Dillan Stengel

Spider silk proteins (spidroins) are large, block-copolymer-like proteins that must remain soluble at high concentration in the spinning dope while being primed for rapid fiber formation. Understanding how these intrinsically disordered proteins organize in solution is key to explaining the transformation from soluble dope to solid fibers with exceptional strength and toughness. Here, we show that major ampullate (Ma) spidroins from the black widow spider form dynamic ensembles that include metastable tubular substructures. Multiscale molecular dynamics (MD) simulations reveal compact, anisotropic monomers with tubular geometries ∼3–4 nm in diameter and 50 nm in contour length. Small-angle X-ray scattering (SAXS) ensemble fitting confirms that a minority population of tubular conformers is required to reproduce experimental scattering profiles. Complementary atomistic MD and solution NMR chemical shift and relaxation analyses show that these tubular conformers are enriched in β-turn and bend motifs, maintaining local flexibility while promoting overall compaction. Mutational simulations further demonstrate that alternating poly(Ala) and Gly-Gly-X sequence patterning drives amphiphilic packing that stabilizes the tubular morphology. Together, these findings reveal that spider silk proteins form dynamic, disordered ensembles with sequence-encoded tubular substructure. This model reconciles SAXS and NMR observations and provides a mechanistic framework for how amphiphilic patterning, metastability, and disorder collectively enable spider silk proteins to remain soluble yet preorganized for hierarchical self-assembly into one of nature's toughest materials.

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 ¹H and solid-state ²H NMR measurements of encapsulation complexes of fluorene(-d₂), fluoranthene(-d₁₀), and pyrene(-d₁₀) 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.

Significance: The true physical form of the proteins within the silk glands of spiders that permits storage at very high concentrations rather than as precipitated material prior to being transformed into solid silk fibers remains one of the fundamental mysteries that has limited our ability to produce artificial silks of the quality of natural silks. Here we determine that spider silk proteins are stored as complex micellar nanoparticles composed of assembled subdomains. When extruded during the silk spinning process, these subdomains undergo fibrillization while remaining assembled in micelles. Knowledge of the nanostructured protein assemblies in the dope is critical to the basic understanding of the spinning process and to our ability to mimic the natural material properties in synthetic analogues.

Solid-State NMR results on ¹³ C-Ala/Ser and ¹³ C-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).