Unfolded Monomers Research Articles

The Synthesis of a 2D Ultra-Large Protein Supramolecular Nanofilm by Chemoselective Thiol-Disulfide Exchange and its Emergent Functions.

The design and scalable synthesis of robust 2D biological ultrathin films with a tunable structure and function and the ability to be easily transferred to a range of substrates remain key challenges in chemistry and materials science. Herein, we report the use of the thiol-disulfide exchange reaction in the synthesis of a macroscopic 2D ultrathin proteinaceous film with the potential for large-scale fabrication and on-demand encapsulation/release of functional molecules. The reaction between the Cys6-Cys127 disulfide bond of lysozyme and cysteine is chemo- and site-selective. The partially unfolded lysozyme-cysteine monomers aggregate at the air/water or solid/liquid interface to form an ultra-large 2D nanofilm (900 cm2 ) with about 100 % optical transparency. This material adheres to a wide range of substrates and encapsulates and releases a range of molecules without significantly affecting activity.

Molecular Mechanisms of the Ultra-Strong Inhibition Effect of Oxidized Carbon Dots on Human Insulin Fibrillation.

Amyloid fibrillation of protein is associated with a great variety of pathologic conditions. The aggregation of protein is a complicated process with multisteps, whereas most of the inhibitors with elaborately designed structures can show an inhibition effect only on the nucleation stages of protein fibrillation. Herein, oxidized carbon dots (CDs) were achieved to study the relationship between the surface properties of CDs and their inhibition effect on human insulin (HI) fibrillation. More oxygen-containing function groups can be obtained after oxidation reaction of CDs, such as -OH and -COOH. The results show that 10-1 CDs (the mass ratios of CD/KMnO4 is 10:1), with the highest carboxyl group content, possess the best inhibition ability. All the nucleation, growth, and final phases can be retarded by 10-1 CDs, which have been studied in detail by fluorescence spectra. However, CDs without oxidation can show only a weak inhibition effect on the nucleation stage. The 10-1 CDs is demonstrated to binding with HI monomers much stronger than that of CDs by isothermal titration calorimetry (ITC). Moreover, molecular dynamics simulations (MD) studies indicate that CDs with more carboxyl groups can show stronger affinities with native or unfolded HI monomers, which may be mainly derived from the active binding sites of histidine residues (His5 and His10) on B-chain through electrostatic interaction. Because the unfolding of B-chain in HI is prior to that of A-chain in our MD simulations, the later aggregation of HI can be inhibited effectively by the stronger binding forces between 10 and 1 CDs and the B-chain of HI.

Capillary zone electrophoresis-native mass spectrometry for the quality control of intact therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies (mAbs) are complex glycoproteins and ensuring their safety, efficacy and quality is still challenging. Indeed, during their manufacturing process, they are exposed to several stresses that can lead to their denaturation, misfolding or dimerization. We report here a new method based on capillary electrophoresis coupled to native mass spectrometry (MS) with a sheath liquid interface to analyze an intact therapeutic mAb, Infliximab, under non-denaturing conditions that preserve its conformational heterogeneity as well as self-association without inducing further unfolding / denaturation. For capillary zone electrophoresis (CZE) separation, a triple layer coating using polybrene-dextran sulfate-polybrene was employed. A sheath liquid composed of isopropanol - water - acetic acid with a flow rate of 10 μL min−1 and mild MS conditions allowed optimal signal intensities. A specific mass spectrum was obtained for each Infliximab conformation in a “stressed” formulated preparation. This is the first time that within a single analysis different conformational states, i.e. native and unfolded monomers as well as dimers are simultaneously detected. The results and the lack of analytical bias arising from the CZE-MS conditions were confirmed by using atomic force microscopy (AFM) as an orthogonal technique. A middle-up approach combined to CZE-MS analysis of the stressed samples suggested that the dimer formation involved mostly Fab-Fab interactions.

Structural, thermodynamic and catalytic characterization of an ancestral triosephosphate isomerase reveal early evolutionary coupling between monomer association and function.

Function, structure, and stability are strongly coupled in obligated oligomers, such as triosephosphate isomerase (TIM). However, little is known about how this coupling evolved. To address this question, five ancestral TIMs (ancTIMs) in the opisthokont lineage were inferred. The encoded proteins were purified and characterized, and spectroscopic and hydrodynamic analysis indicated that all are folded dimers. The catalytic efficiency of ancTIMs is very high and all dissociate into inactive and partially unfolded monomers. The placement of catalytic residues in the three-dimensional structure, as well as the enthalpy-driven binding signature of the oldest ancestor (TIM63) resemble extant TIMs. Although TIM63 dimers dissociate more readily than do extant TIMs, calorimetric data show that the free ancestral subunits are folded to a greater extent than their extant counterparts are, suggesting that full catalytic proficiency was established in the dimer before the stability of the isolated monomer eroded. Notably, the low association energy in ancTIMs is compensated for by a high activation barrier, and by a significant shift in the dimer-monomer equilibrium induced by ligand binding. Our results indicate that before the animal and fungi lineages diverged, TIM was an obligated oligomer with substrate binding properties and catalytic efficiency that resemble that of extant TIMs. Therefore, TIM function and association have been strongly coupled at least for the last third of biological evolution on earth. DATABASES: PDB Entry: 6NEE. ENZYMES: Triosephosphate isomerase 5.3.1.1, Glycerol-3-phosphate dehydrogenase 1.1.1.8.

Topological Analysis of Transthyretin Disassembly Mechanism: Surface-Induced Dissociation Reveals Hidden Reaction Pathways.

The proposed mechanism of fibril formation of transthyretin (TTR) involves self-assembly of partially unfolded monomers. However, the mechanism(s) of disassembly to monomer and potential intermediates involved in this process are not fully understood. In this study, native mass spectrometry and surface-induced dissociation (SID) are used to investigate the TTR disassembly mechanism(s) and the effects of temperature and ionic strength on the kinetics of TTR complex formation. Results from the SID of hybrid tetramers formed during subunit exchange provide strong evidence for a two-step mechanism whereby the tetramer dissociates to dimers that then dissociate to monomers. Also, the SID results uncovered a hidden pathway in which a specific topology of the hybrid tetramer is directly produced by assembly of dimers in the early steps of TTR disassembly. Implementation of SID to dissect protein topology during subunit exchange provides unique opportunities to gain unparalleled insight into disassembly pathways.

Structure and conformational stability of the triosephosphate isomerase from Zea mays. Comparison with the chemical unfolding pathways of other eukaryotic TIMs

We studied the structure, function and thermodynamic properties for the unfolding of the Triosephosphate isomerase (TIM) from Zea mays (ZmTIM). ZmTIM shows a catalytic efficiency close to the diffusion limit. Native ZmTIM is a dimer that dissociates upon dilution into inactive and unfolded monomers. Its thermal unfolding is irreversible with a Tm of 61.6 ± 1.4 °C and an activation energy of 383.4 ± 11.5 kJ mol−1. The urea-induced unfolding of ZmTIM is reversible. Transitions followed by catalytic activity and spectroscopic properties are monophasic and superimposable, indicating that ZmTIM unfolds/refolds in a two-state behavior with an unfolding ΔG°(H20) = 99.8 ± 5.3 kJ mol−1. This contrasts with most other studied TIMs, where folding intermediates are common. The three-dimensional structure of ZmTIM was solved at 1.8 Å. A structural comparison with other eukaryotic TIMs shows a similar number of intramolecular and intermolecular interactions. Interestingly the number of interfacial water molecules found in ZmTIM is lower than those observed in most TIMs that show folding intermediates. Although with the available data, there is no clear correlation between structural properties and the number of equilibrium intermediates in the unfolding of TIM, the identification of such structural properties should increase our understanding of folding mechanisms.

Nonproductive Binding Modes as a Prominent Feature of Aβ40 Fiber Elongation: Insights from Molecular Dynamics Simulation

The formation of amyloid fibers has been implicated in a number of neurodegenerative diseases. The growth of amyloid fibers is strongly thermodynamically favorable, but kinetic traps exist where the incoming monomer binds in an incompatible conformation that blocks further elongation. Unfortunately, this process is difficult to follow experimentally at the atomic level. It is also too complex to simulate in full detail and to date has been explored either through coarse-grained simulations, which may miss many important interactions, or full atomic simulations, in which the incoming peptide is constrained to be near the ideal fiber geometry. Here we use an alternate approach starting from a docked complex in which the monomer is from an experimental NMR structure of one of the major conformations in the unbound ensemble, a largely unstructured peptide with the central hydrophobic region in a 310 helix. A 1000 ns full atomic simulation in explicit solvent shows the formation of a metastable intermediate by sequential, concerted movements of both the fiber and the monomer. A Markov state model shows that the unfolded monomer is trapped at the end of the fiber in a set of interconverting antiparallel β-hairpin conformations. The simulation here may serve as a model for the binding of other non-β-sheet conformations to amyloid fibers.

Polymerization pathway of mammalian nonmuscle myosin 2s

The three mammalian nonmuscle myosin 2 (NM2) monomers, like all class 2 myosin monomers, are hexamers of two identical heavy (long) chains and two pairs of light (short) chains bound to the heavy chains. The heavy chains have an N-terminal globular motor domain (head) with actin-activated ATPase activity, a lever arm (neck) to which the two light chains bind, and a coiled-coil helical tail. Monomers polymerize into bipolar filaments, with globular heads at each end separated by a bare zone, by antiparallel association of their coiled-coil tails. NM2 filaments are highly dynamic in situ, frequently disassembling and reassembling at different locations within the cell where they are essential for multiple biological functions. Therefore, it is important to understand the mechanisms of filament polymerization and depolymerization. Monomers can exist in two states: folded and unfolded. It has been thought that unfolded monomers form antiparallel dimers that assemble into bipolar filaments. We now show that polymerization in vitro proceeds from folded monomers to folded antiparallel dimers to folded antiparallel tetramers that unfold forming antiparallel bipolar tetramers. Folded dimers and tetramers then associate with the unfolded tetramer and unfold, forming a mature bipolar filament consisting of multiple unfolded tetramers with an entwined bare zone. We also demonstrate that depolymerization is essentially the reverse of the polymerization process. These results will advance our understanding of NM2 filament dynamics in situ.

Effect of Various Polyols on the Acid-Denatured States of Champedak Galactose-Binding Lectin.

Champedak galactose-binding (CGB) lectin is a tetrameric protein with noncovalently bound monomers, isolated from Artocarpus integer fruit seeds. We had previously reported existence of a structured monomer and an unfolded monomer of CGB lectin at pH 2.5 and pH 1.5, respectively. Polyols are known to induce significant refolding in denatured proteins and stabilize proteins against environmental stresses. Studies on the effect of various polyols on the acid-denatured states of CGB lectin are lacking. The objective of this study was to investigate the effects of four different polyols, namely, ethylene glycol, erythritol, xylitol and sorbitol on the acid-denatured states of CGB lectin. CGB lectin was subjected to acid denaturation at pH 2.5 and pH 1.5, both in the absence and presence of 30% (w/v) polyols, i.e. ethylene glycol, erythritol, xylitol and sorbitol. Thermal denaturation of the acid-denatured states was also studied in the absence and presence of these polyols. Different spectroscopic probes such as tryptophan fluorescence, ANS fluorescence and far-UV CD spectral signal were used to monitor structural changes in the acid-denatured states of CGB lectin in the presence of polyols. Presence of erythritol, xylitol and sorbitol in the incubation mixture was found to stabilize the lectin at both pH 2.5 and pH 1.5, as evident from the burial of the hydrophobic clusters and decreased polarity around Trp residues. These polyols also stabilized the acid-denatured states of CGB lectin against thermal denaturation by shifting the thermal transition curves towards higher temperatures. Exposure of the acid-denatured states of CGB lectin, obtained at pH 2.5 and pH 1.5 to 61°C and 51°C, respectively, induced formation of non-native β-structures, compared to that present at 25°C, and this phenomenon was significantly suppressed in the presence of these polyols. Based on the spectral data, both sorbitol and erythritol appeared to exude better stabilizing effect. On the other hand, ethylene glycol was shown to destabilize the aciddenatured states of CGB lectin. Thermal stabilization of the lectin was noticed in the presence of erythritol, xylitol and sorbitol at both pH 2.5 and pH 1.5. These polyols also stabilize the secondary and tertiary structures of the acid-denatured CGB lectin at 25°C. Ethylene glycol was proved to be a destabilizer of the acid-denatured CGB lectin.

Similar structural stabilities of 3-isopropylmalate dehydrogenases from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 and its atmospheric congener S. oneidensis strain MR-1

We previously found that the enzymatic activity of 3-isopropylmalate dehydrogenase from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 (SbIPMDH) was pressure-tolerant up to 100 MPa, but that from its atmospheric congener S. oneidensis strain MR-1 (SoIPMDH) was pressure-sensitive. Such characteristics were determined by only one amino acid residue at position 266, serine (SoIPMDH) or alanine (SbIPMDH) [Y. Hamajima et al. Extremophiles 20: 177, 2016]. In this study, we investigated the structural stability of these enzymes. At pH 7.6, SoIPMDH was slightly more stable against hydrostatic pressure than SbIPMDH, contrary to the physiological pressures of their normal environments. Pressure unfolding of these IPMDHs followed a two-state unfolding model between a native dimer and two unfolded monomers, and the dimer structure was pressure-tolerant up to 200 MPa, employing a midpoint pressure of 245.3 ± 0.1 MPa and a volume change of −225 ± 24 mL mol−1 for the most unstable mutant, SbIPMDH A266S. Thus, their pressure-dependent activity did not originate from structural perturbations such as unfolding or dimer dissociation. Conversely, urea-induced unfolding of these IPMDHs followed a three-state unfolding model, including a dimer intermediate. Interestingly, the first transition was strongly pH-dependent but pressure-independent; however, the second transition showed the opposite pattern. Obtained volume changes due to urea-induced unfolding were almost equal for both IPMDHs, approximately +10 and −30 mL mol−1 for intermediate formation and dimer dissociation, respectively. These results indicated that both IPMDHs have similar structural stability, and a pressure-adaptation mechanism was provided for only the enzymatic activity of SbIPMDH.

Monomerization and aggregation of β-lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~55Å, ~29Å and ~37Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57kcalmol−1 and 8.93kcalmol−1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~77Å and ~117Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69kcalmol−1 and 9.09kcalmol−1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.

The Mechanism of HdeA Unfolding and Chaperone Activation

HdeA is a periplasmic chaperone that is rapidly activated upon shifting the pH to acidic conditions. This activation is thought to involve monomerization of HdeA. There is evidence that monomerization and partial unfolding allow the chaperone to bind to proteins denatured by low pH, thereby protecting them from aggregation. We analyzed the acid-induced unfolding of HdeA using NMR spectroscopy and fluorescence measurements, and obtained experimental evidence suggesting a complex mechanism in HdeA's acid-induced unfolding pathway, as previously postulated from molecular dynamics simulations. Counterintuitively, dissociation constant measurements show a stabilization of the HdeA dimer upon exposure to mildly acidic conditions. We provide experimental evidence that protonation of Glu37, a glutamate residue embedded in a hydrophobic pocket of HdeA, is important in controlling HdeA stabilization and thus the acid activation of this chaperone. Our data also reveal a sharp transition from folded dimer to unfolded monomer between pH3 and pH 2, and suggest the existence of a low-populated, partially folded intermediate that could assist in chaperone activation or function. Overall, this study provides a detailed experimental investigation into the mechanism by which HdeA unfolds and activates.

Characterizations of the Interactions between Escherichia coli Periplasmic Chaperone HdeA and Its Native Substrates during Acid Stress.

The bacterial acid-resistant chaperone HdeA is a "conditionally disordered" protein that functions at low pH when it undergoes a transition from a well-folded dimer to an unfolded monomer. The dimer dissociation and unfolding processes result in exposure of hydrophobic surfaces that allows binding to a broad range of client proteins. To fully elucidate the chaperone mechanism of HdeA, it is crucial to understand how the activated HdeA interacts with its native substrates during acid stress. Herein, we present a nuclear magnetic resonance study of the pH-dependent HdeA-substrate interactions. Our results show that the activation of HdeA is not only induced by acidification but also regulated by the presence of unfolded substrates. The variable extent of unfolding of substrates differentially regulates the HdeA-substrate interaction, and the binding further affects the HdeA conformation. Finally, we show that HdeA binds its substrates heterogeneously, and the "amphiphilic" model for HdeA-substrate interaction is discussed.

Fate of a Stressed Therapeutic Antibody Tracked by Fluorescence Correlation Spectroscopy: Folded Monomers Survive Aggregation.

Antibodies are therapeutic proteins that are becoming indispensable for the treatment of serious diseases. Their efficacies depend on their folded structures, the loss of which, through unfolding or aggregation, should be closely monitored during their manufacture, storage, and dosing for safe usage. In downstream processes, exposure of the crude antibody solution to acid, followed by neutralization, is an established standard protocol for antibody purification and inactivation of viruses in antibody preparations. Nevertheless, this treatment also triggers an unwanted side reaction, i.e., antibody aggregation. The aggregates continuously evolve at neutral pH. It remains unclear whether the aggregates progressively incorporate the native, folded, monomers into themselves, enabling aggregate growth as seen in amyloid fibrils. In the present study, the diffusion of fluorescently labeled monoclonal humanized immunoglobulin G1 monomers in aggregates produced by pH-shift stress was tracked by fluorescence correlation spectroscopy. This method was used in addition to monitoring aggregate formation by dynamic light scattering. The diffusing velocities of the monomers indicated that the folded monomers were not involved in aggregate formation, in contrast with unfolded monomers. On the basis of the results, we propose a bifurcated pathway for the refolding and aggregation of antibodies triggered by a pH shift from acidic to neutral. In the scheme, the refolded monomers survive until their unfolding. The insights in this study will contribute to the manufacture of aggregation-resistant therapeutic antibodies.

Mechanism of Human Apohemoglobin Unfolding.

Removal of heme from human hemoglobin (Hb) results in formation of an apoglobin heterodimer. Titration of this apodimer with guanidine hydrochloride (GdnHCl) leads to biphasic unfolding curves indicating two distinct steps. Initially, the heme pocket unfolds and generates a dimeric intermediate in which ∼50% of the original helicity is lost, but the α1β1 interface is still intact. At higher GdnHCl concentrations, this intermediate dissociates into unfolded monomers. This structural interpretation was verified by comparing GdnHCl titrations for adult human hemoglobin A (HbA), recombinant fetal human hemoglobin (HbF), recombinant Hb cross-linked with a single glycine linker between the α chains, and recombinant Hbs with apolar heme pocket mutations that markedly stabilize native conformations in both subunits. The first phase of apoHb unfolding is independent of protein concentration, little affected by genetic cross-linking, but significantly shifted toward higher GdnHCl concentrations by the stabilizing distal pocket mutations. The second phase depends on protein concentration and is shifted to higher GdnHCl concentrations by genetic cross-linking. This model for apoHb unfolding allowed us to quantitate subtle differences in stability between apoHbA and apoHbF, which suggest that the β and γ heme pockets have similar stabilities, whereas the α1γ1 interface is more resistant to dissociation than the α1β1 interface.

Chaperone SurA Interacts with Unfolded Membrane Protein Clients via Delocalized Interface

The trafficking of outer membrane proteins (OMPs) through the periplasm of gram-negative bacteria is a prerequisite to the folding of the protein into the membrane and subsequent outer membrane integrity. Critical for this biogenesis process of OMPs in Escherichia coli is SurA, a periplasmic prolyl-isomerase that has been implicated in binding unfolded OMPs (uOMPs) in the periplasm. We aim to understand how SurA interacts with various uOMP clients to prevent their aggregation in the periplasm. We utilized analytical ultracentrifugation to determine that full-length SurA is predominantly monomeric at physiological concentrations. We additionally probed the binding surface of monomeric SurA for uOMP clients using chemical crosslinking under experimental conditions in which the client OMP is an unfolded monomer. These indicated that the N-terminal and 1st peptidyl-prolyl isomerase domains of SurA are involved in a delocalized binding interface with the uOMP client. We have combined these data with complementary solution studies to construct a structural model in which the uOMP is expanded when bound to SurA. Altogether, these studies provide insight into the non-canonical protein-protein interactions formed between chaperones and unfolded clients that are important for the accurate biogenesis of the unfolded client proteins.

Examination of the Oligomerization Mechanism of SOD1 In Vitro and in Live Cells

The dimeric β-barrel protein Cu, Zn superoxide dismutase (SOD1) is an optimal model system for investigating the molecular basis for toxicity in ALS. Mutations throughout the β-barrel sequence are causative for ALS and implicated in the gain-of-function toxicity. The occurrence of mutations at dozens of sites suggests a common mechanism of toxicity for SOD1 disease causing variants. Previous biophysical and structural data on SOD1 and its variants have established a fairly detailed understanding of its folding energy landscape. A key finding is that ALS-variants of SOD1 lead to enhanced populations of both folded and unfolded monomeric forms of SOD1. An unresolved question, however, is the mechanism of oligomerization and toxicity of SOD1 variants. To address this question, we present an aggregation assay that matches physiological conditions as much as possible, avoiding denaturants, fluorinated alcohols and agitation. Our results, based on the analysis of the WT protein and several variants, suggest that the oligomerization rate is correlated with the population of the unfolded monomer. Furthermore, although a secondary nucleation mechanism has been proposed as a key step in SOD1 aggregation our results, obtained without agitation, are best fitted with a crystallization-like nucleation model without fragmentation. We test the consistency of in vitro and live cell data by performing fluorescence correlation spectroscopy (FCS) measurements on live HEK293 cells expressing EGFP fusion constructs of WT and disease causing SOD1 variants. Preliminary quantitative modeling of the FCS data suggest consistency in the propagation rates between in vitro and live cell data. The implication of the unfolded monomer of SOD1 suggests that small-molecules stabilizing the dimeric native state can be used as a potential therapeutic strategy.This research was supported by NIH grant GM54836.

In Vitro Studies of the Folding and Assembly Mechanism of Hemoglobin

Deriving the pathway for human hemoglobin assembly is important both for developing treatments for hemoglobinopathies and for designing robust acellular hemoglobin oxygen carriers. The adult human hemoglobin (HbA) comprises two globin heterodimers, (α1β1)(α2β2), with each subunit containing a heme group for coordination of oxygen and having a tertiary structure very similar to the monomeric paralog, myoglobin.In order to determine the complete folding mechanism, we have measured and analyzed guanidinium-induced unfolding curves for both apo- and holo-hemoglobin. This approach expands on previous work done in our lab with myoglobin and has allowed us to deconvolute the globin unfolding pathway and the heme-binding effects for the various folding states. When heme is extracted from human Hb, the resulting apohemoglobin dissociates to an α1β1 heterodimer. Unfolding occurs in two major phases. The first involves melting of the heme pockets to generate a dimeric molten globule, followed by dissociation into almost completely unfolded monomers. Structural identification of these phases was achieved by comparing the unfolding curves for native HbA, recombinant HbF, a N-to-C termini linked α Hb variant, and a series of Hb mutants with large, hydrophobic residues replacing the distal histidine and valine in the α and β heme pockets.Previously, we showed that monomeric apoglobin stability, and not hemin affinity, is the determining factor for myoglobin expression. In the case of hemoglobin, the individual monomers are unstable, but associate strongly to form a stable molten globule dimeric state, which binds hemin and facilitates the overall assembly of the hemoglobin.Supported by the NIH Grant HL110900 and by Grant C-0612 from Robert A. Welch Foundation.

Theory of Amyloid Fibril Nucleation from Folded Proteins

We present a theoretical model for the nucleation of amyloid fibrils. In our model we use helix-coil theory to describe the equilibrium between a soluble native state and an aggregation-prone unfolded state. We then extend the theory to include oligomers with β-sheet cores and calculate the free energy of these states using estimates for the energies of H-bonds, steric zipper interactions, and the conformational entropy cost of forming secondary structure. We find that states with fewer than ~10 β-strands are unstable relative to the dissociated state and three β-strands is the highest free energy state. We then use a modified version of Classical Nucleation Theory to compute the nucleation rate of fibrils from a supersaturated solution of monomers, dimers, and trimers. The nucleation rate has a non-monotonic dependence on denaturant concentration reflecting the competing effects of destabilizing the fibril and increasing the concentration of unfolded monomers. We estimate heterogeneous nucleation rates and discuss the application of our model to secondary nucleation.

α‐Synuclein Oligomers: A Study in Diversity

AbstractA critical step in Parkinson's disease (PD) is the formation of toxic α‐synuclein oligomers (αSOs). In vitro αSOs are formed by self‐assembly of α‐synuclein at high concentrations, or by the addition of, for example, dopamine, lipids, ethanol, or metal ions. These αSOs are structurally distinct from the unfolded monomer and aggregated β‐sheet fibrils. Nevertheless, the literature reports a wide variety of αSO shapes, sizes, and proposed toxic mechanisms. This heterogeneous character makes it difficult to form a unifying picture. Here, we present an overview of the different αSO species made in vitro, providing a tool for better comparison of different protocols and the ensuing αSOs, and emphasizing the striking versatility in the appearance and properties of these critical species. We also summarize what is known of the biological activities of different αSOs. Despite a large and increasing level of insight into αSO effects in vitro, we still lack strong insight into the structures and sizes of αSO species formed in vivo. Once this is established, it may be possible to generate more uniform protocols that could stimulate further efforts to develop viable PD biomarker assays and therapies.