Conformations of a low-complexity protein in homogeneous and phase-separated frozen solutions.

Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below a temperature T. To investigate whether local conformational distributions are detectably different in the homogeneous (i.e.

Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions.

Unlabelled: Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below temperatures T in the 20-40° C range. To investigate whether local conformational distributions are detectably different in the homogeneous and phase-separated states of FUS-LC, we performed solid state nuclear magnetic resonance (ssNMR) measurements on solutions that were frozen on sub-millisecond time scales after equilibration at temperatures well above (50° C) or well below (4° C) T. Measurements were performed at 25 K with signal enhancements from dynamic nuclear polarization.

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.

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-β 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-β 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-β peptide (Aβ and Aβ).

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.

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.

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 assay for RNP formation, and by examining mutant proteins in a viral assembly assay.

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

Misfolded Aβ 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 Aβ strains (termed 2F and 3F) using in vitro and in vivo assays.

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

Self-assembly of amyloid-β 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-β (Aβ40) that provide structural information for oligomers that form on time scales from 0.7 ms to 1.

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

Fibrils formed by the 42-residue amyloid-β peptide (Aβ42), a main component of amyloid deposits in Alzheimer's disease (AD), are known to be polymorphic, i.e., to contain multiple possible molecular structures.

Time-resolved solid state NMR of biomolecular processes with millisecond time resolution.

We review recent efforts to develop and apply an experimental approach to the structural characterization of transient intermediate states in biomolecular processes that involve large changes in molecular conformation or assembly state. This approach depends on solid state nuclear magnetic resonance (ssNMR) measurements that are performed at very low temperatures, typically 25-30 K, with signal enhancements from dynamic nuclear polarization (DNP). This approach also involves novel technology for initiating the process of interest, either by rapid mixing of two solutions or by a rapid inverse temperature jump, and for rapid freezing to trap intermediate states.

Nitroxide-based triradical dopants for efficient low-temperature dynamic nuclear polarization in aqueous solutions over a broad pH range.

Dynamic nuclear polarization (DNP) can provide substantial sensitivity enhancements in solid state nuclear magnetic resonance (ssNMR) measurements on frozen solutions, thereby enabling experiments that would otherwise be impractical. Previous work has shown that nitroxide-based triradical compounds are particularly effective as dopants in DNP-enhanced measurements at moderate magic-angle spinning frequencies and moderate magnetic field strengths, generally leading to a more rapid build-up of nuclear spin polarizations under microwave irradiation than the more common biradical dopants at the same electron spin concentrations. Here we report the synthesis and DNP performance at 25 K and 9.

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

Although amyloid plaques composed of fibrillar amyloid-β (Aβ) 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 Aβ fibrils from brain tissue of nondemented elderly individuals with high amyloid loads differ from structures of Aβ fibrils from AD tissue.

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

Previous studies have shown that racemic mixtures of 40- and 42-residue amyloid-β peptides (d,l-Aβ40 and d,l-Aβ42) form amyloid fibrils with accelerated kinetics and enhanced stability relative to their homochiral counterparts (l-Aβ40 and l-Aβ42), suggesting a "chiral inactivation" approach to abrogating the neurotoxicity of Aβ oligomers (Aβ-CI). Here we report a structural study of d,l-Aβ40 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-Aβ40 fibrils that resemble those in known l-Aβ40 fibril structures.

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

Amyloid-β (Aβ) 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 Aβ peptides (Aβ40), 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.

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 Ca concentrations through its calcium-dependent binding affinities to numerous target proteins. Binding of two Ca 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.

Succinyl-DOTOPA: An effective triradical dopant for low-temperature dynamic nuclear polarization with high solubility in aqueous solvent mixtures at neutral pH.

We report the synthesis of the nitroxide-based triradical compound succinyl-DOTOPA and the characterization of its performance as a dopant for dynamic nuclear polarization (DNP) experiments in frozen solutions at low temperatures. Compared with previously described DOTOPA derivatives, succinyl-DOTOPA has substantially greater solubility in glycerol/water mixtures with pH > 4 and therefore has wider applicability. Solid state nuclear magnetic resonance (ssNMR) measurements at 9.

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.

Coexisting order and disorder within a common 40-residue amyloid-β fibril structure in Alzheimer's disease brain tissue.

Fibrils formed by 40- and 42-residue amyloid-β (Aβ40 and Aβ42) peptides exhibit molecular-level structural polymorphisms. A recent screen of fibrils derived from brain tissue of Alzheimer's disease patients revealed a single predominant Aβ40 polymorph. We present solid state nuclear magnetic resonance (ssNMR) data that define its coexisting structurally ordered and disordered segments.

Depletion of amyloid-β peptides from solution by sequestration within fibril-seeded hydrogels.

Aggregation of amyloid-β (Aβ) peptides in brain tissue leads to neurodegeneration in Alzheimer's disease (AD). Regardless of the kinetics or detailed mechanisms of Aβ aggregation, aggregation can only occur if Aβ concentrations exceed their local equilibrium solubility values. We propose that excess Aβ peptides can be removed from supersaturated solutions, including solutions in biological fluids, by the addition of hydrogels that are seeded with Aβ fibril fragments.

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.

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

Aggregation of amyloid-β 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-β peptides (Aβ40 and Aβ42) are polymorphic, with variations in molecular structure that depend on fibril growth conditions. Recent experiments suggest that variations in amyloid-β 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.

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

Self-assembly of amyloid-β (Aβ) 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 Aβ 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.

Modeling an in-register, parallel "iowa" aβ fibril structure using solid-state NMR data from labeled samples with rosetta.

Determining the structures of amyloid fibrils is an important first step toward understanding the molecular basis of neurodegenerative diseases. For β-amyloid (Aβ) fibrils, conventional solid-state NMR structure determination using uniform labeling is limited by extensive peak overlap. We describe the characterization of a distinct structural polymorph of Aβ using solid-state NMR, transmission electron microscopy (TEM), and Rosetta model building.