Different membrane order measurement techniques are not mutually consistent.

"Membrane order" is a term commonly used to describe the elastic and mechanical properties of the lipid bilayer, though its exact meaning is somewhat context- and method dependent. These mechanical properties of the membrane control many cellular functions and are measured using various biophysical techniques. Here, we ask if the results obtained from various techniques are mutually consistent. Such consistency cannot be assumed a priori because these techniques probe different spatial locations and different spatial and temporal scales. We evaluate the change of membrane order induced by serotonin using nine different techniques in lipid bilayers of three different compositions. Serotonin is an important neurotransmitter present at 100s of mM concentrations in neurotransmitter vesicles, and therefore its interaction with the lipid bilayer is biologically relevant. Our measurement tools include fluorescence of lipophilic dyes (Nile Red, Laurdan, TMA-DPH, DPH), whose properties are a function of membrane order; atomic force spectroscopy, which provides a measure of the force required to indent the lipid bilayer; 2H solid-state NMR spectroscopy, which measures the molecular order of the lipid acyl chain segments; fluorescence correlation spectroscopy, which provides a measure of the diffusivity of the probe in the membrane; and Raman spectroscopy, where spectral intensity ratios are affected by acyl chain order. We find that different measures often do not correlate with each other and sometimes even yield conflicting results. We conclude that no probe provides a general measure of membrane order and that any inference based on the change of membrane order measured by a particular probe may be unreliable.

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.

Rational Design of Protein-Specific Folding Modifiers.

Protein-folding can go wrong in vivo and in vitro, with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a "xenonucleus", which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, Förster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 ± 5%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.

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.

Correction of Systematic Bias in Single Molecule Photobleaching Measurements.

Single molecule photobleaching is a powerful technique to measure the number of fluorescent units in subresolution molecular complexes, such as in toxic protein oligomers associated with amyloid diseases. However, photobleaching can occur before the sample is appropriately placed and focused. Such "prebleaching" can introduce a strong systematic bias toward smaller oligomers. Quantitative correction of prebleaching is known to be an ill-posed problem, limiting the utility of the technique. Here, we provide an experimental solution to improve its reliability. We chemically construct multimeric standards to estimate the prebleaching probability, B. We show that B can be used as a constraint to reliably correct the statistics obtained from a known distribution of standard oligomers. Finally, we apply this method to the data obtained from a heterogeneous oligomeric solution of human islet amyloid polypeptide. Our results show that photobleaching can critically skew the estimation of oligomeric distributions, so that low abundance monomers display a much higher apparent abundance. In summary, any inference from photobleaching experiments with B > 0.1 is likely to be unreliable, but our method can be used to quantitatively correct possible errors.

A Spectroscopic Marker for Structural Transitions Associated with Amyloid-ß Aggregation.

An amyloid aggregate evolves through a series of intermediates that have different secondary structures and intra- and intermolecular contacts. The structural parameters of these intermediates are important determinants of their toxicity. For example, the early oligomeric species of the amyloid-ß (Aß) peptide have been implicated as the most cytotoxic species in Alzheimer's disease but are difficult to identify because of their dynamic and transitory nature. Conventional aggregation monitors such as the fluorescent dye thioflavin T report on only the overall transition of the soluble species to the final amyloid fibrillar aggregated state. Here, we show that the fluorescent dye bis(triphenylphosphonium) tetraphenylethene (TPE-TPP) identifies at least three distinct aggregation intermediates of Aß. Some atomic-level features of these intermediates are known from solid state nuclear magnetic resonance spectroscopy. Hence, the TPE-TPP fluorescence data may be interpreted in terms of these Aß structural transitions. Steady state fluorescence and lifetime characteristics of TPE-TPP distinguish between the small oligomeric species (emission wavelength maximum, λmax = 465 nm; average fluorescence lifetime, τFl measured at 420 nm = 3.58 ± 0.04 ns), the intermediate species (λmax = 452 nm; τFl = 3.00 ± 0.03 ns), and the fibrils (λmax = 406 nm; τFl = 5.19 ± 0.08 ns). Thus, TPE-TPP provides a ready diagnostic for differentiating between the various, including the toxic, Aß aggregates and potentially can be utilized to screen for amyloid aggregation inhibitors.

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.

Structural characteristics of oligomers formed by pyroglutamate-modified amyloid ß peptides studied by solid-state NMR.

Neuronal plaques of amyloid ß (Aß) peptides of varying length carrying different posttranslational modifications represent a molecular hallmark of Alzheimer's disease. It is believed that transient oligomeric Aß assemblies associating in early fibrillation events represent particularly cytotoxic peptide aggregates. Also, N-terminally truncated (in position 3 or 11) and pyroglutamate modified peptides exhibited an increased toxicity compared to the wildtype. In the current study, the molecular structure of oligomeric species of pGlu3-Aß(3-40) and pGlu11-Aß(11-40) was investigated using solid-state NMR spectroscopy. On the secondary structure level, for both modified peptides a large similarity between oligomers and mature fibrils of the modified peptides was found mainly based on 13C NMR chemical shift data. Some smaller structural differences were detected in the vicinity of the respective modification site. Also, the crucial early folding molecular contact between residues Phe19 and Leu34 could be observed for the oligomers of both modified peptide species. Therefore, it has to be concluded that the major secondary structure elements of Aß are already present in oligomers of pGlu3-Aß(3-40) and pGlu11-Aß(11-40). These posttranslationally modified peptides arrange in a similar fashion as observed for wild type Aß(1-40).

Spontaneous Fluctuations Can Guide Drug Design Strategies for Structurally Disordered Proteins.

Structure-based "rational" drug design strategies fail for diseases associated with intrinsically disordered proteins (IDPs). However, structural disorder allows large-amplitude spontaneous intramolecular dynamics in a protein. We demonstrate a method that exploits this dynamics to provide quantitative information about the degree of interaction of an IDP with other molecules. A candidate ligand molecule may not bind strongly, but even momentary interactions can be expected to perturb the fluctuations. We measure the amplitude and frequency of the equilibrium fluctuations of fluorescently labeled small oligomers of hIAPP (an IDP associated with type II diabetes) in a physiological solution, using nanosecond fluorescence cross-correlation spectroscopy. We show that the interterminal distance fluctuates at a characteristic time scale of 134 ± 10 ns, and 6.4 ± 0.2% of the population is in the "closed" (quenched) state at equilibrium. These fluctuations are affected in a dose-dependent manner by a series of small molecules known to reduce the toxicity of various amyloid peptides. The degree of interaction increases in the following order: resveratrol < epicatechin ∼ quercetin < Congo red < epigallocatechin 3-gallate. Such ordering can provide a direction for exploring the chemical space for finding stronger-binding ligands. We test the biological relevance of these measurements by measuring the effect of these molecules on the affinity of hIAPP for lipid vesicles and cell membranes. We find that the ability of a molecule to modulate intramolecular fluctuations correlates well with its ability to lower membrane affinity. We conclude that structural disorder may provide new avenues for rational drug design for IDPs.

Major Reaction Coordinates Linking Transient Amyloid-ß Oligomers to Fibrils Measured at Atomic Level.

The structural underpinnings for the higher toxicity of the oligomeric intermediates of amyloidogenic peptides, compared to the mature fibrils, remain unknown at present. The transient nature and heterogeneity of the oligomers make it difficult to follow their structure. Here, using vibrational and solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, we show that freely aggregating Aß40 oligomers in physiological solutions have an intramolecular antiparallel configuration that is distinct from the intermolecular parallel ß-sheet structure observed in mature fibrils. The intramolecular hydrogen-bonding network flips nearly 90°, and the two ß-strands of each monomeric unit move apart, to give rise to the well-known intermolecular in-register parallel ß-sheet structure in the mature fibrils. Solid-state nuclear magnetic resonance distance measurements capture the interstrand separation within monomer units during the transition from the oligomer to the fibril form. We further find that the D23-K28 salt-bridge, a major feature of the Aß40 fibrils and a focal point of mutations linked to early onset Alzheimer's disease, is not detectable in the small oligomers. Molecular dynamics simulations capture the correlation between changes in the D23-K28 distance and the flipping of the monomer secondary structure between antiparallel and parallel ß-sheet architectures. Overall, we propose interstrand separation and salt-bridge formation as key reaction coordinates describing the structural transition of the small Aß40 oligomers to fibrils.

Curcumin Dictates Divergent Fates for the Central Salt Bridges in Amyloid-ß40 and Amyloid-ß42.

There are three specific regions in the Amyloid beta (Aß) peptide sequence where variations cause enhanced toxicity in Alzheimer's disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn2+ and curcumin on Aß40, and compare these with their previously reported effects on Aß42. Aß42 and Aß40 differ only near the C-terminus, where curcumin interacts, while Zn2+ interacts near the N-terminus. Therefore, this comparison should help us differentiate the effect of modulating the C- and the N-termini. We find that curcumin allows fibril-like structures containing the salt bridge to emerge in the mature Aß40 aggregates, but not in Aß42. In contrast, we find no difference in the effects of Zn+2 on Aß40 and Aß42. In the presence of Zn+2, both of these fail to form proper fibrils, and the salt bridge remains disrupted. These results indicate that modulations of the Aß termini can determine the fate of a salt bridge far away in the sequence, and this has significant consequences for Aß toxicity. We also infer that small molecules can alter oligomer-induced toxicity by modulating the aggregation pathway, without substantially changing the final product of aggregation.

Development of multifunctional heterocyclic Schiff base as a potential metal chelator: a comprehensive spectroscopic approach towards drug discovery.

Amyloid-ß peptides and their metal-associated aggregated states have been implicated in the pathogenesis of Alzheimer's disease. The present paper epitomises the design and synthesis of a small, neutral, lipophilic benzothiazole Schiff base (E)-2-((6-chlorobenzo[d]thiazol-2-ylimino)methyl)-5-diethylamino)phenol (CBMDP), and explores its multifunctionalty as a potential metal chelator/fluorophore using UV-visible absorption, steady-state fluorescence, single molecule fluorescence correlation spectroscopic (FCS) techniques which is further corroborated by in silico studies. Some pharmaceutically relevant properties of the synthesized compound have also been calculated theoretically. Steady-state fluorescence and single molecule FCS reveal that the synthesized CBMDP not only recognizes oligomeric Aß40, but could also be used as an amyloid-specific extrinsic fluorophore as it shows tremendous increase in its emission intensity in the presence of Aß40. Molecular docking exercise and MD simulation reveal that CBMDP localizes itself in the crucial amyloidogenic and copper-binding region of Aß40 and undergoes a strong binding interaction via H-bonding and π-π stacking. It stabilizes the solitary α-helical Aß40 monomer by retaining the initial conformation of the Aß central helix and mostly interacts with the hydrophilic N-terminus and the α-helical region spanning from Ala-2 to Val-24. CBMDP exhibits strong copper as well as zinc chelation ability and retards the rapid copper-induced aggregation of amyloid peptide. In addition, CBMDP shows radical scavenging activity which enriches its functionality. Overall, the consolidated in vitro and in silico results obtained for the synthesized molecule could provide a rational template for developing new multifunctional agents.

Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-ß18-35 and Makes It Soluble.

Aß self-assembles into parallel cross-ß fibrillar aggregates, which is associated with Alzheimer's disease pathology. A central hairpin turn around residues 23-29 is a defining characteristic of Aß in its aggregated state. Major biophysical properties of Aß, including this turn, remain unaltered in the central fragment Aß18-35. Here, we synthesize a single deletion mutant, ΔG25, with the aim of sterically hindering the hairpin turn in Aß18-35. We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of Aß18-35, suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe(19) and Leu(34) residues, observed in full-length Aß and Aß18-35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel ß-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel ß-sheet. We conclude that the self-assembly of Aß is critically dependent on the hairpin turn and on the contact between the Phe(19) and Leu(34) regions, making them potentially sensitive targets for Alzheimer's therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid ß (1-40).

A small library of rationally designed amyloid ß [Aß(1-40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aß(1-40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid-state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aß(1-40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aß fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross-ß structure.

Curcumin alters the salt bridge-containing turn region in amyloid ß(1-42) aggregates.

Amyloid ß (Aß) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aß fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid ß(1-42) (Aß42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23-Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aß42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn(2+) ions, another agent known to disrupt the fibrils and alter Aß42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aß42, which is stabilized both by curcumin and by Zn(2+.)

Rapid, cell-free assay for membrane-active forms of amyloid-ß.

Small oligomers of amyloid beta (Aß) are suspected to be the key to Alzheimer's disease (AD). However, identifying these toxic species in the background of other similar but nontoxic Aß aggregates has remained a challenge. Recent studies indicate that Aß undergoes a global structural transition in an early step of aggregation. This transition is marked by a strong increase in its affinity for cell membranes, which suggests that the resultant oligomers could be the key to Aß toxicity. Here we use this increased membrane affinity to develop a rapid, quantitative, cell-free assay for these bioactive oligomers. It uses fluorescence correlation spectroscopy of fluorescently labeled Aß and requires only 30 s of measurement time. We also describe a simpler (though less rapid) assay based on the same principles, which uses a dialysis step followed by conventional fluorescence spectroscopy. Our results potentially provide a much-needed high-throughput assay for AD drug development.

pH changes the aggregation propensity of amyloid-ß without altering the monomer conformation.

Decoupling conformational changes from aggregation will help us understand amyloids better. Here we attach Alzheimer's amyloid-ß(1-40) monomers to silver nanoparticles, preventing their aggregation, and study their conformation under aggregation-favoring conditions using SERS. Surprisingly, the α-helical character of the peptide remains unchanged between pH 10.5 and 5.5, while the solubility changes >100×. Amyloid aggregation can therefore start without significant conformational changes.

Significant structural differences between transient amyloid-ß oligomers and less-toxic fibrils in regions known to harbor familial Alzheimer's mutations.

Small oligomers of the amyloidâ€…ß (Aß) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimer's disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solid-state NMR methods. The smallest Aß40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Aß mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.

Phospholipid Membranes as Chemically and Functionally Tunable Materials.

The sheet-like lipid bilayer is the fundamental structural component of all cell membranes. Its building blocks are phospholipids and cholesterol. Their amphiphilic structure spontaneously leads to the formation of a bilayer in aqueous environment. Lipids are not just structural elements. Individual lipid species, the lipid membrane structure, and lipid dynamics influence and regulate membrane protein function. An exciting field is emerging where the membrane-associated material properties of different bilayer systems are used in designing innovative solutions for widespread applications across various fields, such as the food industry, cosmetics, nano- and biomedicine, drug storage and delivery, biotechnology, nano- and biosensors, and computing. Here, the authors summarize what is known about how lipids determine the properties and functions of biological membranes and how this has been or can be translated into innovative applications. Based on recent progress in the understanding of membrane structure, dynamics, and physical properties, a perspective is provided on how membrane-controlled regulation of protein functions can extend current applications and even offer new applications.