Phase Networks of Cross-β Peptide Assemblies

Recent evidence suggests that simple peptides can access diverse amphiphilic phases, and that these structures underlie the robust and widely distributed assemblies implicated in nearly 40 protein misfolding diseases. Here we exploit a minimal nucleating core of the Aβ peptide of Alzheimer’s disease to map its morphologically accessible phases that include stable intermolecular molten particles, fibers, twisted and helical ribbons, and nanotubes. Analyses with both fluorescence lifetime imaging microscopy (FLIM) and transmission electron microscopy provide evidence for liquid–liquid phase separations, similar to the coexisting dilute and dense protein-rich liquid phases so critical for the liquid–solid transition in protein crystallization. We show that the observed particles are critical for transitions to the more ordered cross-β peptide phases, which are prevalent in all amyloid assemblies, and identify specific conditions that arrest assembly at the phase boundaries. We have identified a size dependence of the particles in order to transition to the para-crystalline phase and a width of the cross-β assemblies that defines the transition between twisted fibers and helically coiled ribbons. These experimental results reveal an interconnected network of increasing molecularly ordered cross-β transitions, greatly extending the initial computational models for cross-β assemblies

Kinetic Intermediates in Amyloid Assembly

In contrast to an expected Ostwald-like ripening of amyloid assemblies, the nucleating core of the Dutch mutant of the Aβ peptide of Alzheimer’s disease assembles through a series of conformational transitions. Structural characterization of the intermediate assemblies by isotope-edited IR and solid-state NMR reveals unexpected strand orientation intermediates and suggests new nucleation mechanisms in a progressive assembly pathway

Characterization of a Mixture of CO₂ Adsorption Products in Hyperbranched Aminosilica Adsorbents by ¹³C Solid-State NMR

Hyperbranched amine polymers (HAS) grown from the mesoporous silica SBA-15 (hereafter “SBA-15–HAS”) exhibit large capacities for CO₂ adsorption. We have used static in situ and magic-angle spinning (MAS) ex situ ¹³C nuclear magnetic resonance (NMR) to examine the adsorption of CO₂ by SBA-15–HAS. ¹³C NMR distinguishes the signal of gas-phase ¹³CO₂ from that of the chemisorbed species. HAS polymers possess primary, secondary, and tertiary amines, leading to multiple chemisorption reaction outcomes, including carbamate (RnNCOO^–), carbamic acid (RnNCOOH), and bicarbonate (HCO₃^–) moieties. Carbamates and bicarbonate fall within a small ¹³C chemical shift range (162–166 ppm), and a mixture was observed including carbamic acid and carbamate, the former disappearing upon evacuation of the sample. By examining the ¹³C–¹⁴N dipolar coupling through low-field (<i>B</i>₀ = 3 T) ¹³C­{¹H} cross-polarization MAS NMR, carbamate is confirmed through splitting of the ¹³C resonance. A third species that is either bicarbonate or a second carbamate is evident from bimodal <i>T</i>₂ decay times of the ∼163 ppm peak, indicating the presence of two species comprising that single resonance. The mixture of products suggests that (1) the presence of amines and water leads to bicarbonate being present and/or (2) the multiple types of amine sites in HAS permit formation of chemically distinct carbamates

Design of Asymmetric Peptide Bilayer Membranes

Energetic insights emerging from the structural characterization of peptide cross-β assemblies have enabled the design and construction of robust asymmetric bilayer peptide membranes. Two peptides differing only in their N-terminal residue, phosphotyrosine vs lysine, coassemble as stacks of antiparallel β-sheets with precisely patterned charged lattices stabilizing the bilayer leaflet interface. Either homogeneous or mixed leaflet composition is possible, and both create nanotubes with dense negative external and positive internal solvent exposed surfaces. Cross-seeding peptide solutions with a preassembled peptide nanotube seed leads to domains of different leaflet architecture within single nanotubes. Architectural control over these cross-β assemblies, both across the bilayer membrane and along the nanotube length, provides access to highly ordered asymmetric membranes for the further construction of functional mesoscale assemblies

Spectroscopic Characterization of Adsorbed ¹³CO₂ on 3‑Aminopropylsilyl-Modified SBA15 Mesoporous Silica

Multiple chemisorption products are found from the interaction of CO₂ with the solid-amine sorbent, 3-aminopropyl silane (APS), bound to mesoporous silica (SBA15) using solid-state NMR and FTIR spectroscopy. We employed a combination of both ¹⁵N­{¹³C} rotational-echo double-resonance (REDOR) NMR and ¹³C­{¹⁵N} REDOR to determine the chemical identity of these products. ¹⁵N­{¹³C} REDOR measurements are consistent with a single ¹³C–¹⁵N pair and distance of 1.45 Å. In contrast, both ¹³C­{¹⁵N} REDOR and ¹³C CPMAS are consistent with multiple ¹³C products. ¹³C CPMAS shows two neighboring resonances, whose chemical shifts are consistent with carbamate (at 165 ppm) and carbamic acid. The ¹³C­{¹⁵N} REDOR experiments resonant at 165 ppm show an incomplete buildup of the REDOR data to ∼90% of the expected maximum. We conclude this 10% missing intensity corresponds to a ¹³C NMR species that resonates at the identical chemical shift but that is not in dipolar contact with ¹⁵N. These data are consistent with the presence of bicarbonate, HCO₃^–, since it is commonly observed at ∼165 ppm and lacks ¹⁵N for dipolar coupling

Neurofibrillar Tangle Surrogates: Histone H1 Binding to Patterned Phosphotyrosine Peptide Nanotubes

Living cells contain a range of densely phosphorylated surfaces, including phospholipid membranes, ribonucleoproteins, and nucleic acid polymers. Hyperphosphorylated surfaces also accumulate in neurodegenerative diseases as neurofibrillar tangles. We have synthesized and structurally characterized a precisely patterned phosphotyrosine surface and establish this assembly as a surrogate of the neuronal tangles by demonstrating its high-affinity binding to histone H1. This association with nucleic acid binding proteins underscores the role such hyperphosphorylated surfaces may play in disease and opens functional exploration into protein–phosphorylated surface interactions in a wide range of other complex assemblies

Rational Design of Helical Nanotubes from Self-Assembly of Coiled-Coil Lock Washers

Design of a structurally defined helical assembly is described that involves recoding of the amino acid sequence of peptide <b>GCN4-pAA</b>. In solution and the crystalline state, <b>GCN4-pAA</b> adopts a 7-helix bundle structure that resembles a supramolecular lock washer. Structurally informed mutagenesis of the sequence of <b>GCN4-pAA</b> afforded peptide <b>7HSAP1</b>, which undergoes self-association into a nanotube via noncovalent interactions between complementary interfaces of the coiled-coil lock-washer structures. Biophysical measurements conducted in solution and the solid state over multiple length scales of structural hierarchy are consistent with self-assembly of nanotube structures derived from 7-helix bundle subunits. The dimensions of the supramolecular assemblies are similar to those observed in the crystal structure of <b>GCN4-pAA</b>. Fluorescence studies of the interaction of <b>7HSAP1</b> with the solvatochromic fluorophore PRODAN indicated that the nanotubes could encapsulate shape-appropriate small molecules with high binding affinity

Rational Design of Helical Nanotubes from Self-Assembly of Coiled-Coil Lock Washers

Design of a structurally defined helical assembly is described that involves recoding of the amino acid sequence of peptide <b>GCN4-pAA</b>. In solution and the crystalline state, <b>GCN4-pAA</b> adopts a 7-helix bundle structure that resembles a supramolecular lock washer. Structurally informed mutagenesis of the sequence of <b>GCN4-pAA</b> afforded peptide <b>7HSAP1</b>, which undergoes self-association into a nanotube via noncovalent interactions between complementary interfaces of the coiled-coil lock-washer structures. Biophysical measurements conducted in solution and the solid state over multiple length scales of structural hierarchy are consistent with self-assembly of nanotube structures derived from 7-helix bundle subunits. The dimensions of the supramolecular assemblies are similar to those observed in the crystal structure of <b>GCN4-pAA</b>. Fluorescence studies of the interaction of <b>7HSAP1</b> with the solvatochromic fluorophore PRODAN indicated that the nanotubes could encapsulate shape-appropriate small molecules with high binding affinity