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Deposition of extracellular Amyloid Beta (Aβ) and intracellular tau fibrils in post-mortem brains remains the only way to conclusively confirm cases of Alzheimer's Disease (AD). Substantial evidence, though, implicates small globular oligomers instead of fibrils as relevant biomarkers of, and critical contributors to, the clinical symptoms of AD. Efforts to verify and utilize amyloid oligomers as AD biomarkers in vivo have been limited by the near-exclusive dependence on conformation-selective antibodies for oligomer detection. While antibodies have yielded critical evidence for the role of both Aβ and tau oligomers in AD, they are not suitable for imaging amyloid oligomers in vivo. Therefore, it would be desirable to identify a set of oligomer-selective small molecules for subsequent development into Positron Emission Tomography (PET) probes. Using a kinetics-based screening assay, we confirm that the triarylmethane dye Crystal Violet (CV) is oligomer-selective for Aβ42 oligomers (AβOs) grown under near-physiological solution conditions in vitro. In postmortem brains of an AD mouse model and human AD patients, we demonstrate that A11 antibody-positive oligomers but not Thioflavin S (ThioS)-positive fibrils colocalize with CV staining, confirming in vitro results. Therefore, our kinetic screen represents a robust approach for identifying new classes of small molecules as candidates for oligomer-selective dyes (OSDs). Such OSDs, in turn, provide promising starting points for the development of PET probes for pre-mortem imaging of oligomer deposits in humans. [ABSTRACT FROM AUTHOR]

In contrast to prevalent strategies which make use of β-sheet mimetics to block Aβ fibrillar growth, in this study, we designed a series of sulfonyl-γ-AApeptide helices that targeted the crucial α-helix domain of Aβ13-26 and stabilized Aβ conformation to avoid forming the neurotoxic Aβ oligomeric β-sheets. Biophysical assays such as amyloid kinetics and TEM demonstrated that the Aβ oligomerization and fibrillation could be greatly prevented and even reversed in the presence of sulfonyl-γ-AApeptides in a sequence-specific and dose-dependent manner. The studies based on circular dichroism, Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) spectra unambiguously suggested that the sulfonyl-γ-AApeptide Ab-6 could bind to the central region of Aβ42 and induce α-helix conformation in Aβ. Additionally, Electrospray ionisation-ion mobility spectrometry–mass spectrometry (ESI-IMS-MS) was employed to rule out a colloidal mechanism of inhibitor and clearly supported the capability of Ab-6 for inhibiting the formation of Aβ aggregated forms. Furthermore, Ab-6 could rescue neuroblastoma cells by eradicating Aβ-mediated cytotoxicity even in the presence of pre-formed Aβ aggregates. The confocal microscopy demonstrated that Ab-6 could still specifically bind Aβ42 and colocalize into mitochondria in the cellular environment, suggesting the rescue of cell viability might be due to the protection of mitochondrial function otherwise impaired by Aβ42 aggregation. Taken together, our studies indicated that sulfonyl-γ-AApeptides as helical peptidomimetics could direct Aβ into the off-pathway helical secondary structure, thereby preventing the formation of Aβ oligomerization, fibrillation and rescuing Aβ induced cell cytotoxicity. [ABSTRACT FROM AUTHOR]

Elastin‐like polypeptides (ELPs) are peptide‐based biomaterials with residue sequence (VPGXG)n where X is any residue except proline. ELPs are a useful modality for delivering biologically active proteins (growth factors, protease inhibitors, anti‐inflammatory peptides, etc.) as fusion proteins (ELP‐FP). ELP‐FPs are particularly cost‐effective because they can be rapidly purified using Inverse Temperature Cycling (ITC) via the reversible formation and precipitation of entropically driven aggregates above a transition temperature (Tt). When ELP fusion proteins (ELP‐FPs) contain significant charge density at physiological pH, electrostatic repulsion between them severely inhibits aggregate formation. The literature does not currently describe methods for purifying ELP‐FPs containing charged proteins on either side of the ELP sequence as fusion partners without organic solvents. Here, the isoelectric point (pI) of ELP‐FPs is discussed as a means of neutralizing surface charges on ELP‐FPs and increasing ITC yield to dramatically high levels. We use pI‐based phase separation (pI‐BPS) to purify ELP‐FPs containing cationic and anionic fusion proteins. We report a dramatic increase in protein yield when using pI‐BPS for purification of ELP‐FPs. Proteins purified by this method also retain the functional activity of the protein present in the ELP‐FP. Techniques developed here enable significant diversification of possible fusion proteins delivered by ELPs as ELP‐FPs by allowing them to be produced and purified at higher quantities and yields. [ABSTRACT FROM AUTHOR]

The deposition of dense fibril plaques represents the pathological hallmark for a multitude of human disorders, including many neurodegenerative diseases. Fibril plaques are predominately composed of amyloid fibrils, characterized by their underlying cross beta-sheet architecture. Research into the mechanisms of amyloid formation has mostly focused on characterizing and modeling the growth of individual fibrils and associated oligomers from their monomeric precursors. Much less is known about the mechanisms causing individual fibrils to assemble into ordered fibrillar suprastructures. Elucidating the mechanisms regulating this "secondary" self-assembly into distinct suprastructures is important for understanding how individual protein fibrils form the prominent macroscopic plaques observed in disease. Whether and how amyloid fibrils assemble into either 2D or 3D supramolecular structures also relates to ongoing efforts on using amyloid fibrils as substrates or scaffolds for self-assembling functional biomaterials. Here, we investigated the conditions under which preformed amyloid fibrils of a lysozyme assemble into larger superstructures as a function of charge screening or pH. Fibrils either assembled into three-dimensional gel clusters or two-dimensional fibril sheets. The latter displayed optical birefringence, diagnostic of amyloid plaques. We presume that pH and salt modulate fibril charge repulsion, which allows anisotropic fibril–fibril attraction to emerge and drive the transition from 3D to 2D fibril self-assembly. [ABSTRACT FROM AUTHOR]

Aggregation of tau protein in the brain is associated with a class of neurodegenerative diseases known as tauopathies. FK506 binding protein 51 kDa (FKBP51, encoded by FKBP5) forms a mature [...]

Amyloid‐β peptides (Aβ) assemble into both rigid amyloid fibrils and metastable oligomers termed AβO or protofibrils. In Alzheimer's disease, Aβ fibrils constitute the core of senile plaques, but Aβ protofibrils may represent the main toxic species. Aβ protofibrils accumulate at the exterior of senile plaques, yet the protofibril–fibril interplay is not well understood. Applying chemical kinetics and atomic force microscopy to the assembly of Aβ and lysozyme, protofibrils are observed to bind to the lateral surfaces of amyloid fibrils. When utilizing Aβ variants with different critical oligomer concentrations, the interaction inhibits the autocatalytic proliferation of amyloid fibrils by secondary nucleation on the fibril surface. Thus, metastable oligomers antagonize their replacement by amyloid fibrils both by competing for monomers and blocking secondary nucleation sites. The protofibril—fibril interaction governs their temporal evolution and potential to exert specific toxic activities. [ABSTRACT FROM AUTHOR]

Intrinsic protein fluorescence is inextricably linked to the near-UV autofluorescence of aromatic amino acids. Here we show that a novel deep-blue autofluorescence (dbAF), previously thought to emerge as a result of protein aggregation, is present at the level of monomeric proteins and even poly- and single amino acids. Just as its aggregation-related counterpart, this autofluorescence does not depend on aromatic residues, can be excited at the long wavelength edge of the UV and emits in the deep blue. Differences in dbAF excitation and emission peaks and intensities from proteins and single amino acids upon changes in solution conditions suggest dbAF’s sensitivity to both the chemical identity and solution environment of amino acids. Autofluorescence comparable to dbAF is emitted by carbonyl-containing organic solvents, but not those lacking the carbonyl group. This implicates the carbonyl double bonds as the likely source for the autofluorescence in all these compounds. Using beta-lactoglobulin and proline, we have measured the molar extinction coefficients and quantum yields for dbAF in the monomeric state. To establish its potential utility in monitoring protein biophysics, we show that dbAF emission undergoes a red-shift comparable in magnitude to tryptophan upon thermal denaturation of lysozyme, and that it is sensitive to quenching by acrylamide. Carbonyl dbAF therefore provides a previously neglected intrinsic optical probe for investigating the structure and dynamics of amino acids, proteins and, by extension, DNA and RNA. [ABSTRACT FROM AUTHOR]

Poly( N-isopropylacrylamide) or p-NIPAAM gels undergo a prominent deswelling transition near physiological temperatures. Using passive microrheology, we have investigated the viscoelastic response of p-NIPAAM gels over a frequency range not accessible to bulk rheological measurements. Overall, NIPAAM gels moderately shear stiffen with increasing frequency. More intriguingly, sample viscosity rapidly declines with increasing frequency before leveling off near the solvent viscosity. The frequency for this crossover coincides with the emergence of fast gel modes seen in dynamic light scattering (DLS) from the gel. Furthermore, we monitored viscoelastic responses on approach to the deswelling transition. Intrinsic light scattering indicates that experimental conditions are not near the critical point and that the deswelling transition is second order in nature. Nevertheless, the corresponding elastic and viscous moduli of p-NIPAAM displayed power-law decreases with temperature. These changes with temperature were independent of probe frequency. Power law exponents, however, are sensitive to details of the sample preparation suggesting that these viscoelastic responses vary with gel structure. Correlating our microrheological measurements with DLS from the gel matrix itself, we find that several of the observed microrheological features are closely related to the intrinsic dynamics of the p-NIPAAM gels. In particular, the transition from gel- to solvent dominated dissipation coincides with a transition from fast to slow gel modes. Combining microrheology with intrinsic light scattering, therefore, provides a compelling approach to probe rheological responses and correlate them to the underlying network dynamics. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci., 2013 [ABSTRACT FROM AUTHOR]

Formation of large protein fibrils with a characteristic cross b-sheet architecture is the key indicator for a wide variety of systemic and neurodegenerative amyloid diseases. Recent experiments have strongly implicated oligomeric intermediates, transiently formed during fibril assembly, as critical contributors to cellular toxicity in amyloid diseases. At the same time, amyloid fibril assembly can proceed along different assembly pathways that might or might not involve such oligomeric intermediates. Elucidating the mechanisms that determine whether fibril formation proceeds along non-oligomeric or oligomeric pathways, therefore, is important not just for understanding amyloid fibril assembly at the molecular level but also for developing new targets for intervening with fibril formation. We have investigated fibril formation by hen egg white lysozyme, an enzyme for which human variants underlie non-neuropathic amyloidosis. Using a combination of static and dynamic light scattering, atomic force microscopy and circular dichroism, we find that amyloidogenic lysozyme monomers switch between three different assembly pathways: from monomeric to oligomeric fibril assembly and, eventually, disordered precipitation as the ionic strength of the solution increases. Fibril assembly only occurred under conditions of net repulsion among the amyloidogenic monomers while net attraction caused precipitation. The transition from monomeric to oligomeric fibril assembly, in turn, occurred as salt-mediated charge screening reduced repulsion among individual charged residues on the same monomer. We suggest a model of amyloid fibril formation in which repulsive charge interactions are a prerequisite for ordered fibril assembly. Furthermore, the spatial extent of non-specific charge screening selects between monomeric and oligomeric assembly pathways by affecting which subset of denatured states can form suitable intermolecular bonds and by altering the energetic and entropic requirements for the initial intermediates emerging along the monomeric vs. oligomeric assembly path. [ABSTRACT FROM AUTHOR]

Molecular chaperones regulate the aggregation of a number of proteins that pathologically misfold and accumulate in neurodegenerative diseases. Identifying ways to manipulate these proteins in disease models is an area of intense investigation; however, the translation of these results to the mammalian brain has progressed more slowly. In this study, we investigated the ability of one of these chaperones, heat shock protein 27 (Hsp27), to modulate tau dynamics. Recombinant wild-type Hsp27 and a genetically altered version of Hsp27 that is perpetually pseudo-phosphorylated (3×S/D) were generated. Both Hsp27 variants interacted with tau, and atomic force microscopy and dynamic light scattering showed that both variants also prevented tau filament formation. However, extrinsic genetic delivery of these two Hsp27 variants to tau transgenic mice using adeno-associated viral particles showed that wild-type Hsp27 reduced neuronal tau levels, whereas 3×S/D Hsp27 was associated with increased tau levels. Moreover, rapid decay in hippocampal long-term potentiation (LTP) intrinsic to this tau transgenic model was rescued by wild-type Hsp27 overexpression but not by 3×S/D Hsp27. Because the 3×S/D Hsp27 mutant cannot cycle between phosphorylated and dephosphorylated states, we can conclude that Hsp27 must be functionally dynamic to facilitate tau clearance from the brain and rescue LTP; however, when this property is compromised, Hsp27 may actually facilitate accumulation of soluble tau intermediates. [ABSTRACT FROM AUTHOR]

Using millisecond time-resolved optical recordings of transmembrane voltage and intraterminal calcium, we have determined how activity-dependent changes in the population action potential are related to a concurrent modulation of calcium transients in the neurohypophysis. We find that repetitive stimulation dramatically alters the amplitude of the population action potential and significantly increases its temporal dispersion. The population action potentials and the calcium transients exhibit well correlated frequency--dependent amplitude depression, with broadening of the action potential playing only a limited role. High-speed camera recordings indicate that the magnitude of the spike modulation is uniform throughout the neurohypophysis, thereby excluding propagation failure as the underlying mechanism. In contrast, temporal dispersion and latency of the population spike do increase with distance from the stimulation site. This increase is enhanced during repeated stimulation and by raising the stimulation frequency. Changes in Ca influx directly affect the decline in population spike amplitude, consistent with electrophysiological measurements of the local loss of excitability in nerve terminals and varicosities, mediated by a Ca-activated K conductance. Our observations suggest a model of "stuttering conduction": repeated action potential stimulation causes excitability failures limited to nerve terminals and varicosities, which account for the rapid decline in the population spike amplitude. These failures, however, do not block action potential propagation but generate the cumulative increases in spike latency. [ABSTRACT FROM AUTHOR]

Amyloid-formation by the islet amyloid polypeptide (IAPP), produced by the β-cells in the human pancreas, has been associated with the development of type II diabetes mellitus (T2DM). The human plasma-protein transthyretin (TTR), a well-known amyloid-inhibiting protein, is interestingly also expressed within the IAPP producing β-cells. In the present study, we have characterized the ability of TTR to interfere with IAPP amyloid-formation, both in terms of its intrinsic stability as well as with regard to the effect of TTR-stabilizing drugs. The results show that TTR can prolong the lag-phase as well as impair elongation in the course of IAPP-amyloid formation. We also show that the interfering ability correlates inversely with the thermodynamic stability of TTR, while no such correlation was observed as a function of kinetic stability. Furthermore, we demonstrate that the ability of TTR to interfere is maintained also at the low pH environment within the IAPP-containing granules of the pancreatic β-cells. However, at both neutral and low pH, the addition of TTR-stabilizing drugs partly impaired its efficacy. Taken together, these results expose mechanisms of TTR-mediated inhibition of IAPP amyloid-formation and highlights a potential therapeutic target to prevent the onset of T2DM. [ABSTRACT FROM AUTHOR]

Deposits of fibrils formed by disease-specific proteins are the molecular hallmark of such diverse human disorders as Alzheimer's disease, type II diabetes, or rheumatoid arthritis. Amyloid fibril formation by structurally and functionally unrelated proteins exhibits many generic characteristics, most prominently the cross β-sheet structure of their mature fibrils. At the same time, amyloid formation tends to proceed along one of two separate assembly pathways yielding either stiff monomeric filaments or globular oligomers and curvilinear protofibrils. Given the focus on oligomers as major toxic species, the very existence of an oligomer-free assembly pathway is significant. Little is known, though, about the structure of the various intermediates emerging along different pathways and whether the pathways converge towards a common or distinct fibril structures. Using infrared spectroscopy we probed the structural evolution of intermediates and late-stage fibrils formed during in vitro lysozyme amyloid assembly along an oligomeric and oligomer-free pathway. Infrared spectroscopy confirmed that both pathways produced amyloid-specific β-sheet peaks, but at pathway-specific wavenumbers. We further found that the amyloid-specific dye thioflavin T responded to all intermediates along either pathway. The relative amplitudes of thioflavin T fluorescence responses displayed pathway-specific differences and could be utilized for monitoring the structural evolution of intermediates. Pathway-specific structural features obtained from infrared spectroscopy and Thioflavin T responses were identical for fibrils grown at highly acidic or at physiological pH values and showed no discernible effects of protein hydrolysis. Our results suggest that late-stage fibrils formed along either pathway are amyloidogenic in nature, but have distinguishable structural fingerprints. These pathway-specific fingerprints emerge during the earliest aggregation events and persist throughout the entire cascade of aggregation intermediates formed along each pathway. [ABSTRACT FROM AUTHOR]

Assembly of amyloid fibrils and small globular oligomers is associated with a significant number of human disorders that include Alzheimer's disease, senile systemic amyloidosis, and type II diabetes. Recent findings implicate small amyloid oligomers as the dominant aggregate species mediating the toxic effects in these disorders. However, validation of this hypothesis has been hampered by the dearth of experimental techniques to detect, quantify, and discriminate oligomeric intermediates from late-stage fibrils, in vitro and in vivo. We have shown that the onset of significant oligomer formation is associated with a transition in thioflavin T kinetics from sigmoidal to biphasic kinetics. Here we showed that this transition can be exploited for screening fluorophores for preferential responses to oligomer over fibril formation. This assay identified crystal violet as a strongly selective oligomer-indicator dye for lysozyme. Simultaneous recordings of amyloid kinetics with thioflavin T and crystal violet enabled us to separate the combined signals into their underlying oligomeric and fibrillar components. We provided further evidence that this screening assay could be extended to amyloid-β peptides under physiological conditions. Identification of oligomer-selective dyes not only holds the promise of biomedical applications but provides new approaches for unraveling the mechanisms underlying oligomer versus fibril formation in amyloid assembly. [ABSTRACT FROM AUTHOR]

In contrast to prevalent strategies which make use of β-sheet mimetics to block Aβ fibrillar growth, in this study, we designed a series of sulfonyl-γ-AApeptide helices that targeted the crucial α-helix domain of Aβ13-26 and stabilized Aβ conformation to avoid forming the neurotoxic Aβ oligomeric β-sheets. Biophysical assays such as amyloid kinetics and TEM demonstrated that the Aβ oligomerization and fibrillation could be greatly prevented and even reversed in the presence of sulfonyl-γ-AApeptides in a sequence-specific and dose-dependent manner. The studies based on circular dichroism, Two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) spectra unambiguously suggested that the sulfonyl-γ-AApeptide Ab-6 could bind to the central region of Aβ 42 and induce α-helix conformation in Aβ. Additionally, Electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) was employed to rule out a colloidal mechanism of inhibitor and clearly supported the capability of Ab-6 for inhibiting the formation of Aβ aggregated forms. Furthermore, Ab-6 could rescue neuroblastoma cells by eradicating Aβ-mediated cytotoxicity even in the presence of pre-formed Aβ aggregates. The confocal microscopy demonstrated that Ab-6 could still specifically bind Aβ 42 and colocalize into mitochondria in the cellular environment, suggesting the rescue of cell viability might be due to the protection of mitochondrial function otherwise impaired by Aβ 42 aggregation. Taken together, our studies indicated that sulfonyl-γ-AApeptides as helical peptidomimetics could direct Aβ into the off-pathway helical secondary structure, thereby preventing the formation of Aβ oligomerization, fibrillation and rescuing Aβ induced cell cytotoxicity., Competing Interests: Competing interests statement:The authors declare no competing interest.

Amyloid Diseases involve the growth of disease specific proteins into amyloid fibrils and their deposition in protein plaques. Amyloid fibril formation is typically preceded by oligomeric intermediates. Despite significant efforts, the specific role fibrils or oligomers play in the etiology of any given amyloid disease remains controversial. In neurodegenerative disease, though, amyloid oligomers are widely considered critical contributors to disease symptoms. Aside from oligomers as inevitable on-pathway precursors of fibril formation, there is significant evidence for off-pathway oligomer formation competing with fibril growth. The distinct mechanisms and pathways of oligomer formation directly affect our understanding under which conditions oligomers emerge in vivo, and whether their formation is directly coupled to, or distinct from, amyloid fibril formation. In this review, we will discuss the basic energy landscapes underlying the formation of on-pathway vs. off-pathway oligomers, their relation to the related amyloid aggregation kinetics, and their resulting implications for disease etiology. We will review evidence on how differences in the local environment of amyloid assembly can dramatically shift the relative preponderance of oligomers vs. fibrils. Finally, we will comment on gaps in our knowledge of oligomer assembly, of their structure, and on how to assess their relevance to disease etiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Muschol and Hoyer.)

There is compelling evidence that small oligomeric aggregates, emerging during the assembly of amyloid fibrils and plaques, are important molecular pathogens in many amyloid diseases. While significant progress has been made in revealing the mechanisms underlying fibril growth, understanding how amyloid oligomers fit into the fibril assembly process, and how they contribute to the pathogenesis of amyloid diseases, has remained elusive. Commonly, amyloid oligomers are considered to be metastable, early-stage precursors to fibril formation that are either on- or off-pathway from fibril growth. In addition, amyloid oligomers have been reported to colocalize with late-stage fibrils and plaques. Whether these early and late-stage oligomer species are identical or distinct, and whether both are relevant to pathogenesis remains unclear. Here we report on the formation of two distinct oligomer species of lysozyme, formed either during the early or late-stages of in vitro fibril growth. We further observe that the pH change from in vitro growth conditions to cell media used for toxicity studies induced distinct mesoscopic precipitates, two of which resemble either diffuse or neuritic plaques seen in Alzheimer's histology. Our biophysical characterization indicates that both oligomer species share morphological and tinctorial features considered characteristic for amyloid oligomers. At the same time, their sizes, morphologies, their immunostaining, detailed tinctorial profiles and, most prominently, their biological activity are clearly distinct from each other. Probing the conditions promoting the formation of these two distinct oligomer species suggests distinct roles of charge interactions, hydrophobicity and monomer flexibility in directing oligomer assembly., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Amyloid-β peptides (Aβ) assemble into both rigid amyloid fibrils and metastable oligomers termed AβO or protofibrils. In Alzheimer's disease, Aβ fibrils constitute the core of senile plaques, but Aβ protofibrils may represent the main toxic species. Aβ protofibrils accumulate at the exterior of senile plaques, yet the protofibril-fibril interplay is not well understood. Applying chemical kinetics and atomic force microscopy to the assembly of Aβ and lysozyme, protofibrils are observed to bind to the lateral surfaces of amyloid fibrils. When utilizing Aβ variants with different critical oligomer concentrations, the interaction inhibits the autocatalytic proliferation of amyloid fibrils by secondary nucleation on the fibril surface. Thus, metastable oligomers antagonize their replacement by amyloid fibrils both by competing for monomers and blocking secondary nucleation sites. The protofibril-fibril interaction governs their temporal evolution and potential to exert specific toxic activities., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Fascin is an actin bundling protein that cross-links individual actin filaments into straight, compact, and stiff bundles, which are crucial for the formation of filopodia, stereocillia, and other finger-like membrane protrusions. The dysregulation of fascin has been implicated in cancer metastasis, hearing loss, and blindness. Here we identified monoubiquitination as a novel mechanism that regulates fascin bundling activity and dynamics. The monoubiquitination sites were identified to be Lys 247 and Lys 250 , two residues located in a positive charge patch at the actin binding site 2 of fascin. Using a chemical ubiquitination method, we synthesized chemically monoubiquitinated fascin and determined the effects of monoubiquitination on fascin bundling activity and dynamics. Our data demonstrated that monoubiquitination decreased the fascin bundling EC 50 , delayed the initiation of bundle assembly, and accelerated the disassembly of existing bundles. By analyzing the electrostatic properties on the solvent-accessible surface of fascin, we proposed that monoubiquitination introduced steric hindrance to interfere with the interaction between actin filaments and the positively charged patch at actin binding site 2. We also identified Smurf1 as a E3 ligase regulating the monoubiquitination of fascin. Our findings revealed a previously unidentified regulatory mechanism for fascin, which will have important implications for the understanding of actin bundle regulation under physiological and pathological conditions., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Self-assembly of proteins and peptides into amyloid fibrils involves multiple distinct intermediates and late-stage fibrillar polymorphs. Understanding the conditions and mechanisms that promote the formation of one type of intermediate and polymorph over the other represents a fundamental challenge. Answers to this question are also of immediate biomedical relevance since different amyloid aggregate species have been shown to have distinct pathogenic potencies. One amyloid polymorph that has received comparatively little attention are amyloid spherulites. Here we report that self-assembly of the intrinsically disordered polymer poly(L-glutamic) acid (PLE) can generate amyloid spherulites. We characterize spherulite growth kinetics, as well as the morphological, optical and tinctorial features of this amyloid polymorph previously unreported for PLE. We find that PLE spherulites share both tinctorial and structural characteristics with their amyloid fibril counterparts. Differences in PLE's molecular weight, polydispersity or chemistry could not explain the selective propensity toward either fibril or spherulite formation. Instead, we provide evidence that PLE polymers can exist in either a collapsed globule or an extended random coil conformation. The collapsed globule consistently produces spherulites while the extended coil assembles into disordered fibril bundles. This results suggests that these 2 PLE conformers directly affect the morphology of the resulting macroscopic amyloid assembly.

Self-assembly of proteins into amyloid fibrils plays a key role in a multitude of human disorders that range from Alzheimer's disease to type II diabetes. Compact oligomeric species, observed early during amyloid formation, are reported as the molecular entities responsible for the toxic effects of amyloid self-assembly. However, the relation between early-stage oligomeric aggregates and late-stage rigid fibrils, which are the hallmark structure of amyloid plaques, has remained unclear. We show that these different structures occupy well-defined regions in a peculiar phase diagram. Lysozyme amyloid oligomers and their curvilinear fibrils only form after they cross a salt and protein concentration-dependent threshold. We also determine a boundary for the onset of amyloid oligomer precipitation. The oligomeric aggregates are structurally distinct from rigid fibrils and are metastable against nucleation and growth of rigid fibrils. These experimentally determined boundaries match well with colloidal model predictions that account for salt-modulated charge repulsion. The model also incorporates the metastable and kinetic character of oligomer phases. Similarities and differences of amyloid oligomer assembly to metastable liquid-liquid phase separation of proteins and to surfactant aggregation are discussed.

Intrinsic optical changes (light scattering signals) occur in mammalian nerve terminals during and immediately following the arrival of the action potential. In the neurohypophysis (posterior pituitary gland), the action potential is coupled to calcium-mediated secretion of the neuropeptides oxytocin and vasopressin. This excitation-secretion coupling is intimately related to extremely rapid changes in light scattering. These optical signals provide a millisecond-time-resolved monitor of events in the terminals that follow the arrival of the action potential and the entry of calcium. Light scattering procedures are designed to measure intrinsic optical signals from mammalian nerve terminals. In practice, these signals are remarkably simple to record from any of the mammalian neurohypophyses that have been studied. To date, this approach has been used successfully in mouse, rat, and guinea pig. This protocol provides instrumentation requirements and a method for preparation of the neurohypophysis so that intrinsic optical signals can be measured from nerve terminals. It also includes a discussion of the interpretation of the signals that are obtained.

Developing a comprehensive description of the equilibrium structural ensembles for intrinsically disordered proteins (IDPs) is essential to understanding their function. The p53 transactivation domain (p53TAD) is an IDP that interacts with multiple protein partners and contains numerous phosphorylation sites. Multiple techniques were used to investigate the equilibrium structural ensemble of p53TAD in its native and chemically unfolded states. The results from these experiments show that the native state of p53TAD has dimensions similar to a classical random coil while the chemically unfolded state is more extended. To investigate the molecular properties responsible for this behavior, a novel algorithm that generates diverse and unbiased structural ensembles of IDPs was developed. This algorithm was used to generate a large pool of plausible p53TAD structures that were reweighted to identify a subset of structures with the best fit to small angle X-ray scattering data. High weight structures in the native state ensemble show features that are localized to protein binding sites and regions with high proline content. The features localized to the protein binding sites are mostly eliminated in the chemically unfolded ensemble; while, the regions with high proline content remain relatively unaffected. Data from NMR experiments support these results, showing that residues from the protein binding sites experience larger environmental changes upon unfolding by urea than regions with high proline content. This behavior is consistent with the urea-induced exposure of nonpolar and aromatic side-chains in the protein binding sites that are partially excluded from solvent in the native state ensemble.

Using static and dynamic light scattering we have investigated the effects of either strongly chaotropic, nearly neutral or strongly kosmotropic salt ions on the hydration shell and the mutual hydrodynamic interactions of the protein lysozyme under conditions supportive of protein crystallization. After accounting for the effects of protein interaction and for changes in solution viscosity on protein diffusivity, protein hydrodynamic radii were determined with +/-0.25 A resolution. No changes to the extent of lysozyme hydration were discernible for all salt-types, at any salt concentration and for temperatures between 15-40 degrees C. Combining static with dynamic light scattering, we also investigated salt-induced changes to the hydrodynamic protein interactions. With increased salt concentration, hydrodynamic interactions changed from attractive to repulsive, i.e., in exact opposition to salt-induced changes in direct protein interactions. This anti-correlation was independent of solution temperature or salt identity. Although salt-specific effects on direct protein interactions were prominent, neither protein hydration nor solvent-mediated hydrodynamic interactions displayed any obvious salt-specific effects. We infer that the protein hydration shell is more resistant than bulk water to changes in its local structure by either chaotropic or kosmotropic ions.

The mechanisms linking deposits of insoluble amyloid fibrils to the debilitating neuronal cell death characteristic of neurodegenerative diseases remain enigmatic. Recent findings implicate transiently formed intermediates of mature amyloid fibrils as the principal toxic agent. Hence, determining which intermediate aggregates represent on-pathway precursors or off-pathway side branches is critical for understanding amyloid self-assembly, and for devising therapeutic approaches targeting relevant toxic species. We examined amyloid fibril self-assembly in acidic solutions, using the model protein hen egg-white lysozyme. Combining in situ dynamic light scattering with calibrated atomic-force microscopy, we monitored the nucleation and growth kinetics of multiple transient aggregate species, and characterized both their morphologies and physical dimensions. Upon incubation at elevated temperatures, uniformly sized oligomers formed at a constant rate. After a lag period of several hours, protofibrils spontaneously nucleated. The nucleation kinetics of protofibrils and the tight match of their widths and heights with those of oligomers imply that protofibrils both nucleated and grew via oligomer fusion. After reaching several hundred nanometers in length, protofibrils assembled into mature fibrils. Overall, the amyloid fibril assembly of lysozyme followed a strict hierarchical aggregation pathway, with amyloid monomers, oligomers, and protofibrils forming on-pathway intermediates for assembly into successively more complex structures.

Voltage-sensitive dyes (VSDs) provide a spatially resolved optical read-out of electrical signals in excitable tissues. Several common fluorescent VSDs display electrochromic shifts of their emission spectra, making them suitable candidates for ratiometric measurements of transmembrane voltages. These advantages of VSDs are tempered by tissue-specific shifts to their fluorescence emission. In addition, the optimal electrochromic dye response occurs in wavelength bands distinct from the dye's maximal resting emission. This "action spectrum" can undergo tissue-specific shifts as well. We have developed a technique for in situ measurements of the action spectra of VSDs in intact excitable tissues. Fluorescence emission spectra of VSDs during action-potential depolarization were obtained within a single sweep of a spectrophotometer equipped with a change-coupled device (CCD) array detector. To resolve the subtle electrochromic shifts in voltage-induced dye emission, fluorescence emission spectra measured right before and during field-induced action-potential depolarization were averaged over about 100 trials. Removing white-noise contributions from the spectrometer's CCD detector/amplifier via low-pass filtering in Fourier space, the action spectra of all dyes could be readily determined.