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.

Selective One-Dimensional 13C-13C Spin-Diffusion Solid-State Nuclear Magnetic Resonance Methods to Probe Spatial Arrangements in Biopolymers Including Plant Cell Walls, Peptides, and Spider Silk.

Two-dimensional (2D) and 3D through-space 13C-13C homonuclear spin-diffusion techniques are powerful solid-state nuclear magnetic resonance (NMR) tools for extracting structural information from 13C-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 13C-13C 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 13C-13C spin-diffusion methods described here apply in select cases in which the 1D 13C 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 13C-13C 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 13C-13C spin-diffusion buildup curves and extraction of spin-diffusion time constants TSD, yielding detailed structural information. Specifically, selective 1D DARR difference buildup curves applied to 13C-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.

Fusion of Bipolar Tetraether Lipid Membranes Without Enhanced Leakage of Small Molecules.

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.

2H NMR Reveals Liquid State-Like Dynamics of Arene Guests Inside Hexameric Pyrogallol[4]arene Capsules in the Solid State.

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 1H and solid-state 2H 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 1H 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 2H 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.