Fluorescent labeling or tagging of viral proteins is crucial for understanding molecular virology and guiding antiviral interventions. However, traditional methods using large tags are often invasive, impeding visualization of viral proteins in their native states. We detail a methodology combining an amber-free lentiviral packaging system with amber (TAG stop codon) suppression - genetic code expansion and site-specific bioorthogonal click labeling. Amber suppression enables site-specific incorporation of a non-canonical amino acid (ncAA) bearing strained alkynes or alkenes into a target protein. The ncAA-containing viral protein can then be labeled with a fluorophore conjugated with a click-chemistry-reactive functional group via copper-free click chemistry, allowing single-residue fluorescent labeling with minimal impact on protein structure and function. However, amber stop codons that serve as native termination signals in viral genomes can be inadvertently suppressed during genetic code expansion, leading to protein readthrough and unpredictable experimental outcomes. Here, we provide a step-by-step method for constructing a complete amber-free lentivirus, which overcomes this limitation by providing an amber-free viral background. We then describe a stepwise workflow for single ncAA incorporation into the Omicron spike decorated on amber-free lentiviruses, followed by the attachment of organic fluorophores via click chemistry. The methods described here are, in principle, adaptable to other viral surface glycoproteins, non-surface lentiviral proteins, and pseudotyped viral systems, with appropriate optimization and validation as needed.
The use of sugars with bioorthogonal functionalities introduced into living cells or added enzymatically has proven to be a valuable research tool for defining the functional roles of glycosylation in cellular systems. An alternative chemical approach to introducing bioorthogonal groups to specific sugars is described herein, one that specifically targets biologically important terminal sialic acid isomers attached to glycoconjugates in tissues and cells on slides. The most common sialic acid isomers are attached in either α2,3, α2,6, or α2,8 linkages to glycan termini which confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences using standard mass spectrometry approaches. Herein, a sequential double amidation chemical derivatization strategy is described that results in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3 and α2,8-linked sialic acids in the second amidation step. This allows for targeted detection of cells in tissue or individually on glass slides using mass spectrometry imaging or microscopy approaches, as well as distinct mass shifts from α2,6-linked amidated isomers created in the first amidation reaction step. Use of an alkyne-amine like propargylamine as the second amidation reagent introduces different mass shifts for α2,3-linked and α2,8-linked sialic acids. Use of an azide-amine with a poly-ethylene glycol linker introduces a sterically approachable azide group on α2,3-lined and α2,8-linked sialic acids. This azide group can be targeted for click chemistry reactions with biotin-alkyne for streptavidin-conjugated imaging by histochemical stains and fluorescence. Detailed protocols for several example uses are provided.
Osteoarthritis (OA) is a prevalent degenerative joint disease with an unclear molecular pathogenesis. B4GALT1 has been implicated in various pathological processes, but its role and mechanism in OA remain largely unexplored. The expression of B4GALT1 was examined in OA clinical samples and experimental OA models. In vivo OA models were established by destabilization of the medial meniscus (DMM) in mice, while in vitro OA models were generated by lipopolysaccharide (LPS) stimulation of human chondrocytes. CCK-8, flow cytometry, qRT-PCR, and Western blot were employed to investigate the role of B4GALT1 in chondrocyte viability, apoptosis, and inflammatory response. The B4GALT1-IL-1R1 protein interaction was analyzed by Co-IP, and N-glycosylation was assessed using PNGase F treatment and site-directed mutagenesis. OA progression was further evaluated by SO/FG staining, H&E, and X-ray following intra-articular AAV-mediated B4GALT1 knockdown in DMM mice. B4GALT1 was upregulated in OA samples. Functionally, B4GALT1 overexpression exacerbated LPS-induced chondrocyte apoptosis, decline in cell viability, and inflammatory cytokine imbalance (increased TNF-α and IL-1β, decreased IL-13), while its knockdown reversed these effects. Mechanistically, B4GALT1 directly interacted with IL-1R1 and promoted its N-linked glycosylation, specifically at the N193 site, thereby enhancing IL-1R1 protein stability. In vivo, AAV-mediated knockdown of B4GALT1 attenuated DMM-induced cartilage degeneration, joint space narrowing, and inflammatory responses, concomitant with reduced IL-1R1 protein levels. B4GALT1 is upregulated in OA and promotes disease progression by stabilizing IL-1R1 via N-glycosylation at the N193 site. Targeting B4GALT1 may represent a promising therapeutic strategy for the treatment of OA.
Amyloid fibrils formed by amyloid proteins such as α-synuclein (α-syn), Amyloid-β (Aβ) and Tau are central to the pathology of neurodegenerative diseases like Parkinson's disease (PD) and Alzheimer's disease (AD). Structural elucidation of fibril-ligand interactions is essential for the rational design of imaging probes and therapeutic inhibitors targeting these pathological aggregates. Here, we present a comprehensive cryo-electron microscopy (cryo-EM)-based workflow for modeling small-molecule binding to amyloid fibrils, with a focus on α-syn-ligand complexes. The protocol integrates optimized fibril sample preparation, helical reconstruction, and iterative 2D/3D classification to yield high-resolution density maps suitable for atomic modeling. Ligands are incorporated by generating coordinates from SMILES strings and restraint files from Phenix eLBOW, followed by manual docking and real-space refinement. Using CCA-α-syn complex as a case study, we demonstrate precise ligand placement into specific fibril binding sites (the C-pocket, N-pocket, and a back-surface groove of the fibril core distinct from typical globular protein pockets). Subsequent structural refinement preserved key interaction features, including π-π stacking and side-chain hydrogen bonding. Validation metrics confirm the stereochemical integrity and good model-to-map fit of the final fibril-ligand complex structures. Overall, this workflow enables accurate modeling of ligand engagement with amyloids even at ∼3-4 Å resolution and provides a scalable framework for structure-guided ligand discovery in neurodegenerative disease research.
Site-specific fluorescent tagging enables visualization of protein conformational changes and molecular interactions on native biological assemblies. Using the HIV-1 envelope (Env) glycoprotein as an example, this protocol details a site-specific dual-fluorophore tagging strategy suitable for downstream single-molecule Förster resonance energy transfer (smFRET) studies. Genetic code expansion via amber (TAG stop codon) suppression, combined with click chemistry, offers a minimally invasive route to such labeling. However, the sequential incorporation of noncanonical amino acids (ncAAs) at two introduced amber codons often faces low efficiency and off-target readthrough at naturally occurring amber sites in the viral genome. To mitigate these technical constraints, the methods employ an engineered "intact amber-free HIV-1 provirus" in which endogenous TAG codons in essential viral genes are replaced with TAA. This design allows precise incorporation of ncAA trans-cyclooct-2-en-L-lysine (TCO*A) exclusively at two engineered amber sites within Env during virion production in HEK293T cells, followed by labeling via strain-promoted inverse electron-demand Diels-Alder cycloaddition with tetrazine-conjugated fluorophores. The method supports site-specific dual-click labeling for smFRET analysis and single-site labeling for live-cell imaging. The methods presented here provide stepwise procedures for click-chemistry labeling and visualization of Env on intact virions and can be easily adapted for single- or dual-site biorthogonal tagging of other viral and cellular proteins.
The human placenta is a major site of metabolic transformation, synthesizing and catabolizing a wide range of bioactive compounds and thereby contributing to pregnancy success. Monoamine oxidase (MAO) is an important component of placental catabolic systems, catalyzing the oxidative deamination of biogenic monoamines, including serotonin and catecholamines. We developed a high-throughput fluorimetric assay using six concentrations of kynuramine as substrate to determine the kinetic parameters - maximum velocity (Vmax) and Michaelis affinity constant (Km) - of MAO activity in human placental tissue. Pharmacological experiments with selective MAO-A and MAO-B inhibitors identified MAO-A as the sole catalytically active MAO isoform in human term placenta. We applied the assay to placental samples from 93 women to assess whether maternal overweight/obesity (OWO) and/or gestational diabetes mellitus (GDM) are associated with changes in placental MAO-A kinetic parameters, and to examine the relationship between placental MAOA mRNA levels and MAO-A catalytic capacity. Maternal OWO was not associated with changes in Vmax or Km, nor was GDM associated with changes in Vmax (all p>0.05). However, GDM was associated with a modest increase in Km (p=0.024), indicating reduced substrate affinity. In metabolically healthy pregnancies, placental MAOA mRNA levels correlated positively with Vmax (rp=0.68, p=0.001), while this relationship was absent in placentas from women with OWO and/or GDM (p>0.05). Our findings suggest no alterations in placental monoamine catabolism in pregnancies complicated by maternal OWO, but indicate possible subtle changes in those complicated by GDM. The positive correlation between placental MAOA expression and MAO-A catalytic capacity in metabolically healthy pregnancies supports the use of MAOA mRNA levels as a proxy for MAO-A catalytic activity under physiological conditions. However, metabolic disturbances may disrupt this coupling, underscoring the value of the standardized kinetic assay described here as a robust tool for future studies of placental MAO-A function.
Efficient protein labeling with minimal linkage error is a key requirement for super-resolution fluorescence microscopy. Many commonly used labeling strategies, including antibodies, fluorescent proteins, and self-labeling enzymes are limited by steric hindrance, and therefore they limit labeling density or constrain labeling to protein termini, thereby restricting achievable resolution at the molecular scale. Here, we describe a practical and broadly applicable protocol for site-specific protein labeling based on genetic code expansion and bioorthogonal inverse electron-demand Diels-Alder click chemistry. The method relies on the incorporation of a strained alkene-modified noncanonical amino acid at a defined position within a protein of interest, followed by rapid and selective covalent labeling with tetrazine-conjugated organic fluorophores. This approach enables the attachment of small, bright dyes with minimal linkage error and is compatible with both live-cell and fixed-cell imaging. The protocol provides detailed guidance on the design of suitable click sites, expression of amber mutants in mammalian cells, selection of appropriate tetrazine dyes, and optimization of labeling conditions for super-resolution microscopy, including single-molecule localization microscopy. Critical parameters, common pitfalls, and limitations are discussed to facilitate robust implementation across different protein classes and experimental systems. This workflow supports high-density, stoichiometric labeling and enables molecular-scale imaging of proteins in their native cellular context.
Selective chemical labeling of endogenous proteins in living organisms is an important approach for analyzing intact protein function without genetic manipulation. Recently, we achieved the labeling of neurotransmitter receptors in the living brain by employing ligand-directed chemistry that enables covalent modification of endogenous proteins. By designing labeling reagents usable in the living brain and investigating their administration methods, we successfully labeled multiple major neurotransmitter receptors, including AMPA, NMDA, mGlu1, and GABAA receptors. The covalent labeling properties enabled analysis of fixed brain samples, achieving imaging of endogenous receptors under co-immunolabeling and tissue clearing conditions. Furthermore, by utilizing the pulse-tagging of target receptors, we analyzed the degradation kinetics and translocation dynamics of active receptors. Here, we describe in detail a protocol for the selective covalent labeling of neurotransmitter receptors in the brains of living mice. This protocol provides neurobiologists with a powerful method for visualizing and quantitatively analyzing the function and dynamics of endogenous receptors without genetic manipulation.
Lasso peptides (LaPs) are a structurally unique class of ribosomally synthesized and post-translationally modified peptides (RiPPs), characterized by a threaded rotaxane topology that confers exceptional stability and a broad spectrum of bioactivities. Despite increasing interest in LaPs as antimicrobial and therapeutic agents, accurate structure prediction remains a major challenge due to the scarcity of homologous templates and the failure of mainstream prediction tools to capture their knotted topology. In this chapter, we present LassoPred, a modular, machine learning-guided computational pipeline for high-throughput 3D structure prediction of lasso peptides from sequence alone. LassoPred integrates support vector machine classifiers trained on ESM2 embeddings to annotate critical topological features-namely, the isopeptide ring and plug residues-and a topology-aware structure constructor that assembles and refines atomic models via homology modeling, residue mutation, and energy minimization. The tool is implemented in Python, compatible with AMBER and PyMOL, and accessible via a public web interface, enabling both expert and non-specialist users to submit sequences and retrieve optimized structural models. LassoPred demonstrates near-experimental accuracy while reducing prediction time to minutes, expanding known lasso peptide structural coverage from fewer than 50 experimentally determined structures to over 4000 genome-mined models. The pipeline is further extensible to engineered LaP variants and potentially other RiPP families. By bridging the gap between sequence discovery and structural insight, LassoPred facilitates downstream applications in enzymology, structural biology, synthetic biology, and therapeutic design.
A wide range of distinctive helical filamentous amyloid structures self-assembled from monomeric peptide or protein building blocks are found both in nature and in human disease states. Amyloid nano-fibrils are also being developed and synthetically made as novel peptide-based nanomaterials. In humans, accumulation of a range of amyloid structures, for example those formed from the amyloid-beta peptides in Alzheimer's disease, play a crucial role in the pathology of neurodegenerative and metabolic diseases. The diverse range of amyloid structures found is a manifestation of the amyloid structural polymorphism phenomenon. This is where different filament structures are assembled even from the same peptide or protein precursors. Due to the structural diversity of amyloid fibrils that can be found even in the same sample or the same disease state, an experimental method that allows structural analysis of individual amyloid filaments is required to understand the relationships between the polymorphic structures and the biological and physicochemical properties they elicit. Here, a method with a detailed protocol to analyze the structures of individual amyloid filament assemblies by topological Atomic Force Microscopy (AFM) imaging and Contact-Point Reconstruction AFM (CPR-AFM) image analysis is described. This approach to resolve the 3D shapes of amyloid polymorphs, one individual fibril at a time, allows mapping of the polymorphic landscapes of amyloid assemblies. It serves as an inexpensive, fast and effective experimental tool for individual filament level structural analysis, and offers new, exciting opportunities in elucidating population distributions of heterogeneous amyloid samples, rare amyloid structures within the populations, and structural variations between or within individual filaments. These are all key parts to experimental developments in therapeutic discovery and novel bio-nanomaterials applications.
Biosynthetic gene clusters (BGC) are genomic regions that encode the production of specialized metabolites, including antibiotics, pigments, and toxins. While BGC are traditionally classified into broad categories such as NRPS, PKS, and terpene clusters, these classes often overlook finer relationships among gene clusters that produce structurally or functionally related compounds. Tools like BiG-SCAPE and BiG-SLiCE have been developed to address this issue by organizing BGC into gene cluster families (GCFs). CORASON complements these tools by enabling phylogenetic reconstruction of BGC, identifying conserved core genes, and visualizing GFCs as a continuum of variation in gene presence/absence and sequence identity. Although CORASON is incorporated in BiG-SCAPE visualization, it is also a standalone tool initially designed for bacterial genomes annotated via RAST and implemented through Docker in Linux environments. Here, we demonstrate CORASON's broader applicability using fungal GenBank files and its installation via Conda on Windows. As a case study, we examine metagenome-assembled genomes (MAGs) from Fusarium domesticum, a lesser-known member of the Fusarium genus, which is often present in food-associated microbiomes. Unlike its pathogenic relatives (F. oxysporum, F. graminearum), F. domesticum remains understudied, making it an interesting target for genomic mining. This work expands the accessibility of CORASON for fungal genome analysis and highlights its potential in uncovering novel biosynthetic potential in overlooked microbial taxa.
Nature uses disulfide bonds and some unusual inter-strand chemical crosslinks to rigidify protein structure and achieve specific function. Inspired by Nature, protein scientists have employed genetic code expansion technology to introduce latent electrophilic amino acids into protein structure for specific crosslinking with nearby nucleophilic residues via the proximity-driven reactions. Herein, we describe the experimental protocol for recombinant production of orthogonal crosslinked monobodies afforded by a genetically encoded β-lactam-lysine (BeLaK) and their characterization by gel electrophoresis and mass spectrometry. When introduced to the N-terminal β-strand of a series of supercharged monobodies, BeLaK enables efficient inter-strand crosslinking with a proximal lysine on a neighboring β-strand. Compared to its non-crosslinked counterpart, a BeLaK-crosslinked, +18-charged monobody showed higher thermal stability and greater cell permeability. The discovery of this BeLaK-crosslinked, rigidified immunoglobulin fold should facilitate the design of cell-permeable monobodies as potential protein-based therapies targeting the intracellular signaling proteins.
Lipids regulate a broad spectrum of cellular functions through spatiotemporally controlled lipid-protein interactions. Dysregulation of lipid metabolism and lipid-mediated signaling is associated with diverse human diseases, including cancer, metabolic disorders, and neurodegenerative conditions. Consequently, selective inhibition of site-specific lipid-protein interactions has emerged as a promising therapeutic strategy to modulate aberrant cell signaling at the membrane interface. Here, we present a streamlined, quantitative workflow for the discovery, characterization, and evaluation of small-molecule inhibitors that disrupt lipid-dependent membrane association and activation of cytosolic signaling proteins. The protocol integrates a high-throughput fluorescence-quenching assay for inhibitor screening, detailed biochemical and cellular target-validation methods, and standardized procedures for in vitro and in vivo assessment of inhibitor potency, specificity, and safety. These general protocols provide a versatile and reproducible platform for developing potent, specific, and mechanistically defined inhibitors targeting a wide range of lipid-binding proteins implicated in disease.
Understanding how phospholipids orchestrate cellular signaling requires tools that preserve their native complexity to reveal their modular function. Here, we introduce a versatile chemical approach to map the phosphatidic acid (PA) interactome directly within membranes of living cells. Using synthetic PA analogues, modified on the glycerol backbone with a bioorthogonal azide group, we generated functional probes that retain natural headgroup and acyl-chain composition and properties. These analogues can be coupled to photoactivatable crosslinkers, allowing covalent capture of transient PA-protein partners upon UV illumination. We present here approaches to validate their integration and functionality through recruitment assays using PA-binding sensor and ERK1/2 phosphorylation readouts. Applying this framework in neurosecretory PC12 cells, we identified specific PA-binding partners across distinct secretory states, revealing both acyl chain-dependent and activity-specific interaction patterns. This methodology bridges lipid chemistry and cell biology by enabling dynamic, species-specific exploration of phospholipid signaling networks. Beyond neurosecretion, the approach provides a broadly applicable platform for dissecting lipid-mediated processes across diverse physiological and pathological contexts, from membrane trafficking to disease-associated lipid signaling.
Bioorthogonal reactions enable selective biomolecular labeling in complex biological environments with minimal perturbation, and activatable (caged) reagents further provide spatial and temporal control of labeling. In this chapter, we apply a molecular engineering framework to cyclopropene-tetrazine ligation by treating cyclopropene reactivity as a tunable property that can be programmed through substituent electronics, scaffold architecture, and caging-group installation. Building on established activatable cyclopropene designs, we focus on strategies aimed at increasing ligation kinetics through structural constraint of an azaspirocyclopropene framework. Although the targeted ring-contracted scaffold was not sufficiently stable for isolation and direct evaluation in tetrazine ligation, these studies enabled access to functionalized 4-azaspiro[2.3]hexanes from dibromo intermediates. This scaffold offers a practical entry point to piperidine bioisosteres and expands the synthetic utility of the platform beyond molecular labeling. Overall, this work highlights how molecular-level design decisions govern stability and reactivity in activatable cyclopropenes and outlines actionable principles for engineering next-generation tetrazine-ligation partners.
Bioorthogonal chemical reporters have revolutionized the study of glycosylation by enabling the selective labeling, visualization, and enrichment of glycans in living systems. Monosaccharide analogs bearing small, inert bioorthogonal tags such as azides, alkynes, cyclopropenes or sydnones, are metabolically incorporated into glycoconjugates by the cell's endogenous glycosyltransferases (GTs). Subsequent chemoselective ligation (e.g., strain-promoted alkyne-azide cycloaddition or inverse electron-demand Diels-Alder reactions) permits downstream detection or pull-down with minimal perturbation to cellular physiology. This chapter demonstrates the impact of a sydnone bioorthogonal chemical reporter on sialyltransferases (STs) substrate tolerance and catalytic efficiency. While many STs display surprising promiscuity toward modified CMP-sialic acid donors, incorporation efficiencies vary widely depending on the position and steric demand of the bioorthogonal handle, and can alter kinetic parameters, and influence donor/acceptor specificity. Herein, we present detailed protocols for assessing the impact of bioorthogonal chemical reporters, with a focus on 1,3-dipoles, on the activity of recombinant human sialyltransferases. The methods include qualitative evaluation by in-gel fluorescence imaging and quantitative analysis through determination of enzyme-specific kinetic parameters. These protocols can be extended to other glycosyltransferases collectively expanding the enzymatic toolkit for glycobiology, enabling additional mechanistic studies in the interrogation of glycosylation dynamics and GT function.
The ability of native and engineered nucleic acid-processing enzymes to incorporate clickable nucleotide substrates has greatly advanced bioorthogonal labeling of nucleic acids, overcoming the limitations of conventional solid-phase oligonucleotide (ON) synthesis. In this chemoenzymatic approach, template-dependent polymerases routinely enable the incorporation of nucleotides bearing small reactive handles. The resulting nucleic acids undergo chemoselective reactions, such as azide-alkyne cycloaddition, inverse-electron-demand Diels-Alder, or Staudinger ligation, to install desired functionalities. Alternatively, the promiscuity of template-independent transferases, such as terminal uridylyl transferase (TUTase), provides access to site-specific labeling of RNA ONs at the 3'-end. In this methods chapter, we detail protocols for incorporating azide-modified UTP analogs into short RNA ONs and highly structured CRISPR guide RNAs (sgRNAs) using the terminal uridylyl transferase SpCID1. We describe methods to control the enzyme's incessant incorporation behavior and enable subsequent click functionalization of the RNAs. Finally, we demonstrate remodeling of the CRISPR system via synthesis of azide-modified sgRNAs, which when complexed with dCas9, recruit azide groups to specific gene targets for post-hybridization functionalization.
Among the many strategies for bioconjugation, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is a particularly powerful click chemistry tool due to its rapid kinetics and the commercial availability of a wide range of conjugates. Although CuAAC has been widely adopted for labeling biomolecules on living cell surfaces, intracellular labeling in live cells remains challenging. The elevated copper concentration needed to drive the CuAAC reaction efficiently can compromise cell death, restricting intracellular CuAAC labeling to cross-linked cell systems. To address this limitation, inCu-click, an intracellular CuAAC platform that utilizes a DNA-conjugated ligand (inCu-click ligand) to localize and concentrate copper ions at the intended reaction site, was developed. The inCu-click system enables efficient click chemistry under reduced intracellular copper levels, without the need for additional copper salts. This design supports liposomal delivery of the ligand into cells for intracellular CuAAC labeling. Using this platform, we demonstrate reliable and robust fluorescent labeling of nascent phospholipids and alkyne drugs in live cells with minimal impact on viability. Collectively, inCu-click provides a practical, broadly applicable platform for real-time visualization of biomolecular dynamics in complex live-cell environments.
Prenylation is a lipid post-translational modification (PTM) on proteins that plays a critical role in regulating the interaction of proteins with the membrane. Approximately 2 % of proteins in cells are prenylated. Dysregulation of prenylation has been implicated in several diseases where it affects protein localization and function of these proteins in the cell. Ras proteins are members of a broad family of small GTPases that are prenylated and therefore able to localize to the cell membrane and turn on downstream signaling. Because of these roles, inhibiting the prenylation of oncogenic Ras proteins may serve as a method to control Ras mutation-related cancers. To understand the mechanism of Ras protein prenylation and subsequent interactions, chemically modified forms of Ras can be particularly useful. In this chapter, a platform to prepare modified Ras proteins using expressed protein ligation is described. The workflow involves the expression and purification of a truncated Ras thioester protein, the synthesis of a hypervariable region peptide, and the subsequent ligation of these two fragments to obtain the full-length protein.
Extracellular plaques comprised of amyloid-β (Aβ) are a defining hallmark of Alzheimer's disease. However, it remains debated whether these insoluble fibrils or the soluble oligomers that precede them are the toxic species responsible for pathology. Aβ oligomers are difficult to study by conventional biophysical techniques because of their transient nature and heterogeneous size. While Aβ40 oligomers are invisible to solution nuclear magnetic resonance (NMR) spectroscopy due to their slow tumbling, the intrinsically disordered monomeric form is readily detected by solution NMR. Under appropriate conditions, oligomers rapidly form at atmospheric pressure and can be dissociated by the application of ∼2.5 kbar hydrostatic pressure. By incorporating pressure jumps within NMR experiments, features of the NMR-invisible oligomers can be probed indirectly by detecting the NMR-visible monomer. This chapter discusses operation of a home-built pressure-jump apparatus and its application to measuring oligomeric relaxation times, chemical shifts, and 1H-1H NOE transfers. Aβ40 oligomers have particularly short 15N T2 relaxation times and long T1 relaxation times, especially for residues V18-A21 and I31-L34, indicating that these oligomers contain highly ordered regions that tumble slowly in solution. 1H-1H NOEs further reveal that Aβ40 oligomers contain hydrogen bonds between V18 and M35, between F20 and G33, and between E22 and I31. Zn2+ ions accelerate oligomer formation and promote homogeneous, rigid assemblies with reduced internal motion. Some of the technology development was carried out using Amelotin, an intrinsically disordered protein that also forms pressure-sensitive oligomers but is less prone to irreversible fibril formation and more amenable to large-scale perdeuterated expression.