Proline-Tyrosine Ring Interactions in Black Widow Dragline Silk Revealed by Solid-State Nuclear Magnetic Resonance and Molecular Dynamics Simulations.

Selective one-dimensional 13C-13C spin-diffusion solid-state nuclear magnetic resonance (SSNMR) provides evidence for CH/π ring packing interactions between Pro and Tyr residues in 13C-enriched Latrodectus hesperus dragline silk. The secondary structure of Pro-containing motifs in dragline spider silks consistently points to an elastin-like type II ß-turn conformation based on 13C chemical shift analysis. 13C-13C 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.

Tyrosine's Unique Role in the Hierarchical Assembly of Recombinant Spider Silk Proteins: From Spinning Dope to Fibers.

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-ß-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-ß-sheet domains that contain Tyr.

Biphasic nature of lipid bilayers assembled on silica nanoparticles and evidence for an interdigitated phase.

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 Lα bilayer phase, the molecular organization of lipids assembled on mesoscale (4-100 nm diameter) nanoparticles is scarce. Here, DSC, TEM and 2H and 31P solid-state NMR are implemented to probe the organization of 1,2-dipalmitoyl-d54-glycero-3-phosphocholine (DMPC-d54) assembled on mesoscale silica nanoparticles illustrating a significant deviation from Lα 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).

Investigating the Atomic and Mesoscale Interactions that Facilitate Spider Silk Protein Pre-Assembly.

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 ß-sheet forming domains in fibers, are perturbed by urea, suggesting that these regions may be important for silk protein pre-assembly stabilization.

Hierarchical spidroin micellar nanoparticles as the fundamental precursors of spider silks.

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.

Probing the binding modes and dynamics of histidine on fumed silica surfaces by solid-state NMR.

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.

Experimental Methods for Characterizing the Secondary Structure and Thermal Properties of Silk Proteins.

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.

Thiol-Triggered Release of Intraliposomal Content from Liposomes Made of Extremophile-Inspired Tetraether Lipids.

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 IC50 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.

Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum-Stable Membranes with Low Leakage.

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.

Effect of Headgroups on Small-Ion Permeability across Archaea-Inspired Tetraether Lipid Membranes.

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.

Solid-State NMR Characterization of Mixed Phosphonic Acid Ligand Binding and Organization on Silica Nanoparticles.

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.

Thermal and Structural Properties of Silk Biomaterials Plasticized by Glycerol.

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 ß-sheet formation in the dried silk materials, but not to the extent of methanol treatment. Fast scanning calorimetry suggested that ß-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 ß-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.

Processing of meteoritic organic materials as a possible analog of early molecular evolution in planetary environments.

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.

Secondary Structure Adopted by the Gly-Gly-X Repetitive Regions of Dragline Spider Silk.

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-ß-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 ß-turns, 310-helicies, and coil structures with a negligible population of α-helix observed.

Cyclohexane Rings Reduce Membrane Permeability to Small Ions in Archaea-Inspired Tetraether Lipids.

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.

Probing the Impact of Acidification on Spider Silk Assembly Kinetics.

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 ß-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.

Molecular dynamics of spider dragline silk fiber investigated by 2H MAS NMR.

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 (ß-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 ß-sheet region and 31-helicies. ß-Sheet subregion 1 ascribed to the poly(Ala) core exhibits slower dynamics, while ß-sheet subregion 2 present in the interfacial, primarily poly(Gly-Ala) region that links the ß-sheets to disordered 31-helical motifs, exhibits faster motions when the silk is supercontracted. Particularly notable is the observation of microsecond backbone motions for ß-sheet subregion 2 and 31-helicies. It is proposed that these microsecond backbone motions lead to hydrogen-bond disruption in ß-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.

Probing lipid-cholesterol interactions in DOPC/eSM/Chol and DOPC/DPPC/Chol model lipid rafts with DSC and (13)C solid-state NMR.

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.

Reversible assembly of ß-sheet nanocrystals within caddisfly silk.

Nuclear magnetic resonance (NMR) and X-ray diffraction (XRD) experiments reveal the structural importance of divalent cation-phosphate complexes in the formation of ß-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 ß-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.

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.