![Schematic of the experimental setup. (a) One Aβ molecule immobilized on the mica substrate is picked up by another Aβ molecule functionalized on the AFM tip. Putative interaction segments within a dimer are highlighted in pink bands. A rupture event occurs when a misfolded dimer is torn apart. For clarity, only one linker molecule is drawn on AFM tip and substrate, and the sizes of objects in the scheme are not to scale. N and C denote N−terminus and C−terminus, respectively. (b) Typical force−distance curve that shows either no interactions (black) or specific interactions (red). In the case of specific interactions, breaking a misfolded Aβ dimer resulted in a rupture distance at ~ 48 nm and a rupture force at ~ 73 pN. A vertical black arrow indicates the rupture position. For clarity, only the retraction curves are shown. Y offset was performed with the red line but not with the black line. A typical histogram of rupture force distribution at a loading rate of 6215 pN/s for Aβ40 (c). The most probable rupture force for Aβ40 was 57.0 pN. A typical histogram of rupture force distribution at a loading rate of 6068 pN/s for [VPV]Aβ40 (d). The most probable rupture force for [VPV]Aβ40 was 83.6 pN. The solid lines represent the fit of probability density function.](https://www.researchgate.net/profile/David-Teplow/publication/257463822/figure/fig3/AS:293972233736224@1447099850835/Schematic-of-the-experimental-setup-a-One-Ab-molecule-immobilized-on-the-mica_Q320.jpg)
![The distributions of contour lengths (a) and rupture forces (b) at loading rates of 5000−7000 pN/s for [VPV]Aβ40. The contour length distribution showed a major data cluster at ~ 42 and a comparable cluster at ~ 57 nm. The most probable rupture force was 79.3 ± 1.5 pN. The scatter distribution of rupture forces with varying contour lengths is shown in (c). The total number of data was 280. Error is shown by ± SEM.](https://www.researchgate.net/profile/David-Teplow/publication/257463822/figure/fig2/AS:293972233736214@1447099850697/The-distributions-of-contour-lengths-a-and-rupture-forces-b-at-loading-rates-of_Q320.jpg)
![The contour length distributions for Aβ40, [VPV]Aβ40, Aβ42, [VPV]Aβ42, and [pP]Aβ42 dimers are shown in (a), (b), (c), (d) and (e), respectively. The interaction segments are aligned according to the contour length distribution. The dotted lines denote the distribution profiles and peaks are approximated according to the profiles. Notably, there are some uncertainties in length estimation. The arrows indicate interaction segments.](https://www.researchgate.net/profile/David-Teplow/publication/257463822/figure/fig4/AS:293972237930540@1447099851805/The-contour-length-distributions-for-Ab40-VPVAb40-Ab42-VPVAb42-and-pPAb42_Q320.jpg)
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Mechanism of amyloid β−protein dimerization determined using single−molecule AFM force spectroscopy
Abstract and Figures
Aβ42 and Aβ40 are the two primary alloforms of human amyloid β-protein (Aβ). The two additional C-terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments.
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... For instance, atomic force microscopy (AFM) has previously been used to study oligomers [242]. AFM methods generally focus on dimerization studies, by placing a monomer in a surface and evaluating how it interacts with a monomer placed in the cantilever [243][244][245][246][247][248]. AFM can also be used as a surface imaging technique [249], having been used for secondary structure imaging when coupled with infrared spectroscopy [250]. ...
Amyloid proteins are species whose aggregation has been associated with various neurodegenerative diseases. While intrinsically disordered in their monomeric form, they tend to aggregate into highly ordered β-sheet rich fibrils. Nevertheless, many studies indicate that intermediate species known as amyloid oligomers, which are linked to neurotoxicity, may be the connection between amyloid proteins and their pathology. However, oligomers are very transient, non-covalently bound, heterogeneous, and at a very low concentration relative to monomers and fibrils. Due to this, oligomers are challenging to study with conventional methods. and developing and optimizing methods for analyzing oligomers is crucial for the advancement of the amyloid field. In this thesis, we aim to optimize two oligomer analysis methods and use them to improve our understanding of the amyloid system. On one hand, we optimize the Photo-induced cross-linking of unmodified proteins (PICUP) for the study of αSyn, and we use it to identify transient interactions within and between α-synuclein monomers in solution, bound to lipid membranes, and in fibrils. On the other hand, we use microfluid free flow electrophoresis (μFFE) for the study of Aβ42 oligomer populations. Doing so, we learn how oligomer population is affected by the protein production source as well as sheer forces, and we show that amyloid fibrils do not only catalyze oligomer formation but also play a key role in oligomer dissociation.
... [44][45][46][47] Such studies have also explored how disordered monomers interact to generate misfolded oligomers. [47][48][49] The transience and heterogeneity of IDP structures makes them challenging to characterize experimentally, however, motivating the integration of SMFS measurements with computational simulations to extend the reach of both experiment and modeling, as done for Ab 50 and asynuclein. 51 SMFS measurements like those described above all share some key characteristics. ...
The use of force probes to induce unfolding and refolding of single molecules through the application of mechanical tension, known as single-molecule force spectroscopy (SMFS), has proven to be a powerful tool for studying the dynamics of protein folding. Here we provide an overview of what has been learned about protein folding using SMFS, from small, single-domain proteins to large, multi-domain proteins. We highlight the ability of SMFS to measure the energy landscapes underlying folding, to map complex pathways for native and non-native folding, to probe the mechanisms of chaperones that assist with native folding, to elucidate the effects of the ribosome on co-translational folding, and to monitor the folding of membrane proteins.
Fibril formation resulting from protein aggregation is a hallmark of a large group of neurodegenerative human diseases, including Alzheimer's disease, type 2 diabetes, amyotrophic lateral sclerosis, and Parkinson's disease, among many others. Key factors governing protein fibril formation have been identified over the past decades to elucidate various facets of misfolding and aggregation. However, surprisingly little is known about how and why fibril structure is achieved, and it remains a fundamental problem in molecular biology. In this review, we discuss the relationship between fibril formation kinetics and various characteristics, including sequence, mutations, monomer secondary structure, mechanical stability of the fibril state, aromaticity, hydrophobicity, charge, and population of fibril-prone conformations in the monomeric state.
Soluble oligomers produced during protein aggregation have been thought to be toxic species causing various diseases. Characterization of these oligomers is difficult because oligomers are a heterogeneous mixture, which is not readily separable, and may appear transiently during aggregation. Single-molecule spectroscopy can provide valuable information by detecting individual oligomers, but there have been various problems in determining the size and concentration of oligomers. In this work, we use a newly-developed method to analyze single-molecule fluorescence burst data of freely diffusing molecules in solution based on molecular diffusion theory and maximum likelihood method. We demonstrate that the photon count rate, diffusion time, population, and FRET efficiency can be accurately determined from simulated data and the experimental data of a known oligomerization system, the tetramerization domain of p53. We used this method to characterize the oligomers of the 42-residue amyloid β peptide (Aβ42). Combining peptide incubation in a plate reader and single-molecule free diffusion experiments allows for the detection of stable oligomers appearing at various stages of aggregation. We find that the average size of these oligomers is 70-mer and their overall population is very low, less than 1 nM, in the early and middle stages of aggregation of 1 μM Aβ42 peptide. Based on their average size and long diffusion time, we predict the oligomers have a highly elongated rod-like shape.
Histidine behaviors (including tautomeric and protonation behaviors, and integration on ε, δ, or p states) have been linked to protein folding and misfolding. However, the histidine behaviors of Aβ(1-42) are unconfirmed, which is the key point to understanding the pathogenesis of Alzheimer's disease. In the current study, 19 replica exchange molecular dynamics (REMD) simulations were performed to investigate the impact of histidine on the structural properties in protonation evolution stages one, two, and three. In contrast to the deprotonated (εεε) state, our current findings demonstrate that any protonated state will promote the formation of the β-sheet structure. The sheet-rich structures of (pεε), (δεp), (pεp), and (ppp) have the same basic characteristics of three-strand structures between the N-terminus, central hydrophobic core (CHC), and C-terminus. We found that the (pεε) (probability of 77.7%) and (δεp) (probability of 60.2%) prefer the abundant conformation over the other systems with higher regularity antiparallel β-sheet structure characteristics. Further H-bonding results indicate that H6 and H14 are more essential than H13. In addition, the Pearson correlation coefficient analysis revealed that the experimental result coincided with our simulated (δpε) system. The current study aids in the better understanding of the mechanisms of histidine behaviors, providing a fresh outlook on protein folding and misfolding.
Understanding biological interactions at a molecular level grants valuable information relevant to improving medical treatments and outcomes. Among the suite of technologies available, Atomic Force Microscopy (AFM) is unique in its ability to quantitatively probe forces and receptor-ligand interactions in real-time. The ability to assess the formation of supramolecular bonds and intermediates in real-time on surfaces and living cells generates important information relevant to understanding biological phenomena. Combining AFM with fluorescence-based techniques allows for an unprecedented level of insight not only concerning the formation and rupture of bonds, but understanding medically relevant interactions at a molecular level. As the ability of AFM to probe cells and more complex models improves, being able to assess binding kinetics, chemical topographies, and garner spectroscopic information will likely become key to developing further improvements in fields such as cancer, nanomaterials, and virology. The rapid response to the COVID-19 crisis, producing information regarding not just receptor affinities, but also strain-dependent efficacy of neutralizing nanobodies, demonstrates just how viable and integral to the pre-clinical development of information AFM techniques are in this era of medicine.
Although controversial, the amyloid cascade hypothesis remains central to the Alzheimer's disease (AD) field and posits amyloid‐beta (Aβ) as the central factor initiating disease onset. In recent years, there has been an increase in emphasis on studying the role of low molecular weight aggregates, such as oligomers, which are suggested to be more neurotoxic than fibrillary Aβ. Other Aβ isoforms, such as truncated Aβ, have also been implicated in disease. However, developing a clear understanding of AD pathogenesis has been hampered by the complexity of Aβ biochemistry in vitro and in vivo. This review explores factors contributing to the lack of consistency in experimental approaches taken to model Aβ aggregation and toxicity and provides an overview of the different techniques available to analyse Aβ, such as electron and atomic force microscopy, nuclear magnetic resonance spectroscopy, dye‐based assays, size exclusion chromatography, mass spectrometry and SDS‐PAGE. The review also explores how different types of Aβ can influence Aβ aggregation and toxicity, leading to variation in experimental outcomes, further highlighting the need for standardisation in Aβ preparations and methods used in current research.
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Since the discovery of insulin, a century ago, the repertoire of therapeutic polypeptides targeting diabetes - and now also obesity - have increased substantially. The focus on quality has shifted from impure and unstable preparations of animal insulin to highly pure, homologous recombinant insulin, along with other peptide-based hormones and analogues such as amylin analogues (pramlintide, davalintide, cagrilintide), glucagon and glucagon-like peptide-1 receptor agonists (GLP-1, liraglutide, exenatide, semaglutide). Proper formulation, storage, manipulation and usage by professionals and patients are required in order to avoid agglomeration into high molecular weight products (HMWP), either amorphous or amyloid, which could result in potential loss of biological activity and short- or long-term immune reaction and silent inactivation. In this narrative review, we present perspective of the aggregation of therapeutic polypeptides used in diabetes and other metabolic diseases, covering the nature and mechanisms, analytical techniques, physical and chemical stability, strategies aimed to hamper the formation of HMWP, and perspectives on future biopharmaceutical developments.
Amyloid-β peptide (Aβ) aggregation, one of the major hallmarks of Alzheimer’s disease (AD), has been commonly recognized as the critical culprit in AD etiology. However, the exact mechanism of Aβ aggregation in AD is still not fully understood. Moreover, development of methods capable of probing the aggregation without any perturbations has yet to be achieved. To address this challenge, we herein report a label-free fluorescent probe (BTS) for dynamic in situ visualization of Aβ40 aggregation. BTS is able to bind to all forms of Aβ40 with high binding affinity, accompanied with similar fluorescence enhancement. Intriguingly, BTS does not affect Aβ40 aggregation, probably due to its unique binding mode targeting the N-terminus of Aβ40 as the irresponsible region for aggregation. Importantly, with the prominent fluorescence performances including high selectivity and sensitivity (168 nM) as well as large Stokes shift (185 nm), BTS can veritably monitor entire Aβ aggregation process through fluorescence imaging. The probe would provide a promising tool for exploring Aβ aggregation-related pathogenesis and treatment of AD.
Oligomerization of the 42-residue peptide Aβ42 plays a key role in the pathogenesis of Alzheimer disease. Despite great academic and medical interest, the structures of these oligomers have not been well characterized. Site-directed spin labeling combined with electron paramagnetic resonance spectroscopy is a powerful approach for studying structurally ill-defined systems, but its application in amyloid oligomer structure study has not been systematically explored. Here we report a comprehensive structural study on a toxic Aβ42 oligomer, called globulomer, using site-directed spin labeling complemented by other techniques. Transmission electron microscopy shows that these oligomers are globular structures with diameters of ∼7–8 nm. Circular dichroism shows primarily β-structures. X-ray powder diffraction suggests a highly ordered intrasheet hydrogen-bonding network and a heterogeneous intersheet packing. Residue-level mobility analysis on spin labels introduced at 14 different positions shows a structured state and a disordered state at all labeling sites. Side chain mobility analysis suggests that structural order increases from N- to C-terminal regions. Intermolecular distance measurements at 14 residue positions suggest that C-terminal residues Gly-29–Val-40 form a tightly packed core with intermolecular distances in a narrow range of 11.5–12.5 Å. These intermolecular distances rule out the existence of fibril-like parallel in-register β-structures and strongly suggest an antiparallel β-sheet arrangement in Aβ42 globulomers.
Background: Aβ42 oligomers underlie neurotoxicity in Alzheimer disease, but their molecular structures are unknown.
Results: Electron paramagnetic resonance studies reveal intermolecular distances at 11.5–12.5 Å for C-terminal region of Aβ42.
Conclusion: Aβ42 oligomers consist of a tightly packed C-terminal region that adopts antiparallel structures.
Significance: This work provides insights into the structures of Aβ42 oligomers and helps understand their oligomerization mechanism and toxicity.
Amyloid -protein (A) is linked to neuronal injury and death in
Alzheimer's disease (AD). Of particular relevance for elucidating the
role of A in AD is new evidence that oligomeric forms of A are potent
neurotoxins that play a major role in neurodegeneration and the strong
association of the 42-residue form of A, A42, with the disease. Detailed
knowledge of the structure and assembly dynamics of A thus is important
for the development of properly targeted AD therapeutics. Recently, we
have shown that A oligomers can be cross-linked efficiently, and their
relative abundances quantified, by using the technique of photo-induced
cross-linking of unmodified proteins (PICUP). Here, PICUP,
size-exclusion chromatography, dynamic light scattering, circular
dichroism spectroscopy, and electron microscopy have been combined to
elucidate fundamental features of the early assembly of A40 and A42.
Carefully prepared aggregate-free A40 existed as monomers, dimers,
trimers, and tetramers, in rapid equilibrium. In contrast, A42
preferentially formed pentamer/hexamer units (paranuclei) that assembled
further to form beaded superstructures similar to early protofibrils.
Addition of Ile-41 to A40 was sufficient to induce formation of
paranuclei, but the presence of Ala-42 was required for their further
association. These data demonstrate that A42 assembly involves formation
of several distinct transient structures that gradually rearrange into
protofibrils. The strong etiologic association of A42 with AD may thus
be a result of assemblies formed at the earliest stages of peptide
oligomerization.
Misfolding and aggregation of the amyloid β-protein (Aβ) are hallmarks of Alzheimer's disease. Both processes are dependent on the environmental conditions, including the presence of divalent cations, such as Cu(2+). Cu(2+) cations regulate early stages of Aβ aggregation, but the molecular mechanism of Cu(2+) regulation is unknown. In this study we applied single molecule AFM force spectroscopy to elucidate the role of Cu(2+) cations on interpeptide interactions. By immobilizing one of two interacting Aβ42 molecules on a mica surface and tethering the counterpart molecule onto the tip, we were able to probe the interpeptide interactions in the presence and absence of Cu(2+) cations at pH 7.4, 6.8, 6.0, 5.0, and 4.0. The results show that the presence of Cu(2+) cations change the pattern of Aβ interactions for pH values between pH 7.4 and pH 5.0. Under these conditions, Cu(2+) cations induce Aβ42 peptide structural changes resulting in N-termini interactions within the dimers. Cu(2+) cations also stabilize the dimers. No effects of Cu(2+) cations on Aβ-Aβ interactions were observed at pH 4.0, suggesting that peptide protonation changes the peptide-cation interaction. The effect of Cu(2+) cations on later stages of Aβ aggregation was studied by AFM topographic images. The results demonstrate that substoichiometric Cu(2+) cations accelerate the formation of fibrils at pH 7.4 and 5.0, whereas no effect of Cu(2+) cations was observed at pH 4.0. Taken together, the combined AFM force spectroscopy and imaging analyses demonstrate that Cu(2+) cations promote both the initial and the elongation stages of Aβ aggregation, but protein protonation diminishes the effect of Cu(2+).
We elongated individual poly(ethylene-glycol) (PEG) molecules tethered at one end to an AFM cantilever. We observed the resistive force as a function of elongation in different solvents. In all cases the molecular response was found to be fully reversible and thus in thermodynamic equilibrium. In hexadecane the stretched PEG acts like an ideal entropy spring and can be well described as a freely jointed chain. In water we observed marked deviations in the transition region from entropic to enthalpic elasticity, indicating the deformation of a supra-structure within the polymer. An analysis based on elastically coupled Markovian two-level systems agrees well with recent ab initio calculations predicting that PEG in water forms a non-planar supra-structure which is stabilized by water bridges. We obtained a binding free energy of 3.0 ± 0.3 kT .
Using homonuclear (1)H NOESY spectra, with chemical shifts, (3)JH(N)H(α) scalar couplings, residual dipolar couplings, and (1)H-(15)N NOEs, we have optimized and validated the conformational ensembles of the amyloid-β 1-40 (Aβ40) and amyloid-β 1-42 (Aβ42) peptides generated by molecular dynamics simulations. We find that both peptides have a diverse set of secondary structure elements including turns, helices, and antiparallel and parallel β-strands. The most significant difference in the structural ensembles of the two peptides is the type of β-hairpins and β-strands they populate. We find that Aβ42 forms a major antiparallel β-hairpin involving the central hydrophobic cluster residues (16-21) with residues 29-36, compatible with known amyloid fibril forming regions, whereas Aβ40 forms an alternative but less populated antiparallel β-hairpin between the central hydrophobic cluster and residues 9-13, that sometimes forms a β-sheet by association with residues 35-37. Furthermore, we show that the two additional C-terminal residues of Aβ42, in particular Ile-41, directly control the differences in the β-strand content found between the Aβ40 and Aβ42 structural ensembles. Integrating the experimental and theoretical evidence accumulated over the last decade, it is now possible to present monomeric structural ensembles of Aβ40 and Aβ42 consistent with available information that produce a plausible molecular basis for why Aβ42 exhibits greater fibrillization rates than Aβ40.
The misfolding and self-assembly of the amyloid-beta (Aβ) peptide into aggregates is a molecular signature of the development of Alzheimer's disease but molecular mechanisms of the peptide aggregation remain unknown. Here, we combined Atomic Force Microscopy (AFM) and Molecular Dynamics (MD) simulations to characterize misfolding process of an Aβ peptide. Dynamic force spectroscopy analysis showed that the peptide forms stable dimers with the lifetime of ~1 s. During MD simulations isolated monomers gradually adopt essentially similar non-structured conformations independent from the initial structure. However, when two monomers approach their structure changes dramatically and the conformational space for the two monomers become restricted. The arrangement of monomers in antiparallel orientation leads to the cooperative formation of β-sheet conformation. Interactions, including hydrogen bonds, salt bridges and weakly polar interactions of side chains stabilize the structure of the dimer. Under the applied force, the dimer, as during the AFM experiments, dissociates in a cooperative manner. Thus, misfolding of the Aβ peptide proceeds via the loss of conformational flexibility and formation of stable dimers suggesting their key role in the subsequent Aβ aggregation process.
Self-assembly of the 40/42 amino acid Aβ peptide is a key player in Alzheimer's disease. Aβ40 is the most prevalent species, while Aβ42 is the most toxic. It has been suggested that the amino acids 21-30 could nucleate the folding of Aβ monomer and a bent in this region could be the rate-limiting step in Aβ fibril formation. In this study, we review our current understanding of the computer-predicted conformations of amino acids 23-28 in the monomer of Aβ(21-30) and the monomers Aβ40 and Aβ42. On the basis of new simulations on dimers of full-length Aβ, we propose that the ratelimiting step involves the formation of a multimeric β-sheet spanning the central hydrophobic core (residues 17-21).