Molecular structure of ß-amyloid fibrils in Alzheimer's disease brain tissue.

In vitro, ß-amyloid (Aß) peptides form polymorphic fibrils, with molecular structures that depend on growth conditions, plus various oligomeric and protofibrillar aggregates. Here, we investigate structures of human brain-derived Aß fibrils, using seeded fibril growth from brain extract and data from solid-state nuclear magnetic resonance and electron microscopy. Experiments on tissue from two Alzheimer's disease (AD) patients with distinct clinical histories showed a single predominant 40 residue Aß (Aß40) fibril structure in each patient; however, the structures were different from one another. A molecular structural model developed for Aß40 fibrils from one patient reveals features that distinguish in-vivo- from in-vitro-produced fibrils. The data suggest that fibrils in the brain may spread from a single nucleation site, that structural variations may correlate with variations in AD, and that structure-specific amyloid imaging agents may be an important future goal.

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Structures of brain-derived 42-residue amyloid-ß fibril polymorphs with unusual molecular conformations and intermolecular interactions.

Fibrils formed by the 42-residue amyloid-ß peptide (Aß42), a main component of amyloid deposits in Alzheimer's disease (AD), are known to be polymorphic, i.e., to contain multiple possible molecular structures. Previous studies of Aß42 fibrils, including fibrils prepared entirely in vitro or extracted from brain tissue and using solid-state NMR (ssNMR) or cryogenic electron microscopy (cryo-EM) methods, have found polymorphs with differences in amino acid sidechain orientations, lengths of structurally ordered segments, and contacts between cross-ß subunit pairs within a single filament. Despite these differences, Aß42 molecules adopt a common S-shaped conformation in all previously described high-resolution Aß42 fibril structures. Here we report two cryo-EM-based structures of Aß42 fibrils that are qualitatively different, in samples derived from AD brain tissue by seeded growth. In type A fibrils, residues 12 to 42 adopt a ν-shaped conformation, with both intra-subunit and intersubunit hydrophobic contacts to form a compact core. In type B fibrils, residues 2 to 42 adopt an υ-shaped conformation, with only intersubunit contacts and internal pores. Type A and type B fibrils have opposite helical handedness. Cryo-EM density maps and molecular dynamics simulations indicate intersubunit K16-A42 salt bridges in type B fibrils and partially occupied K28-A42 salt bridges in type A fibrils. The coexistence of two predominant polymorphs, with differences in N-terminal dynamics, is supported by ssNMR data, as is faithful propagation of structures from first-generation to second-generation brain-seeded Aß42 fibril samples. These results demonstrate that Aß42 fibrils can exhibit a greater range of structural variations than seen in previous studies.

Two structurally defined Aß polymorphs promote different pathological changes in susceptible mice.

Misfolded Aß is involved in the progression of Alzheimer's disease (AD). However, the role of its polymorphic variants or conformational strains in AD pathogenesis is not fully understood. Here, we study the seeding properties of two structurally defined synthetic misfolded Aß strains (termed 2F and 3F) using in vitro and in vivo assays. We show that 2F and 3F strains differ in their biochemical properties, including resistance to proteolysis, binding to strain-specific dyes, and in vitro seeding. Injection of these strains into a transgenic mouse model produces different pathological features, namely different rates of aggregation, formation of different plaque types, tropism to specific brain regions, differential recruitment of Aß40 /Aß42 peptides, and induction of microglial and astroglial responses. Importantly, the aggregates induced by 2F and 3F are structurally different as determined by ssNMR. Our study analyzes the biological properties of purified Aß polymorphs that have been characterized at the atomic resolution level and provides relevant information on the pathological significance of misfolded Aß strains.

Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca2+-ligated calmodulin.

Recent advances in rapid mixing and freeze quenching have opened the path for time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state NMR of protein-substrate interactions. DEER, in conjunction with phase memory time filtering to quantitatively extract species populations, permits monitoring time-dependent probability distance distributions between pairs of spin labels, while solid-state NMR provides quantitative residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein. Here, we demonstrate the power of these combined approaches to unravel the kinetic and structural pathways in the binding of the intrinsically disordered peptide substrate (M13) derived from myosin light-chain kinase to the universal eukaryotic calcium regulator, calmodulin. Global kinetic analysis of the data reveals coupled folding and binding of the peptide associated with large spatial rearrangements of the two domains of calmodulin. The initial binding events involve a bifurcating pathway in which the M13 peptide associates via either its N- or C-terminal regions with the C- or N-terminal domains, respectively, of calmodulin/4Ca2+ to yield two extended "encounter" complexes, states A and A*, without conformational ordering of M13. State A is immediately converted to the final compact complex, state C, on a timescale τ ≤ 600 µs. State A*, however, only reaches the final complex via a collapsed intermediate B (τ ∼ 1.5 to 2.5 ms), in which the peptide is only partially ordered and not all intermolecular contacts are formed. State B then undergoes a relatively slow (τ ∼ 7 to 18 ms) conformational rearrangement to state C.

Experimental Evidence for Millisecond-Timescale Structural Evolution Following the Microsecond-Timescale Folding of a Small Protein.

Prior work has shown that small proteins can fold (i.e., convert from unstructured to structured states) within 10 µs. Here we use time-resolved solid state nuclear magnetic resonance (ssNMR) methods to show that full folding of the 35-residue villin headpiece subdomain (HP35) requires a slow annealing process that has not been previously detected. ^{13}C ssNMR spectra of frozen HP35 solutions, acquired with a variable time τ_{e} at 30 °C after rapid cooling from 95 °C and before rapid freezing, show changes on the 3-10 ms timescale, attributable to slow rearrangements of protein sidechains during τ_{e}.

Structural differences in amyloid-ß fibrils from brains of nondemented elderly individuals and Alzheimer's disease patients.

Although amyloid plaques composed of fibrillar amyloid-ß (Aß) assemblies are a diagnostic hallmark of Alzheimer's disease (AD), quantities of amyloid similar to those in AD patients are observed in brain tissue of some nondemented elderly individuals. The relationship between amyloid deposition and neurodegeneration in AD has, therefore, been unclear. Here, we use solid-state NMR to investigate whether molecular structures of Aß fibrils from brain tissue of nondemented elderly individuals with high amyloid loads differ from structures of Aß fibrils from AD tissue. Two-dimensional solid-state NMR spectra of isotopically labeled Aß fibrils, prepared by seeded growth from frontal lobe tissue extracts, are similar in the two cases but with statistically significant differences in intensity distributions of cross-peak signals. Differences in solid-state NMR data are greater for 42-residue amyloid-ß (Aß42) fibrils than for 40-residue amyloid-ß (Aß40) fibrils. These data suggest that similar sets of fibril polymorphs develop in nondemented elderly individuals and AD patients but with different relative populations on average.

Molecular structure of a prevalent amyloid-ß fibril polymorph from Alzheimer's disease brain tissue.

Amyloid-ß (Aß) fibrils exhibit self-propagating, molecular-level polymorphisms that may contribute to variations in clinical and pathological characteristics of Alzheimer's disease (AD). We report the molecular structure of a specific fibril polymorph, formed by 40-residue Aß peptides (Aß40), that is derived from cortical tissue of an AD patient by seeded fibril growth. The structure is determined from cryogenic electron microscopy (cryoEM) images, supplemented by mass-per-length (MPL) measurements and solid-state NMR (ssNMR) data. Previous ssNMR studies with multiple AD patients had identified this polymorph as the most prevalent brain-derived Aß40 fibril polymorph from typical AD patients. The structure, which has 2.8-Å resolution according to standard criteria, differs qualitatively from all previously described Aß fibril structures, both in its molecular conformations and its organization of cross-ß subunits. Unique features include twofold screw symmetry about the fibril growth axis, despite an MPL value that indicates three Aß40 molecules per 4.8-Å ß-sheet spacing, a four-layered architecture, and fully extended conformations for molecules in the central two cross-ß layers. The cryoEM density, ssNMR data, and MPL data are consistent with ß-hairpin conformations for molecules in the outer cross-ß layers. Knowledge of this brain-derived fibril structure may contribute to the development of structure-specific amyloid imaging agents and aggregation inhibitors with greater diagnostic and therapeutic utility.

Structural variation in amyloid-ß fibrils from Alzheimer's disease clinical subtypes.

Aggregation of amyloid-ß peptides into fibrils or other self-assembled states is central to the pathogenesis of Alzheimer's disease. Fibrils formed in vitro by 40- and 42-residue amyloid-ß peptides (Aß40 and Aß42) are polymorphic, with variations in molecular structure that depend on fibril growth conditions. Recent experiments suggest that variations in amyloid-ß fibril structure in vivo may correlate with variations in Alzheimer's disease phenotype, in analogy to distinct prion strains that are associated with different clinical and pathological phenotypes. Here we investigate correlations between structural variation and Alzheimer's disease phenotype using solid-state nuclear magnetic resonance (ssNMR) measurements on Aß40 and Aß42 fibrils prepared by seeded growth from extracts of Alzheimer's disease brain cortex. We compared two atypical Alzheimer's disease clinical subtypes-the rapidly progressive form (r-AD) and the posterior cortical atrophy variant (PCA-AD)-with a typical prolonged-duration form (t-AD). On the basis of ssNMR data from 37 cortical tissue samples from 18 individuals, we find that a single Aß40 fibril structure is most abundant in samples from patients with t-AD and PCA-AD, whereas Aß40 fibrils from r-AD samples exhibit a significantly greater proportion of additional structures. Data for Aß42 fibrils indicate structural heterogeneity in most samples from all patient categories, with at least two prevalent structures. These results demonstrate the existence of a specific predominant Aß40 fibril structure in t-AD and PCA-AD, suggest that r-AD may relate to additional fibril structures and indicate that there is a qualitative difference between Aß40 and Aß42 aggregates in the brain tissue of patients with Alzheimer's disease.

Application of millisecond time-resolved solid state NMR to the kinetics and mechanism of melittin self-assembly.

Common experimental approaches for characterizing structural conversion processes such as protein folding and self-assembly do not report on all aspects of the evolution from an initial state to the final state. Here, we demonstrate an approach that is based on rapid mixing, freeze-trapping, and low-temperature solid-state NMR (ssNMR) with signal enhancements from dynamic nuclear polarization (DNP). Experiments on the folding and tetramerization of the 26-residue peptide melittin following a rapid pH jump show that multiple aspects of molecular structure can be followed with millisecond time resolution, including secondary structure at specific isotopically labeled sites, intramolecular and intermolecular contacts between specific pairs of labeled residues, and overall structural order. DNP-enhanced ssNMR data reveal that conversion of conformationally disordered melittin monomers at low pH to α-helical conformations at neutral pH occurs on nearly the same timescale as formation of antiparallel melittin dimers, about 6 to 9 ms for 0.3 mM melittin at 24 °C in aqueous solution containing 20% (vol/vol) glycerol and 75 mM sodium phosphate. Although stopped-flow fluorescence data suggest that melittin tetramers form quickly after dimerization, ssNMR spectra show that full structural order within melittin tetramers develops more slowly, in ∼60 ms. Time-resolved ssNMR is likely to find many applications to biomolecular structural conversion processes, including early stages of amyloid formation, viral capsid formation, and protein-protein recognition.

Constraints on the Structure of Fibrils Formed by a Racemic Mixture of Amyloid-ß Peptides from Solid-State NMR, Electron Microscopy, and Theory.

Previous studies have shown that racemic mixtures of 40- and 42-residue amyloid-ß peptides (d,l-Aß40 and d,l-Aß42) form amyloid fibrils with accelerated kinetics and enhanced stability relative to their homochiral counterparts (l-Aß40 and l-Aß42), suggesting a "chiral inactivation" approach to abrogating the neurotoxicity of Aß oligomers (Aß-CI). Here we report a structural study of d,l-Aß40 fibrils, using electron microscopy, solid-state nuclear magnetic resonance (NMR), and density functional theory (DFT) calculations. Two- and three-dimensional solid-state NMR spectra indicate molecular conformations in d,l-Aß40 fibrils that resemble those in known l-Aß40 fibril structures. However, quantitative measurements of 13C-13C and 15N-13C distances in selectively labeled d,l-Aß40 fibril samples indicate a qualitatively different supramolecular structure. While cross-ß structures in mature l-Aß40 fibrils are comprised of in-register, parallel ß-sheets, our data indicate antiparallel ß-sheets in d,l-Aß40 fibrils, with alternation of d and l molecules along the fibril growth direction, i.e., antiparallel "rippled sheet" structures. The solid-state NMR data suggest the coexistence of d,l-Aß40 fibril polymorphs with three different registries of intermolecular hydrogen bonds within the antiparallel rippled sheets. DFT calculations support an energetic preference for antiparallel alignments of the ß-strand segments identified by solid-state NMR. These results provide insight into the structural basis for Aß-CI and establish the importance of rippled sheets in self-assembly of full-length, naturally occurring amyloidogenic peptides.

Millisecond Time-Resolved Solid-State NMR Reveals a Two-Stage Molecular Mechanism for Formation of Complexes between Calmodulin and a Target Peptide from Myosin Light Chain Kinase.

Calmodulin (CaM) mediates a wide range of biological responses to changes in intracellular Ca2+ concentrations through its calcium-dependent binding affinities to numerous target proteins. Binding of two Ca2+ ions to each of the two four-helix-bundle domains of CaM results in major conformational changes that create a potential binding site for the CaM binding domain of a target protein, which also undergoes major conformational changes to form the complex with CaM. Details of the molecular mechanism of complex formation are not well established, despite numerous structural, spectroscopic, thermodynamic, and kinetic studies. Here, we report a study of the process by which the 26-residue peptide M13, which represents the CaM binding domain of skeletal muscle myosin light chain kinase, forms a complex with CaM in the presence of excess Ca2+ on the millisecond time scale. Our experiments use a combination of selective 13C labeling of CaM and M13, rapid mixing of CaM solutions with M13/Ca2+ solutions, rapid freeze-quenching of the mixed solutions, and low-temperature solid state nuclear magnetic resonance (ssNMR) enhanced by dynamic nuclear polarization. From measurements of the dependence of 2D 13C-13C ssNMR spectra on the time between mixing and freezing, we find that the N-terminal portion of M13 converts from a conformationally disordered state to an α-helix and develops contacts with the C-terminal domain of CaM in about 2 ms. The C-terminal portion of M13 becomes α-helical and develops contacts with the N-terminal domain of CaM more slowly, in about 8 ms. The level of structural order in the CaM/M13/Ca2+ complexes, indicated by 13C ssNMR line widths, continues to increase beyond 27 ms.

Oncological Safety and Technical Feasibility of Nipple-Sparing Mastectomy for Breast Cancer: The Hong Kong Experience.

BACKGROUND: Nipple-sparing mastectomy (NSM) has gained widespread popularity in recent years. Nonetheless, patient selection, technical consideration and oncological safety of its extension to breast cancer treatment remain uncertain. Few publications have reviewed the application of NSM in Asian populations. METHODS: We retrospectively reviewed 91 women with malignant breast tumours, who underwent 97 NSM in Hong Kong Sanatorium and Hospital from 2009 to 2015. Breast cancer patients who required mastectomy and opted for immediate reconstruction were considered for NSM if they showed no obvious nipple involvement clinically. All breast specimens were subjected to intraoperative pathological examination of the retroareolar tissue to exclude occult tumour infiltration before the final decision of nipple-areola complex (NAC) preservation. Clinical parameters, tumour characteristics and oncological outcomes were analyzed. RESULTS: Carcinoma of the breast accounts for 99.0% of our indications for therapeutic NSM. Almost all NSM were accompanied with immediate reconstruction. Abnormal pathology was shown in retroareolar tissue of ten patients (10.3%), and seven of these NAC were excised due to tumour involvement detected by intraoperative frozen section. Six (6.2%) NSM were complicated with superficial epidermolysis. Yet, there was no delayed NAC excision because of nipple necrosis. Overall NAC preservation rate reached 92.8%. Local and/or distant recurrences occurred in four patients (4.1%) after a mean follow-up of 20.6 months. One NAC recurrence was documented. CONCLUSION: Our series support the oncological safety of NSM after exclusion of neoplastic NAC involvement preliminarily by intraoperative frozen section and definitively by final pathology. Its technical feasibility is well proven by the low nipple necrosis rate.

Fibrillation of ß amyloid peptides in the presence of phospholipid bilayers and the consequent membrane disruption.

Fibrillation of ß amyloid (Aß) peptides and the accumulation of amyloid plaques are considered as an important clinical hallmark to identify Alzheimer's disease (AD). The physiological connection between Aß plaques and the disruption of neuronal cells has not been clearly understood. One hypothesis to explain the Aß neurotoxicity is that the fibrillation process induces disruption to the cellular membrane. We studied the Aß fibrillation process in two biologically relevant conditions with the peptide either pre-incorporated into or externally added to the synthetic phospholipid bilayers. These two sample preparation conditions mimic the physiological membrane proximities of Aß peptides before and after the enzymatic cleavage of amyloid precursor protein (APP). Using thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM), we were able to monitor the kinetics and morphological evolution of fibril formation, which was highly sensitive to the two sample preparation protocols. While the external addition protocol generates long and mature fibrils through normal fibrillation process, the pre-incubation protocol was found to stabilize the immature protofibrils. Fluorescence spectroscopy studies with doubly-labeled phospholipids indicated that there may be a lipid uptake process associated with the fibril formation. Solid state nuclear magnetic resonance (NMR) spectroscopy provided evidence for high resolution structural variations in fibrils formed with different protocols, and in particular the stabilization of long-range contact between N- and C-terminal ß strands. In addition, disruption of phospholipid bilayers was supported by measurements with ³¹P chemical shifts and relaxation time constants.

Successive Stages of Amyloid-ß Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization.

Self-assembly of amyloid-ß (Aß) peptides in human brain tissue leads to neurodegeneration in Alzheimer's disease (AD). Amyloid fibrils, whose structures have been extensively characterized by solid state nuclear magnetic resonance (ssNMR) and other methods, are the thermodynamic end point of Aß self-assembly. Oligomeric and protofibrillar assemblies, whose structures are less well-understood, are also observed as intermediates in the assembly process in vitro and have been implicated as important neurotoxic species in AD. We report experiments in which the structural evolution of 40-residue Aß (Aß40) is monitored by ssNMR measurements on frozen solutions prepared at four successive stages of the self-assembly process. Measurements on transient intermediates are enabled by ssNMR signal enhancements from dynamic nuclear polarization (DNP) at temperatures below 30 K. DNP-enhanced ssNMR data reveal a monotonic increase in conformational order from an initial state comprised primarily of monomers and small oligomers in solution at high pH, to larger oligomers near neutral pH, to metastable protofibrils, and finally to fibrils. Surprisingly, the predominant molecular conformation, indicated by (13)C NMR chemical shifts and by side chain contacts between F19 and L34 residues, is qualitatively similar at all stages. However, the in-register parallel ß-sheet supramolecular structure, indicated by intermolecular (13)C spin polarization transfers, does not develop before the fibril stage. This work represents the first application of DNP-enhanced ssNMR to the characterization of peptide or protein self-assembly intermediates.

Antiparallel ß-sheet architecture in Iowa-mutant ß-amyloid fibrils.

Wild-type, full-length (40- and 42-residue) amyloid ß-peptide (Aß) fibrils have been shown by a variety of magnetic resonance techniques to contain cross-ß structures in which the ß-sheets have an in-register parallel supramolecular organization. In contrast, recent studies of fibrils formed in vitro by the Asp23-to-Asn mutant of 40-residue Aß (D23N-Aß(1-40)), which is associated with early onset neurodegeneration, indicate that D23N-Aß(1-40) fibrils can contain either parallel or antiparallel ß-sheets. We report a protocol for producing structurally pure antiparallel D23N-Aß(1-40) fibril samples and a series of solid state nuclear magnetic resonance and electron microscopy measurements that lead to a specific model for the antiparallel D23N-Aß(1-40) fibril structure. This model reveals how both parallel and antiparallel cross-ß structures can be constructed from similar peptide monomer conformations and stabilized by similar sets of interactions, primarily hydrophobic in nature. We find that antiparallel D23N-Aß(1-40) fibrils are thermodynamically metastable with respect to conversion to parallel structures, propagate less efficiently than parallel fibrils in seeded fibril growth, and therefore must nucleate more efficiently than parallel fibrils in order to be observable. Experiments in neuronal cell cultures indicate that both antiparallel and parallel D23N-Aß(1-40) fibrils are cytotoxic. Thus, our antiparallel D23N-Aß(1-40) fibril model represents a specific "toxic intermediate" in the aggregation process of a disease-associated Aß mutant.

Structure of Amyloid Peptide Ribbons Characterized by Electron Microscopy, Atomic Force Microscopy, and Solid-State Nuclear Magnetic Resonance.

Polypeptides often self-assemble to form amyloid fibrils, which contain cross-ß structural motifs and are typically 5-15 nm in width and micrometers in length. In many cases, short segments of longer amyloid-forming protein or peptide sequences also form cross-ß assemblies but with distinctive ribbon-like morphologies that are characterized by a well-defined thickness (on the order of 5 nm) in one lateral dimension and a variable width (typically 10-100 nm) in the other. Here, we use a novel combination of data from solid-state nuclear magnetic resonance (ssNMR), dark-field transmission electron microscopy (TEM), atomic force microscopy (AFM), and cryogenic electron microscopy (cryoEM) to investigate the structures within amyloid ribbons formed by residues 14-23 and residues 11-25 of the Alzheimer's disease-associated amyloid-ß peptide (Aß14-23 and Aß11-25). The ssNMR data indicate antiparallel ß-sheets with specific registries of intermolecular hydrogen bonds. Mass-per-area values are derived from dark-field TEM data. The ribbon thickness is determined from AFM images. For Aß14-23 ribbons, averaged cryoEM images show a periodic spacing of ß-sheets. The combined data support structures in which the amyloid ribbon growth direction is the direction of intermolecular hydrogen bonds between ß-strands, the ribbon thickness corresponds to the width of one ß-sheet (i.e., approximately the length of one molecule), and the variable ribbon width is a variable multiple of the thickness of one ß-sheet (i.e., a multiple of the repeat distance in a stack of ß-sheets). This architecture for a cross-ß assembly may generally exist within amyloid ribbons.

From Milliseconds to Minutes: Melittin Self-Assembly from Concerted Non-Equilibrium Pressure-Jump and Equilibrium Relaxation Nuclear Magnetic Resonance.

Non-equilibrium kinetics techniques like pressure-jump nuclear magnetic resonance (NMR) are powerful in tracking changes in oligomeric populations and are not limited by relaxation rates for the time scales of exchange that can be probed. However, these techniques are less sensitive to minor, transient populations than are Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. We integrated non-equilibrium pressure-jump and equilibrium CPMG relaxation dispersion data to fully map the kinetic landscape of melittin tetramerization. While monomeric peptides weakly form dimers (Kd,D/M ≈ 26 mM) whose population never exceeds 1.6% at 288 K, dimers associate tightly to form stable tetrameric species (Kd,T/D ≈ 740 nM). Exchange between the monomer and dimer, along with exchange between the dimer and tetramer, occurs on the millisecond time scale. The NMR approach developed herein can be readily applied to studying the folding and misfolding of a wide range of oligomeric assemblies.

Early events in amyloid-ß self-assembly probed by time-resolved solid state NMR and light scattering.

Self-assembly of amyloid-ß peptides leads to oligomers, protofibrils, and fibrils that are likely instigators of neurodegeneration in Alzheimer's disease. We report results of time-resolved solid state nuclear magnetic resonance (ssNMR) and light scattering experiments on 40-residue amyloid-ß (Aß40) that provide structural information for oligomers that form on time scales from 0.7 ms to 1.0 h after initiation of self-assembly by a rapid pH drop. Low-temperature ssNMR spectra of freeze-trapped intermediates indicate that ß-strand conformations within and contacts between the two main hydrophobic segments of Aß40 develop within 1 ms, while light scattering data imply a primarily monomeric state up to 5 ms. Intermolecular contacts involving residues 18 and 33 develop within 0.5 s, at which time Aß40 is approximately octameric. These contacts argue against ß-sheet organizations resembling those found previously in protofibrils and fibrils. Only minor changes in the Aß40 conformational distribution are detected as larger assemblies develop.

Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38±10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.