silica spheres where the lipid bilayer exhibits the typical L α bilayer phase, the molecular organization of lipids assembled on mesoscale (4-100 nm diameter) nanoparticles is scarce. Here, DSC, TEM and 2 H and 31 P solid-state NMR are implemented to probe the organization of 1,2-dipalmitoyl-d 54 - glycero -3-phosphocholine (DMPC-d 54 ) assembled on mesoscale silica nanoparticles illustrating a significant deviation from L α bilayer structure due to the increasing curvature of mesoscale supports. A biphasic system is observed that exhibits a combination of high-curvature, non-lamellar and lamellar phases for mesoscale (<100 nm) supports with evidence of an interdigitated phase on the smallest diameter support (4 nm).

Black widow spider dragline silk is one of nature's high-performance biological polymers, exceeding the strength and toughness of most man-made materials including high tensile steel and Kevlar. Major ampullate (Ma), or dragline silk, is primarily comprised of two spidroin proteins (Sp) stored within the Ma gland. In the native gland environment, the MaSp1 and MaSp2 proteins self-associate to form hierarchical 200-300 nm superstructures despite being intrinsically disordered proteins (IDPs). Here, dynamic light scattering (DLS), three-dimensional (3D) triple resonance solution NMR, and diffusion NMR is utilized to probe the MaSp size, molecular structure, and dynamics of these protein pre-assemblies diluted in 4 M urea and identify specific regions of the proteins important for silk protein pre-assembly. 3D NMR indicates that the Gly-Ala-Ala and Ala-Ala-Gly motifs flanking the poly(Ala) runs, which comprise the β-sheet forming domains in fibers, are perturbed by urea, suggesting that these regions may be important for silk protein pre-assembly stabilization.

Two-dimensional (2D) and 3D through-space 13 C- 13 C homonuclear spin-diffusion techniques are powerful solid-state nuclear magnetic resonance (NMR) tools for extracting structural information from 13 C-enriched biomolecules, but necessarily long acquisition times restrict their applications. In this work, we explore the broad utility and underutilized power of a chemical shift-selective one-dimensional (1D) version of a 2D 13 C- 13 C spin-diffusion solid-state NMR technique. The method, which is called 1D dipolar-assisted rotational resonance (DARR) difference, is applied to a variety of biomaterials including lignocellulosic plant cell walls, microcrystalline peptide fMLF, and black widow dragline spider silk. 1D 13 C- 13 C spin-diffusion methods described here apply in select cases in which the 1D 13 C solid-state NMR spectrum displays chemical shift-resolved moieties. This is analogous to the selective 1D nuclear Overhauser effect spectroscopy (NOESY) experiment utilized in liquid-state NMR as a faster (1D instead of 2D) and often less ambiguous (direct sampling of the time domain data, coupled with increased signal averaging) alternative to 2D NOESY. Selective 1D 13 C- 13 C spin-diffusion methods are more time-efficient than their 2D counterparts such as proton-driven spin diffusion (PDSD) and dipolar-assisted rotational resonance. The additional time gained enables measurements of 13 C- 13 C spin-diffusion buildup curves and extraction of spin-diffusion time constants T SD , yielding detailed structural information. Specifically, selective 1D DARR difference buildup curves applied to 13 C-enriched hybrid poplar woody stems confirm strong spatial interaction between lignin and acetylated xylan polymers within poplar plant secondary cell walls, and an interpolymer distance of ∼0.45-0.5 nm was estimated. Additionally, Tyr/Gly long-range correlations were observed on isotopically enriched black widow spider dragline silks.

Silica nanoparticles can be designed to exhibit a diverse range of morphologies (e.g. non-porous, mesoporous), physical properties (e.g. hydrophobic, hydrophilic) and a wide range of chemical and biomolecular surface functionalizations. In the present work, the adsorption complex of histidine (His) and fumed silica nanoparticles (FSN) is probed using thermal analysis (TGA/DTG) and a battery of solid-state (SS) NMR methods supported by DFT chemical shift calculations. Multinuclear (1H/13C/15N) one- and two-dimensional magic angle spinning (MAS) SSNMR experiments were applied to determine site-specific interactions between His and FSN surfaces as a function of adsorption solution concentration, pH and hydration state. By directly comparing SSNMR observables (linewidth, chemical shift and relaxation parameters) for His-FSN adsorption complexes to various crystalline, amorphous and aqueous His forms, the His structural and dynamic environment on FSN surfaces could be determined at an atomic level. The observed 13C and 15N MAS NMR chemical shifts, linewidths and relaxation parameters show that the His surface layer on FSN has a significant dependence on pH and hydration state. His is highly dynamic on FSN surfaces under acidic conditions (pH 4) as evidenced by sharp resonances with near isotropic chemical shifts regardless of hydration level indicating a non-specific binding arrangement while, a considerably more rigid His environment with defined protonation states is observed at near neutral pH with subtle variations between hydrated and anhydrous complexes. At near neutral pH, less charge repulsion occurs on the FSN surface and His is more tightly bound as evidenced by considerable line broadening likely due to chemical shift heterogeneity and a distribution in hydrogen-bonding strengths on the FSN surface. Multiple His sites exchange with a tightly bound water layer in hydrated samples while, direct interaction with the FSN surface and significant chemical shift perturbations for imidazole ring nitrogen sites and some carbon resonances are observed after drying. The SSNMR data was used to propose an interfacial molecular binding model between His and FSN surfaces under varying conditions setting the stage for future multinuclear, multidimensional SSNMR studies of His-containing peptides on silica nanoparticles and other nanomaterials of interest.

A major challenge in liposomal research is to minimize the leakage of encapsulated cargo from either uncontrolled passive permeability across the liposomal membrane or upon fusion with other membranes. We previously showed that liposomes made from pure Archaea-inspired bipolar tetraether lipids exhibit exceptionally low permeability of encapsulated small molecules due to their capability to form more tightly packed membranes compared to typical monopolar lipids. Here, we demonstrate that liposomes made of synthetic bipolar tetraether lipids can also undergo membrane fusion, which is commonly accompanied by content leakage of liposomes when using typical bilayer-forming lipids. Importantly, we demonstrate calcium-mediated fusion events between liposome made of glycerolmonoalkyl glycerol tetraether lipids with phosphatidic acid headgroups (GMGTPA) occur without liposome content release, which contrasts with liposomes made of bilayer-forming EggPA lipids that displayed ~80% of content release under the same fusogenic conditions. NMR spectroscopy studies of a deuterated analog of GMGTPA lipids reveal the presence of multiple rigid and dynamic conformations, which provide evidence for the possibility of these lipids to form intermediate states typically associated with membrane fusion events. The results support that biomimetic GMGT lipids possess several attractive properties (e.g., low permeability and non-leaky fusion capability) for further development in liposome-based technologies.

The dynamics of guests in molecular encapsulation complexes have been studied extensively in solution, but the corresponding behavior of those guests when the capsules are present in the solid state is not as well understood. Here we report on comparative solution 1 H and solid-state 2 H NMR measurements of encapsulation complexes of fluorene(- d 2 ), fluoranthene(- d 10 ), and pyrene-(- d 10 ) in pyrogallol[4]arene hexamers assembled in the solid state by ball milling. In solution, the 1 H spectra show that these rigid guests tumble and exchange positions quickly within the capsules' interiors, with the exception of pyrene, which has slower tumbling and positional exchange. Static solid-state 2 H NMR using the deuterated guests shows that, when the capsules are in the solid state, their guests retain the liquid state-like dynamics observed for the capsules in solution. When the pyrogallol[4]arene hexamers' pendant decyl groups were substituted with propyl groups, guest dynamics in the solid state were slowed. We propose that these pendant alkyl groups form an interdigitated and dynamic waxy domain surrounding the capsules in the solid state, and that the greater mobility of the decyl groups is translated across the walls of the host, resulting in more rapid guest dynamics in the capsules' interiors., Competing Interests: Conflicts of interest There are no conflicts to declare.

Maintaining membrane integrity is a challenge at extreme temperatures. Biochemical synthesis of membrane-spanning lipids is one adaptation that organisms such as thermophilic archaea have evolved to meet this challenge and preserve vital cellular function at high temperatures. The molecular-level details of how these tethered lipids affect membrane dynamics and function, however, remain unclear. Using synthetic monolayer-forming lipids with transmembrane tethers, here, we reveal that lipid tethering makes membrane permeation an entropically controlled process that helps to limit membrane leakage at elevated temperatures relative to bilayer-forming lipid membranes. All-atom molecular dynamics simulations support a view that permeation through membranes made of tethered lipids reduces the torsional entropy of the lipids and leads to tighter lipid packing, providing a molecular interpretation for the increased transition-state entropy of leakage.

Spider venom contains a number of small peptides that can control the gating properties of a wide range of ion channels with high affinity and specificity. These ion channels are responsible for coordination and control of many bodily functions such as transducing signals into sensory functions, smooth muscle contractions as well as serving as sensors in volume regulation. Hence, these peptides have been the topic of many research efforts in hopes that they can be used as biomedical therapeutics. Several peptides are known to control the gating properties of ion channels by involving the lipid membrane. GsMTx4, originally isolated from the Chilean Rose tarantula (Grammostola rosea), is known to selectively inhibit mechanosensitive ion channels by partitioning into the lipid bilayer. To further understand this indirect gating mechanism, we investigated the interactions between native GsAF2, VsTx1 and a synthetic form of GsMTx4 with model DMPC lipid bilayers using 31 P solid-state NMR, 13 C CP-MAS NMR, NS-TEM and cryo-TEM. The results reveal that these inhibitor cystine knot peptides perforate the DMPC lipid vesicles similarly with some subtle differences and ultimately create small spherical vesicles and anisotropic cylindrical and discoidal vesicles at concentrations near 1.0-1.5 mol% peptide. The anisotropic components align with their long axes along the NMR static B 0 magnetic field, a property that should be useful in future NMR structural investigations of these systems. These findings move us forward in our understanding of how these peptides bind and interact with the lipid bilayer., (Copyright © 2018. Published by Elsevier B.V.)

Silk proteins are biopolymers produced by spinning organisms that have been studied extensively for applications in materials engineering, regenerative medicine, and devices due to their high tensile strength and extensibility. This remarkable combination of mechanical properties arises from their unique semi-crystalline secondary structure and block copolymer features. The secondary structure of silks is highly sensitive to processing, and can be manipulated to achieve a wide array of material profiles. Studying the secondary structure of silks is therefore critical to understanding the relationship between structure and function, the strength and stability of silk-based materials, and the natural fiber synthesis process employed by spinning organisms. However, silks present unique challenges to structural characterization due to high-molecular-weight protein chains, repetitive sequences, and heterogeneity in intra- and interchain domain sizes. Here, experimental techniques used to study the secondary structure of silks, the information attainable from these techniques, and the limitations associated with them are reviewed. Ultimately, the appropriate utilization of a suite of techniques discussed here will enable detailed characterization of silk-based materials, from studying fundamental processing-structure-function relationships to developing commercially useful quality control assessments., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many natural silks produced by spiders and insects are unique materials in their exceptional toughness and tensile strength, while being lightweight and biodegradable-properties that are currently unparalleled in synthetic materials. Myriad approaches have been attempted to prepare artificial silks from recombinant spider silk spidroins but have each failed to achieve the advantageous properties of the natural material. This is because of an incomplete understanding of the in vivo spidroin-to-fiber spinning process and, particularly, because of a lack of knowledge of the true morphological nature of spidroin nanostructures in the precursor dope solution and the mechanisms by which these nanostructures transform into micrometer-scale silk fibers. Herein we determine the physical form of the natural spidroin precursor nanostructures stored within spider glands that seed the formation of their silks and reveal the fundamental structural transformations that occur during the initial stages of extrusion en route to fiber formation. Using a combination of solution phase diffusion NMR and cryogenic transmission electron microscopy (cryo-TEM), we reveal direct evidence that the concentrated spidroin proteins are stored in the silk glands of black widow spiders as complex, hierarchical nanoassemblies (∼300 nm diameter) that are composed of micellar subdomains, substructures that themselves are engaged in the initial nanoscale transformations that occur in response to shear. We find that the established micelle theory of silk fiber precursor storage is incomplete and that the first steps toward liquid crystalline organization during silk spinning involve the fibrillization of nanoscale hierarchical micelle subdomains., Competing Interests: The authors declare no conflict of interest.

Solid-State NMR results on 13C-Ala/Ser and 13C-Val enriched Argiope argentata prey-wrapping silk show that native, freshly spun aciniform silk nanofibers are dominated by α-helical (∼50% total) and random-coil (∼35% total) secondary structures, with minor β-sheet nanocrystalline domains (∼15% total). This is the most in-depth study to date characterizing the protein structural conformation of the toughest natural biopolymer: aciniform prey-wrapping silks.

Pyrogallol[4]arene hexamers are hydrogen-bonded molecular capsules of exceptional kinetic stability that can entrap small molecule guests indefinitely, without exchange, at ambient temperatures. Here, we report on the use of a ball mill to induce self-assembly of the capsule components and the guests in the solid state. Stoichiometric amounts of pyrogallol[4]arene and a guest, which can be an arene, alkane, amine, or carboxylic acid, were milled at 30 Hz for fixed durations, dissolved, and characterization by NMR. Most of the resulting encapsulation complexes were kinetically stable but thermodynamically unstable in solution, and the yield of their formation correlates with the duration of the milling and is related to the structures of guest and host. This method extends the scope of molecular encapsulation, as demonstrated by the preparation of kinetically trapped encapsulation complexes of [2.2]paracyclophane, for which we could find no other method of preparation. To gain mechanistic insights into the solid-state assembly process, we characterized the milled powders using 13 C CP-MAS NMR, we studied the effects of changing the alkane domain of the host, and we examined how dissolution conditions impact on the distribution of observed encapsulation complexes once in solution. The results support a mechanism comprising mechanically induced solid-state reorganization to produce a mixture rich in nearly or fully assembled guest-filled capsules.

Liposomal drug-delivery systems have been used for delivery of drugs to targeted tissues while reducing unwanted side effects. DOXIL, for instance, is a liposomal formulation of the anticancer agent doxorubicin (DOX) that has been used to address problems associated with nonspecific toxicity of free DOX. However, while this liposomal formulation allows for a more-stable circulation of doxorubicin in the body compared to free drug, the efficacy for cancer therapy is reduced in comparison with systemic injections of free drug. A robust liposomal system that can be triggered to release DOX in cancer cells could mitigate problems associated with reduced drug efficacy. In this work, we present a serum-stable, cholesterol-integrated tetraether lipid comprising of a cleavable disulfide bond, {GcGT(S-S)PC-CH}, that is designed to respond to the reducing environment of the cell to trigger the release intraliposomal content upon cellular uptake by cancer cells. A cell viability assay revealed that DOX- loaded liposomes composed of pure GcGT(S-S)PC-CH lipids were ∼20 times more toxic than DOXIL, with an IC 50 value comparable to that of free DOX. The low inherent membrane-leakage properties of GcGT(S-S)PC-CH liposomes in the presence of serum, combined with an intracellular triggered release of encapsulated cargo, represents a promising approach for developing improved drug-delivery formulations for the treatment of cancer and possibly other diseases.

In this work, thermal condensation of alanine adsorbed on fumed silica nanoparticles is investigated using thermal analysis and multiple spectroscopic techniques, including infrared (IR), Raman, and nuclear magnetic resonance (NMR) spectroscopies. Thermal analysis shows that adsorbed alanine can undergo thermal condensation, forming peptide bonds within a short time period and at a lower temperature (∼170 °C) on fumed silica nanoparticle surfaces than that in bulk (∼210 °C). Spectroscopic results further show that alanine is converted to alanine anhydride with a yield of 98.8% during thermal condensation. After comparing peptide formation on solution-derived colloidal silica nanoparticles, it is found that fumed silica nanoparticles show much better efficiency and selectivity than solution-derived colloidal silica nanoparticles for synthesizing alanine anhydride. Furthermore, Raman spectroscopy provides evidence that the high efficiency for fumed silica nanoparticles is likely related to their unique surface features: the intrinsic high population of strained ring structures present at the surface. This work indicates the great potential of fumed silica nanoparticles in synthesizing peptides with high efficiency and selectivity.

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5-30-fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time-dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The molecular interactions of silk materials plasticized using glycerol were studied, as these materials provide options for biodegradable and flexible protein-based systems. Plasticizer interactions with silk were analyzed by thermal, spectroscopic, and solid-state NMR analyses. Spectroscopic analysis implied that glycerol was hydrogen bonded to the peptide matrix, but may be displaced with polar solvents. Solid-state NMR indicated that glycerol induced β-sheet formation in the dried silk materials, but not to the extent of methanol treatment. Fast scanning calorimetry suggested that β-sheet crystal formation in silk-glycerol films appeared to be less organized than in the methanol treated silk films. We propose that glycerol may be simultaneously inducing and interfering with β-sheet formation in silk materials, causing some improper folding that results in less-organized silk II structures even after the glycerol is removed. This difference, along with trace residual glycerol, allows glycerol extracted silk materials to retain more flexibility than methanol processed versions.

Solid-state NMR and molecular dynamics (MD) simulations are presented to help elucidate the molecular secondary structure of poly(Gly-Gly-X), which is one of the most common structural repetitive motifs found in orb-weaving dragline spider silk proteins. The combination of NMR and computational experiments provides insight into the molecular secondary structure of poly(Gly-Gly-X) segments and provides further support that these regions are disordered and primarily non-β-sheet. Furthermore, the combination of NMR and MD simulations illustrate the possibility for several secondary structural elements in the poly(Gly-Gly-X) regions of dragline silks, including β-turns, 3 10 -helicies, and coil structures with a negligible population of α-helix observed., Competing Interests: The authors declare no conflict of interest.

This paper examines the effects of four different polar headgroups on small-ion membrane permeability from liposomes comprised of Archaea-inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane-leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of commercial bilayer-forming 1-palmitoyl-2-oleoyl-sn-glycerol (PO) lipids varied by as much as 32-fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

As ligand functionalization of nanomaterials becomes more complex, methods to characterize the organization of multiple ligands on surfaces is required. In an effort to further the understanding of ligand-surface interactions, a combination of multinuclear ((1)H, (29)Si, (31)P) and multidimensional solid-state nuclear magnetic resonance (NMR) techniques was utilized to characterize the phosphonic acid functionalization of fumed silica nanoparticles using methylphosphonic acid (MPA) and phenylphosphonic acid (PPA). (1)H → (29)Si cross-polarization (CP)-magic angle spinning (MAS) solid-state NMR was used to selectively detect silicon atoms near hydrogen atoms (primarily surface species); these results indicate that geminal silanols are preferentially depleted during the functionalization with phosphonic acids. (1)H → (31)P CP-MAS solid-state NMR measurements on the functionalized silica nanoparticles show three distinct resonances shifted upfield (lower ppm) and broadened compared to the resonances of the crystalline ligands. Quantitative (31)P MAS solid-state NMR measurements indicate that ligands favor a monodentate binding mode. When fumed silica nanoparticles were functionalized with an equal molar ratio of MPA and PPA, the MPA bound the nanoparticle surface preferentially. Cross-peaks apparent in the 2D (1)H exchange spectroscopy (EXSY) NMR measurements of the multiligand sample at short mixing times indicate that the MPA and PPA are spatially close (≤5 Å) on the surface of the nanostructure. Furthermore, (1)H-(1)H double quantum-single quantum (DQ-SQ) back-to-back (BABA) 2D NMR spectra further confirmed that MPA and PPA are strongly dipolar coupled with observation of DQ intermolecular contacts between the ligands. DQ experimental buildup curves and simulations indicate that the average distance between MPA and PPA is no further than 4.2 ± 0.2 Å.

Extremophile archaeal organisms overcome problems of membrane permeability by producing lipids with structural elements that putatively improve membrane integrity compared to lipids from other life forms. Herein, we describe a series of lipids that mimic some key structural features of archaeal lipids, such as: 1) single tethering of lipid tails to create fully transmembrane tetraether lipids and 2) the incorporation of small rings into these tethered segments. We found that membranes formed from pure tetraether lipids leaked small ions at a rate that was about two orders of magnitude slower than common bilayer-forming lipids. Incorporation of cyclopentane rings into the tetraether lipids did not affect membrane leakage, whereas a cyclohexane ring reduced leakage by an additional 40 %. These results show that mimicking certain structural features of natural archaeal lipids results in improved membrane integrity, which may help overcome limitations of many current lipid-based technologies., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Spiders utilize fine adjustment of the physicochemical conditions within its silk spinning system to regulate spidroin assembly into solid silk fibers with outstanding mechanical properties. However, the exact mechanism about which this occurs remains elusive and is still hotly debated. In this study, the effect of acidification on spider silk assembly was investigated on native spidroins from the major ampullate (MA) gland fluid excised from Latrodectus hesperus (Black Widow) spiders. Incubating the protein-rich MA silk gland fluid at acidic pH conditions results in the formation of silk fibers that are 10-100 μm in length and ∼2 μm in diameter as judged by optical and electron microscope methods. The in vitro spider silk assembly kinetics were monitored as a function of pH with a (13)C solid-state MAS NMR approach. The results confirm the importance of acidic pH in the spider silk self-assembly process with observation of a sigmoidal nucleation-elongation kinetic profile. The rates of nucleation and elongation as well as the percentage of β-sheet structure in the grown fibers depend on the pH. These results confirm the importance of an acidic pH gradient along the spinning duct for spider silk formation and provide a powerful spectroscopic approach to probe the kinetics of spider silk formation under various biochemical conditions.

The molecular dynamics of the proteins that comprise spider dragline silk were investigated with solid-state (2)H magic angle spinning (MAS) NMR line shape and spin-lattice relaxation time (T1) analysis. The experiments were performed on (2)H/(13)C/(15)N-enriched N. clavipes dragline silk fibers. The silk protein side-chain and backbone dynamics were probed for Ala-rich regions (β-sheet and 31-helical domains) in both native (dry) and supercontracted (wet) spider silk. In native (dry) silk fibers, the side chains in all Ala containing regions undergo similar fast methyl rotations (>10(9) s(-1)), while the backbone remains essentially static (<10(2) s(-1)). When the silk is wet and supercontracted, the presence of water initiates fast side-chain and backbone motions for a fraction of the β-sheet region and 31-helicies. β-Sheet subregion 1 ascribed to the poly(Ala) core exhibits slower dynamics, while β-sheet subregion 2 present in the interfacial, primarily poly(Gly-Ala) region that links the β-sheets to disordered 31-helical motifs, exhibits faster motions when the silk is supercontracted. Particularly notable is the observation of microsecond backbone motions for β-sheet subregion 2 and 31-helicies. It is proposed that these microsecond backbone motions lead to hydrogen-bond disruption in β-sheet subregion 2 and helps to explain the decrease in silk stiffness when the silk is wet and supercontracted. In addition, water mobilizes and softens 31-helical motifs, contributing to the increased extensibility observed when the silk is in a supercontracted state. The present study provides critical insight into the supercontraction mechanism and corresponding changes in mechanical properties observed for spider dragline silks.

Characterizing the nano-bio interface has been a long-standing endeavor in the quest for novel biosensors, biophotovoltaics, and biocompatible electronic devices. In this context, the present computational work on the interaction of two peptides, A6K (Ac-AAAAAAK-NH2) and A7 (Ac-AAAAAAA-NH2) with semiconducting TiO2 nanoparticles is an effort to understand the peptide-metal oxide nanointerface. These investigations were spurred by recent experimental observations that nanostructured semiconducting metal oxides templated with A6K peptides not only stabilize large proteins like photosystem-I (PS-I) but also exhibit enhanced charge-transfer characteristics. Our results indicate that α-helical structures of A6K are not only energetically more stabilized on TiO2 nanoparticles, but the resulting hybrids also exhibit enhanced electron transfer characteristics. This enhancement can be attributed to substantial changes in the electronic characteristics at the peptide-TiO2 interface. Apart from understanding the mechanism of electron transfer (ET) in peptide-stabilized PS-I on metal oxide nanoparticles, the current work also has implications in the development of novel solar cells and photocatalysts.

(2)H-(13)C HETCOR MAS NMR is performed on (2)H/(13)C/(15)N-Pro enriched A. aurantia dragline silk. Proline dynamics are extracted from (2)H NMR line shapes and T1 in a site-specific manner to elucidate the backbone and side chain molecular dynamics for the MaSp2 GPGXX β-turn regions for spider dragline silk in the dry and wet, supercontracted states.

Nuclear magnetic resonance (NMR) and X-ray diffraction (XRD) experiments reveal the structural importance of divalent cation-phosphate complexes in the formation of β-sheet nanocrystals from phosphorylated serine-rich regions within aquatic silk from caddisfly larvae of the species Hesperophyla consimilis. Wide angle XRD data on native caddisfly silk show that the silk contains a significant crystalline component with a repetitive orthorhombic unit cell aligned along the fiber axis with dimensions of 5.9 Å × 23.2 Å × 17.3 Å. These nanocrystalline domains depend on multivalent cations, which can be removed through chelation with ethylenediaminetetraacetic acid (EDTA). A comparison of wide angle X-ray diffraction data before and after EDTA treatment reveals that the integrated peak area of reflections corresponding to the nanocrystalline regions decreases by 15-25% while that of the amorphous background reflections increases by 20%, indicating a partial loss of crystallinity. (31)P solid-state NMR data on native caddisfly silk also show that the phosphorylated serine-rich motifs transform from a rigid environment to one that is highly mobile and water-solvated after treatment with EDTA. The removal of divalent cations through exchange and chelation has therefore caused a collapse of the β-sheet structure. However, NMR results show that the rigid phosphorus environment is mostly recovered after the silk is re-treated with calcium. The (31)P spin-lattice (T1) relaxation times were measured at 7.6 ± 3.1 and 1 ± 0.5 s for this calcium-recovered sample and the native silk sample, respectively. The shorter (31)P T1 relaxation times measured for the native silk sample are attributed to the presence of paramagnetic iron that is stripped away during EDTA chelation treatment and replaced with diamagnetic calcium.

Embiopterans produce silken galleries and sheets using exceptionally fine silk fibers in which they live and breed. In this study, we use electron microscopy (EM), Fourier-transform infrared (FT-IR) spectroscopy, wide angle X-ray diffraction (WAXD) and solid-state nuclear magnetic resonance (ssNMR) techniques to elucidate the molecular level protein structure of webspinner (embiid) silks. Silks from two species Antipaluria urichi and Aposthonia ceylonica are studied in this work. Electron microscopy images show that the fibers are about 90-100 nm in diameter, making webspinner silks among the finest of all known animal silks. Structural studies reveal that the silk protein core is dominated by β-sheet structures, and that the protein core is coated with a hydrophobic alkane-rich surface coating. FTIR spectra of native embiid silk shows characteristic alkane CH 2 stretchings near 2800-2900 cm -1 , which decrease approximately 50% after washing the silk with 2 : 1 CHCl 3 : MeOH. Furthermore, 13 C ssNMR data shows a significant CH 2 resonance that is strongly affected by the presence of water, supporting the idea that the silk fibers are coated with a hydrocarbon-rich layer. Such a layer is likely used to protect the colonies from rain. FTIR data also suggests that embiid silks are dominated by β-sheet secondary structures similar to spider and silkworm silk fibers. NMR data confirms the presence of β-sheet nanostructures dominated by serine-rich repetitive regions. A deconvolution of the serine Cβ NMR resonance reveals that approximately 70% of all seryl residues exist in a β-sheet structure. This is consistent with WAXD results that suggest webspinner silks are 70% crystalline, which is the highest crystalline fraction reported for any animal silks. The work presented here provides a molecular level structural picture of silk fibers produced by webspinners.

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 β-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 β-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 β-sheet (poly(Gly-Ala)) and 3(1)-helical (poly(Gly-Gly-X(aa))) regions, while serine is present in β-sheet (poly(Gly-Ala-Ser)), 3(1)-helix (poly(Gly-Gly-Ser)), and β-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.

The composition of the Sutter's Mill meteorite insoluble organic material was studied both in toto by solid-state NMR spectroscopy of the powders and by gas chromatography-mass spectrometry analyses of compounds released upon their hydrothermal treatment. Results were compared with those obtained for other meteorites of diverse classifications (Murray, GRA 95229, Murchison, Orgueil, and Tagish Lake) and found to be so far unique in regard to the molecular species released. These include, in addition to O-containing aromatic compounds, complex polyether- and ester-containing alkyl molecules of prebiotic appeal and never detected in meteorites before. The Sutter's Mill fragments we analyzed had likely been altered by heat, and the hydrothermal conditions of the experiments realistically mimic early Earth settings, such as near volcanic activity or impact craters. On this basis, the data suggest a far larger availability of meteoritic organic materials for planetary environments than previously assumed and that molecular evolution on the early Earth could have benefited from accretion of carbonaceous meteorites both directly with soluble compounds and, for a more protracted time, through alteration, processing, and release from their insoluble organic materials.

The amino acid composition of Nephila clavipes dragline silk fiber was determined by conducting ¹H nuclear magnetic resonance (NMR) spectroscopy experiments on acid-hydrolyzed material. N. clavipes dragline silk was found to consist of 43.0±0.6% Gly, 29.3±0.2% Ala, 9.1±0.1% Glx, 4.0±0.1% Leu, 3.3±0.1% Tyr, 3.4±0.2% Ser, 2.7±0.1% Pro, 2.1±0.1% Arg, 1.07±0.05% Asx, 0.96±0.05% Val, 0.48±0.03% Thr, 0.35±0.03% Phe, and 0.28±0.03% Ile. Compared with standard chromatography-based amino acid analysis (AAA), the chemical resolution of NMR allows for an amino acid solution to be characterized without separation and is shown to provide considerably higher precision. This allows for more accurate statistics on the variability of amino acids in spider dragline silk. In general, this ¹H NMR AAA technique is applicable to a large range of proteins and peptides for precise composition characterization, especially when the precise content of a minor component is critical and relatively large amounts of sample are available (microgram to milligram quantities)., (Published by Elsevier Inc.)

The interaction between cholesterol (Chol) and phospholipids in bilayers was investigated for the ternary model lipid rafts, DOPC/eSM/Chol and DOPC/DPPC/Chol, with differential scanning calorimetry (DSC) and (13)C cross polarization magic angle spinning (CP-MAS) solid-state NMR. The enthalpy and transition temperature (Tm) of the Lα liquid crystalline phase transition from DSC was used to probe the thermodynamics of the different lipids in the two systems as a function of Chol content. The main chain (13)C (CH2)n resonance is resolved in the (13)C CP-MAS NMR spectra for the unsaturated (DOPC) and saturated (eSM or DPPC) chain lipid in the ternary lipid raft mixtures. The (13)C chemical shift of this resonance can be used to detect differences in chain ordering and overall interactions with Chol for the different lipid constituents in the ternary systems. The combination of DSC and (13)C CP-MAS NMR results indicate that there is a preferential interaction between SM and Chol below Tm for the DOPC/eSM/Chol system when the Chol content is ≤20mol%. In contrast, no preferential interaction between Chol and DPPC is observed in the DOPC/DPPC/Chol system above or below Tm. Finally, (13)C CP-MAS NMR resolves two Chol environments in the DOPC/eSM/Chol system below Tm at Chol contents >20mol% while, a single Chol environment is observed for DOPC/DPPC/Chol at all compositions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Two-dimensional (2D) (1)H-(13)C heteronuclear correlation (HETCOR) solid-state NMR spectra collected with fast magic angle spinning (MAS) are used in conjunction with density functional theory (DFT) proton chemical shift calculations to determine the hydrogen-bonding strength for ordered β-sheet and disordered 310-helical structures in spider dragline silk. The hydrogen-bond strength is determined to be identical for both structures in spider silk with a 1.83-1.84 Å NH···OC hydrogen-bond distance.

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 (1)H NMR was implemented to evaluate incorporation of (13)C/(15)N-labeled amino acids in hydrolyzed spider dragline silk. To determine site-specific (13)C and (15)N isotope enrichments, an analysis method was developed to fit the (1)H-(13)C and (1)H-(15)N J-splitting (J CH and J NH) (1)H NMR peak patterns of hydrolyzed silk fiber. This is demonstrated for Nephila clavipes spiders, where [U-(13)C3,(15)N]-Ala and [1-(13)C,(15)N]-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.

Adhesive silks spun by aquatic caddisfly (order Trichoptera) larvae are used to build both intricate protective shelters and food harvesting nets underwater. In this study, we use (13)C and (31)P solid-state NMR and wide angle X-ray diffraction (WAXD) as tools to elucidate molecular protein structure of caddisfly larval silk from the species Hesperophylax consimilis . Caddisfly larval silk is a fibroin protein based biopolymer containing mostly repetitive amino acid motifs. NMR and X-ray results provide strong supporting evidence for a structural model in which phosphorylated serine repeats (pSX)4 complex with divalent cations Ca(2+) and Mg(2+) to form rigid nanocrystalline β-sheet structures in caddisfly silk. (13)C NMR data suggests that both phosphorylated serine and neighboring valine residues exist in a β-sheet conformation while glycine and leucine residues common in GGX repeats likely reside in random coil conformations. Additionally, (31)P chemical shift anisotropy (CSA) analysis indicates that the phosphates on phosphoserine residues are doubly ionized, and are charge-stabilized by divalent cations. Positively charged arginine side chains also likely play a role in charge stabilization. Finally, WAXD results finds that the silk is at least 7-8% crystalline, with β-sheet interplane spacings of 3.7 and 4.5 Å.

Two-dimensional (2D) cross-polarization magic angle spinning (CP-MAS) (2)H-(13)C heteronuclear correlation (HETCOR) experiments were utilized to indirectly detect site-specific deuterium MAS powder patterns. The (2)H-(13)C cross-polarization efficiency is orientation-dependent and non-uniform for all crystallites. This leads to difficulty in extracting the correct (2)H MAS quadrupole powder patterns. In order to obtain accurate deuterium line shapes, (13)C spin lock rf field, spin lock rf ramp and CP contact time were carefully calibrated with the assistance of theoretical simulations. The extracted quadrupole patterns for U-[(2)H/(13)C/(15)N]-alanine indicate that the methyl deuterium undergoes classic, three-site jumping in the fast motion regime (10(-8)-10(-12)s) and the methine deuterium has a rigid deuterium powder pattern. For U-[(2)H/(13)C/(15)N]-phenylalanine, indirectly detected deuterium line shapes illustrate that the aromatic ring undergoes 180° flips in the fast motion regime while (2)Hβ and (2)Hα are completely rigid. The experimental deuterium line shapes for U-[(2)H/(13)C/(15)N]-proline reflect that (2)Hβ, (2)Hγ and (2)Hδ are subjected to fast, two-site reorientations at an angle of (15±5)°, (30±5)° and (25±10)° respectively. In addition, an approach that combines a composite inversion pulse with (2)H-(13)C CP-MAS is applied to measure (2)H spin-lattice relaxation times in a site-specific, (13)C-detected fashion., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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 β-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.

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: (GPGGX(1) GPGGX(2))(2) with X(1) /X(2) = 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 (GPGGX(1) GPGGX(2))(2) with X(1) /X(2) = 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., (Copyright © 2011 Wiley Periodicals, Inc.)

Stishovite (SiO(2) with the rutile structure and octahedrally coordinated silicon) is an important high-pressure mineral. It has previously been considered to be essentially anhydrous. In this study, hydrothermal treatment of silica glass and coesite at 350-550 °C near 10 GPa produces stishovite with significant amounts of H(2)O in its structure. A combination of methodologies (X-ray diffraction, thermal analysis, oxide melt solution calorimetry, secondary ion mass spectrometry, infrared and nuclear magnetic resonance spectroscopy) indicate the presence of 1.3 ± 0.2 wt % H(2)O and NMR suggests that the primary mechanism for the H(2)O uptake is a direct hydrogarnet-like substitution of 4H(+) for Si(4+), with the protons clustered as hydroxyls around a silicon vacancy. This substitution is accompanied by a substantial volume decrease for the system (SiO(2) + H(2)O), although the stishovite expands slightly, and it is only slightly unfavorable in energy. Stishovite could thus be a host for H(2)O at convergent plate boundaries, and in other relatively cool high-pressure environments.

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.

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.

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).

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.

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.

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.

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., (Copyright 2009 Elsevier Inc. All rights reserved.)