Lateral Lipid Diffusion Dominates NOESY Cross-Relaxation in Membranes

Lateral Lipid Diffusion Dominates NOESY Cross-Relaxation in Membrane

Structural Evolution of Iowa Mutant β-Amyloid Fibrils from Polymorphic to Homogeneous States under Repeated Seeded Growth

Structural variations in β-amyloid fibrils are potentially important to the toxicity of these fibrils in Alzheimer’s disease (AD). We describe a repeated seeding protocol that selects a homogeneous fibril structure from a polymorphic initial state in the case of 40-residue β-amyloid fibrils with the Asp23-to-Asn, or Iowa, mutation (D23N-Aβ1−40). We use thioflavin T (ThT) fluorescence, transmission electron microscopy (TEM), and solid-state nuclear magnetic resonance (NMR) to track the evolution of fibril structure through multiple generations under this protocol. The data show that (i) repeated seeding selectively amplifies a single D23N-Aβ1−40 fibril structure that can be a minor component of the initial polymorphic state; (ii) the final structure is highly sensitive to growth conditions, including pH, temperature, and agitation; (iii) although the initial state can include fibrils that contain both antiparallel and parallel β-sheets, the final structures contain only parallel β-sheets, suggesting that antiparallel β-sheet structures are thermodynamically and kinetically metastable. Additionally, our data demonstrate that ThT fluorescence enhancements, which are commonly used to monitor amyloid fibril formation, vary strongly with structural variations, even among fibrils comprised of the same polypeptide. Finally, we present a simple mathematical model that describes the structural evolution of fibril samples under repeated seeding

Detection of a Transient Intermediate in a Rapid Protein Folding Process by Solid-State Nuclear Magnetic Resonance

Detection of a Transient Intermediate in a Rapid Protein Folding Process by Solid-State Nuclear Magnetic Resonanc

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 (Aβ14–23 and Aβ11–25). The ssNMR data indicate antiparallel β-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 Aβ14–23 ribbons, averaged cryoEM images show a periodic spacing of β-sheets. The combined data support structures in which the amyloid ribbon growth direction is the direction of intermolecular hydrogen bonds between β-strands, the ribbon thickness corresponds to the width of one β-sheet (i.e., approximately the length of one molecule), and the variable ribbon width is a variable multiple of the thickness of one β-sheet (i.e., a multiple of the repeat distance in a stack of β-sheets). This architecture for a cross-β assembly may generally exist within amyloid ribbons

Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR^†

The 37-residue amylin peptide, also known as islet amyloid polypeptide, forms fibrils that are the main peptide or protein component of amyloid that develops in the pancreas of type 2 diabetes patients. Amylin also readily forms amyloid fibrils in vitro that are highly polymorphic under typical experimental conditions. We describe a protocol for the preparation of synthetic amylin fibrils that exhibit a single predominant morphology, which we call a striated ribbon, in electron microscopy and atomic force microscopy images. Solid-state nuclear magnetic resonance (NMR) measurements on a series of isotopically labeled samples indicate a single molecular structure within the striated ribbons. We use scanning transmission electron microscopy and several types of one- and two-dimensional solid-state NMR techniques to obtain constraints on the peptide conformation and supramolecular structure in these amylin fibrils and to derive molecular structural models that are consistent with the experimental data. The basic structural unit in amylin striated ribbons, which we call the protofilament, contains four layers of parallel β-sheets, formed by two symmetric layers of amylin molecules. The molecular structure of amylin protofilaments in striated ribbons closely resembles the protofilament in amyloid fibrils with a similar morphology formed by the 40-residue β-amyloid peptide that is associated with Alzheimer's disease

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

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

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. However, quantitative measurements of 13C–13C and 15N–13C distances in selectively labeled d,l-Aβ40 fibril samples indicate a qualitatively different supra­molecular structure. While cross-β structures in mature l-Aβ40 fibrils are comprised of in-register, parallel β-sheets, our data indicate antiparallel β-sheets in d,l-Aβ40 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-Aβ40 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 β-strand segments identified by solid-state NMR. These results provide insight into the structural basis for Aβ-CI and establish the importance of rippled sheets in self-assembly of full-length, naturally occurring amyloidogenic peptides

Characterization of β-Sheet Structure in Ure2p₁_-₈₉ Yeast Prion Fibrils by Solid-State Nuclear Magnetic Resonance^†

Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR linewidths that exceed those in solid-state NMR spectra of fibrils formed by residues 218−289 of the HET-s prion protein of Podospora anserina [as originally reported in Siemer, A. B., Ritter, C., Ernst, M., Riek, R., and Meier, B. H. (2005) Angew. Chem., Int. Ed. 44, 2441−2444 and confirmed by measurements reported here] by factors of three or more, indicating a lower degree of structural order at the molecular level in Ure2p1-89 fibrils. The very high degree of structural order in HET-s fibrils indicated by solid-state NMR data is therefore not a universal characteristic of prion proteins, and is likely to be a consequence of the evolved biological function of HET-s in heterokaryon incompatibility. Analysis of cross peak intensities in two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils suggests that certain portions of the amino acid sequence may not participate in a rigid β-sheet structure, possibly including portions of the Asn-rich segment between residues 44 and 76