Single-Molecule Maps of Membrane Insertion by Amyloid-ß Oligomers Predict Their Toxicity.

The interaction of small Amyloid-ß (Aß) oligomers with the lipid membrane is an important component of the pathomechanism of Alzheimer's disease (AD). However, oligomers are heterogeneous in size. How each type of oligomer incorporates into the membrane, and how that relates to their toxicity, is unknown. Here, we employ a single molecule technique called Q-SLIP (Quencher-induced Step Length Increase in Photobleaching) to measure the membrane insertion of each monomeric unit of individual oligomers of Aß42, Aß40, and Aß40-F19-Cyclohexyl alanine (Aß40-F19Cha), and correlate it with their toxicity. We observe that the N-terminus of Aß42 inserts close to the center of the bilayer, the less toxic Aß40 inserts to a shallower depth, and the least toxic Aß40-F19Cha has no specific distribution. This oligomer-specific map provides a mechanistic representation of membrane-mediated Aß toxicity and should be a valuable tool for AD research.

A Toxicogenic Interaction between Intracellular Amyloid-ß and Apolipoprotein-E.

Alzheimer's disease (AD) is associated with the aggregation of amyloid ß (Aß) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aß oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aß-ApoE interaction suppresses these changes and concomitantly reduces Aß toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer's patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aß oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery.

Determining the Stoichiometry of Amyloid Oligomers by Single-Molecule Photobleaching.

Small oligomers are the initial intermediates in the pathway to amyloid fibril formation. They have a distinct identity from the monomers as well as from the protofibrils and the fibrils, both in their structure and in their properties. In many cases, they play a crucial biological role. However, due to their transient nature, they are difficult to characterize. "Oligomer" is a diffuse definition, encompassing aggregates of many different sizes, and this lack of precise definition causes much confusion and disagreement between different research groups. Here, we define the small oligomers as "n"-mers with n < 10, which is the size range in which the amyloid proteins typically exist at the initial phase of the aggregation process. Since the oligomers dynamically interconvert into each other, a solution of aggregating amyloid proteins will contain a distribution of sizes. A precise characterization of an oligomeric solution will, therefore, require quantification of the relative population of each size. Size-based separation methods, such as size-exclusion chromatography, are typically used to characterize this distribution. However, if the interconversion between oligomers of different sizes is fast, this would not yield reliable results. Single-molecule photobleaching (smPB) is a direct method to evaluate this size distribution in a heterogeneous solution without separation. In addition, understanding the mechanism of action of amyloid oligomers requires knowing the affinity of each oligomer type to different cellular components, such as the cell membrane. These measurements are also amenable to smPB. Here we show how to perform smPB, both for oligomers in solution and for oligomers attached to the membrane.

Single Molecule Measurements of the Accessibility of Molecular Surfaces.

An important measure of the conformation of protein molecules is the degree of surface exposure of its specific segments. However, this is hard to measure at the level of individual molecules. Here, we combine single molecule photobleaching (smPB, which resolves individual photobleaching steps of single molecules) and fluorescence quenching techniques to measure the accessibility of individual fluorescently labeled protein molecules to quencher molecules in solution. A quencher can reduce the time a fluorophore spends in the excited state, increasing its photostability under continuous irradiation. Consequently, the photo-bleaching step length would increase, providing a measure for the accessibility of the fluorophore to the solvent. We demonstrate the method by measuring the bleaching step-length increase in a lipid, and also in a lipid-anchored peptide (both labelled with rhodamine-B and attached to supported lipid bilayers). The fluorophores in both molecules are expected to be solvent-exposed. They show a near two-fold increase in the step length upon incubation with 5 mM tryptophan (a quencher of rhodamine-B), validating our approach. A population distribution plot of step lengths before and after addition of tryptophan show that the increase is not always homogenous. Indeed there are different species present with differential levels of exposure. We then apply this technique to determine the solvent exposure of membrane-attached N-terminus labelled amylin (h-IAPP, an amyloid associated with Type II diabetes) whose interaction with lipid bilayers is poorly understood. hIAPP shows a much smaller increase of the step length, signifying a lower level of solvent exposure of its N-terminus. Analysis of results from individual molecules and step length distribution reveal that there are at least two different conformers of amylin in the lipid bilayer. Our results show that our method ("Q-SLIP", Quenching-induced Step Length increase in Photobleaching) provides a simple route to probe the conformational states of membrane proteins at a single molecule level.

Biophysical properties of the isolated spike protein binding helix of human ACE2.

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel ß-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1mod) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.

Altered Membrane Mechanics Provides a Receptor-Independent Pathway for Serotonin Action.

Serotonin, an important signaling molecule in humans, has an unexpectedly high lipid membrane affinity. The significance of this finding has evoked considerable speculation. Here we show that membrane binding by serotonin can directly modulate membrane properties and cellular function, providing an activity pathway completely independent of serotonin receptors. Atomic force microscopy shows that serotonin makes artificial lipid bilayers softer, and induces nucleation of liquid disordered domains inside the raft-like liquid-ordered domains. Solid-state NMR spectroscopy corroborates this data at the atomic level, revealing a homogeneous decrease in the order parameter of the lipid chains in the presence of serotonin. In the RN46A immortalized serotonergic neuronal cell line, extracellular serotonin enhances transferrin receptor endocytosis, even in the presence of broad-spectrum serotonin receptor and transporter inhibitors. Similarly, it increases the membrane binding and internalization of oligomeric peptides. Our results uncover a mode of serotonin-membrane interaction that can potentiate key cellular processes in a receptor-independent fashion.

Membrane affinity of individual toxic protein oligomers determined at the single-molecule level.

Oligomers are the key suspects in protein aggregation-linked diseases, such as Alzheimer's and Type II diabetes, and most likely exert their toxicity by interacting with lipid membranes. However, the "which oligomer" question remains an obstacle in understanding the disease mechanism, as the exact identity of the toxic oligomer(s) is not yet known. Oligomers exist as a mixture of species of different sizes (i.e. as different 'n-mers') in a physiological solution, making it difficult to determine the properties of individual species. Here we demonstrate a method based on single-molecule photo-bleaching (smPB) which can provide an answer to the "which oligomer" question, at least as far as membrane affinity is concerned. We calculate the ratio of the oligomer size distribution of human Islet Amyloid Polypeptide (IAPP) in the aqueous phase and that on a coexisting artificial lipid bilayer, and this measures the relative membrane affinity of individual oligomeric species. A problem with smPB measurements is that they can be very sensitive to pre-measurement bleaching. Here we correct for pre-bleaching using a covalently linked multimeric peptide as a bleaching standard. We find that the order of membrane affinity for IAPP n-mers is trimer > dimer > tetramer ≫ monomer. Our results agree well with the average membrane affinity values of oligomeric and monomeric solutions previously measured with Fluorescence Correlation Spectroscopy. The "which oligomer" question, in the context of membrane affinity, can therefore, be solved quantitatively for any membrane-active toxic protein aggregate.

Ordered and Disordered Segments of Amyloid-ß Drive Sequential Steps of the Toxic Pathway.

While the roles of intrinsically disordered protein domains in driving interprotein interactions are increasingly well-appreciated, the mechanism of toxicity of disease-causing disordered proteins remains poorly understood. A prime example is Alzheimer's disease (AD) associated amyloid beta (Aß). Aß oligomers are highly toxic partially structured peptide assemblies with a distinct ordered region (residues ∼10-40) and a shorter disordered region (residues ∼1-9). Here, we investigate the role of this disordered domain and its relation to the ordered domain in the manifestation of toxicity through a set of Aß fragments and stereoisomers designed for this purpose. We measure their effects on lipid membranes and cultured neurons, probing their toxicity, intracellular distributions, and specific molecular interactions using the techniques of confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, and fluorescence correlation spectroscopy. Remarkably, we find that neither part-Aß10-40 or Aß1-9, is toxic by itself. The ordered part (Aß10-40) is the major determinant of how Aß attaches to lipid bilayers, enters neuronal cells, and localizes primarily in the late endosomal compartments. However, once Aß enters the cell, it is the disordered part (only when it is connected to the rest of the peptide) that has a strong and stereospecific interaction with an unknown cellular component, as demonstrated by distinct changes in the fluorescence lifetime of a fluorophore attached to the N-terminal. This interaction appears to commit Aß to the toxic pathway. Our findings correlate well with Aß sites of familial AD mutations, a significant fraction of which cluster in the disordered region. We conclude that, while the ordered region dictates attachment and cellular entry, the key to toxicity lies in the ordered part presenting the disordered part for a specific cellular interaction.