Exploring the Barriers in the Aggregation of a Hexadecameric Human Prion Peptide through the Markov State Model.

The prefibrillar aggregation kinetics of prion peptides are still an enigma. In this perspective, we employ atomistic molecular dynamics (MD) simulations of the shortest human prion peptide (HPP) (127GYMLGS132) at various temperatures and peptide concentrations and apply the Markov state model to determine the various intermediates and lag phases. Our results reveal that the natural mechanism of prion peptide self-assembly in the aqueous phase is impeded by two significant kinetic barriers with oligomer sizes of 6-9 and 12-13 peptides, respectively. The first one is the aggregation of unstructured lower-order oligomers, and the second is fibril nucleation, which impedes the further growth of prion aggregates. Among these two activation barriers, the second one is found to be dominant irrespective of the increase in temperature and peptide concentration. These lag phases are captured in all three different force-field parameters, namely, GROMOS-54a7, AMBER-99SB-ILDN, and CHARMMS 36m, at different concentrations. The GROMOS-54a7 and AMBER-99SB-ILDN force fields showed a comparatively higher percentage of ß-sheet formation in the metastable aggregate that evolved during the aggregation process. In contrast, the CHARMM-36m force field showed mostly coil or turn conformations. The addition of a novel catecholamine derivative (naphthoquinone dopamine (NQDA)) arrests the aggregation process between the lag phases by increasing the activation barrier for the Lag1 and Lag2 phases in all of the force fields, which further validates the existence of these lag phases. The preferential binding of NQDA with the peptides increases the hydration of peptides and eventually disrupts the organized morphology of prefibrillar aggregates. It reduces the dimer dissociation energy by -24.34 kJ/mol.

Conformational Fluctuations in ß2-Microglubulin Using Markov State Modeling and Molecular Dynamics.

Conformational dynamics in proteins can give rise to aggregation prone states during folding, and these kinetically stable states could form oligomers and aggregates. In this study, we investigate the intermediate states and near-folded states of ß2-microglobulin and their physico-chemical properties using molecular dynamics and Markov state modeling. Analysis of hundreds of microseconds simulation show the importance of the edge strands in the misfolded states that give rise to a high exposure of hydrophobic residues in the core of the protein that could initiate oligomerization and aggregate formation. Our study sheds light on the first step of aggregation of ß2m monomers and gave a better picture of the landscape of protein misfolding and aggregation.

Early aggregation mechanism of Aß16-22 revealed by Markov state models.

Aß16-22 is believed to have critical role in early aggregation of full length amyloids that are associated with the Alzheimer's disease and can aggregate to form amyloid fibrils. However, the early aggregation mechanism is still unsolved. Here, multiple long-term molecular dynamics simulations combining with Markov state model were used to probe the early oligomerization mechanism of Aß16-22 peptides. The identified dimeric form adopted either globular random-coil or extended ß-strand like conformations. The observed dimers of these variants shared many overall conformational characteristics but differed in several aspects at detailed level. In all cases, the most common type of secondary structure was intermolecular antiparallel ß-sheets. The inter-state transitions were very frequent ranges from few to hundred nanoseconds. More strikingly, those states which contain fraction of ß secondary structure and significant amount of extended coiled structures, therefore exposed to the solvent, were majorly participated in aggregation. The assembly of low-energy dimers, in which the peptides form antiparallel ß sheets, occurred by multiple pathways with the formation of an obligatory intermediates. We proposed that these states might facilitate the Aß16-22 aggregation through a significant component of the conformational selection mechanism, because they might increase the aggregates population by promoting the inter-chain hydrophobic and the hydrogen bond contacts. The formation of early stage antiparallel ß sheet structures is critical for oligomerization, and at the same time provided a flat geometry to seed the ordered ß-strand packing of the fibrils. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aß aggregation and will insight into early oligomerization for large ß amyloids.

Charge Regulation during Amyloid Formation of α-Synuclein.

Electrostatic interactions play crucial roles in protein function. Measuring pKa value perturbations upon complex formation or self-assembly of e.g. amyloid fibrils gives valuable information about the effect of electrostatic interactions in those processes. Site-specific pKa value determination by solution NMR spectroscopy is challenged by the high molecular weight of amyloid fibrils. Here we report a pH increase during fibril formation of α-synuclein, observed using three complementary experimental methods: pH electrode measurements in water; colorimetric changes of a fluorescent indicator; and chemical shift changes for histidine residues using solution state NMR spectroscopy. A significant pH increase was detected during fibril formation in water, on average by 0.9 pH units from 5.6 to 6.5, showing that protons are taken up during fibril formation. The pH upshift was used to calculate the average change in the apparent pKaave value of the acidic residues, which was found to increase by at least 1.1 unit due to fibril formation. Metropolis Monte Carlo simulations were performed on a comparable system that also showed a proton uptake due to fibril formation. Fibril formation moreover leads to a significant change in proton binding capacitance. Parallel studies of a mutant with five charge deletions in the C-terminal tail revealed a smaller pH increase due to fibril formation, and a smaller change (0.5 units on average) in the apparent pKaave values of the acidic residues. We conclude that the proton uptake during the fibril formation is connected to the high density of acidic residues in the C-terminal tail of α-synuclein.

Assessment of Amyloid Forming Tendency of Peptide Sequences from Amyloid Beta and Tau Proteins Using Force-Field, Semi-Empirical, and Density Functional Theory Calculations.

A wide variety of neurodegenerative diseases are characterized by the accumulation of protein aggregates in intraneuronal or extraneuronal brain regions. In Alzheimer's disease (AD), the extracellular aggregates originate from amyloid-ß proteins, while the intracellular aggregates are formed from microtubule-binding tau proteins. The amyloid forming peptide sequences in the amyloid-ß peptides and tau proteins are responsible for aggregate formation. Experimental studies have until the date reported many of such amyloid forming peptide sequences in different proteins, however, there is still limited molecular level understanding about their tendency to form aggregates. In this study, we employed umbrella sampling simulations and subsequent electronic structure theory calculations in order to estimate the energy profiles for interconversion of the helix to ß-sheet like secondary structures of sequences from amyloid-ß protein (KLVFFA) and tau protein (QVEVKSEKLD and VQIVYKPVD). The study also included a poly-alanine sequence as a reference system. The calculated force-field based free energy profiles predicted a flat minimum for monomers of sequences from amyloid and tau proteins corresponding to an α-helix like secondary structure. For the parallel and anti-parallel dimer of KLVFFA, double well potentials were obtained with the minima corresponding to α-helix and ß-sheet like secondary structures. A similar double well-like potential has been found for dimeric forms for the sequences from tau fibril. Complementary semi-empirical and density functional theory calculations displayed similar trends, validating the force-field based free energy profiles obtained for these systems.

Aggregation of amyloid peptides into fibrils driven by nanoparticles and their curvature effect.

Fibrillation of amyloid peptides induces human diseases such as Alzheimer's disease, which has become a huge challenge. Some nanoparticles (NPs) could enhance peptide fibrillation by decreasing the lag time, yet how the size and shape of NPs affect amyloid fibrillation as well as the underlying mechanism remains unclear. Here, we investigated amyloid fibrillation on the surface of spherical NPs and cylindrical nanorods (NRs) of different sizes using coarse-grained Monte Carlo simulations. We focused on the curvature effect of NPs/NRs on the adsorption and fibrillation of peptide chains due to the size/shape difference. As the size of the NPs/NRs increases, the number of assembled peptide chains shows a non-monotonic tendency, and there is an optimal size for the highest adsorption. In most cases, the NRs could adsorb more peptides than the NPs of the same diameter due to the lower curvature. The mechanism beneath these observations was elucidated from a thermodynamic point of view. Our findings could provide a physical basis for the adsorption and fibrillation of amyloid peptides on NPs, and guide the design of future curvature-dependent NP-based amyloid treatment.

Statistical Mechanics of Globular Oligomer Formation by Protein Molecules.

The misfolding and aggregation of proteins into linear fibrils is widespread in human biology, for example, in connection with amyloid formation and the pathology of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The oligomeric species that are formed in the early stages of protein aggregation are of great interest, having been linked with the cellular toxicity associated with these conditions. However, these species are not characterized in any detail experimentally, and their properties are not well understood. Many of these species have been found to have approximately spherical morphology and to be held together by hydrophobic interactions. We present here an analytical statistical mechanical model of globular oligomer formation from simple idealized amphiphilic protein monomers and show that this correlates well with Monte Carlo simulations of oligomer formation. We identify the controlling parameters of the model, which are closely related to simple quantities that may be fitted directly from experiment. We predict that globular oligomers are unlikely to form at equilibrium in many polypeptide systems but instead form transiently in the early stages of amyloid formation. We contrast the globular model of oligomer formation to a well-established model of linear oligomer formation, highlighting how the differing ensemble properties of linear and globular oligomers offer a potential strategy for characterizing oligomers from experimental measurements.

Nonproductive Binding Modes as a Prominent Feature of Aß40 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 ß-hairpin conformations. The simulation here may serve as a model for the binding of other non-ß-sheet conformations to amyloid fibers.

Experimental and Computational Protocols for Studies of Cross-Seeding Amyloid Assemblies.

Alzheimer's disease (AD) and type 2 diabetes (T2D) are two common protein aggregation diseases. Compelling evidence has shown a link between AD and T2D, which may derive from interspecies cross-sequence interactions between amyloid-ß peptide (Aß), associated with AD, and human islet amyloid polypeptide (hIAPP), associated with T2D. Herein, we present experimental and computational protocols and tools to study the aggregate structures and kinetics, conformational conversion, and molecular interactions of Aß-hIAPP mixtures. These protocols could be generally applied to other cross-seeding behaviors of amyloid peptides.

Both lipopolysaccharide and lipoteichoic acids potently induce anomalous fibrin amyloid formation: assessment with novel Amytracker™ stains.

In recent work, we discovered that the presence of highly substoichiometric amounts (10-8 molar ratio) of lipopolysaccharide (LPS) from Gram-negative bacteria caused fibrinogen clotting to lead to the formation of an amyloid form of fibrin. We here show that the broadly equivalent lipoteichoic acids (LTAs) from two species of Gram-positive bacteria have similarly (if not more) potent effects. Using thioflavin T fluorescence to detect amyloid as before, the addition of low concentrations of free ferric ion is found to have similar effects. Luminescent conjugated oligothiophene dyes (LCOs), marketed under the trade name Amytracker™, also stain classical amyloid structures. We here show that they too give very large fluorescence enhancements when clotting is initiated in the presence of the four amyloidogens (LPS, ferric ions and two LTA types). The staining patterns differ significantly as a function of both the amyloidogens and the dyes used to assess them, indicating clearly that the nature of the clots formed is different. This is also the case when clotting is measured viscometrically using thromboelastography. Overall, the data provide further evidence for an important role of bacterial cell wall products in the various coagulopathies that are observable in chronic, inflammatory diseases. The assays may have potential in both diagnostics and therapeutics.

Functional amyloids: interrelationship with other amyloids and therapeutic assessment to treat neurodegenerative diseases.

Misfolded ß-sheet structures of proteins leading to neurodegenerative diseases like Alzheimer's disease (AD) and Parkinson's disease (PD) are in the spotlight since long. However, not much was known about the functional amyloids till the last decade. Researchers have become increasingly more concerned with the degree of involvement of these functional amyloids in human physiology. Interestingly, it has been found that the human body is exposed to a tremendous systemic amyloid burden, especially, during aging. Although many findings regarding these functional amyloids come up every day, some questions still remain unanswered like do these functional amyloids directly involve in the fibrillization of amyloid beta (Aß) 42 peptide or enhance the Aß42 aggregation rate; whether functional bacterial amyloids (FuBA) co-localize with the senile plaques of AD or not. A detailed review of the latest status regarding the interrelationship between functional amyloids, pathogenic amyloids and misfolded prions and therapeutic assessment of functional amyloids for the treatment of neurodegenerative diseases can help identify an alternative medication for neurodegeneration. A unique mathematical model is proposed here for alteration of Aß42 aggregation kinetics in AD to carve out the future direction of therapeutic consideration.

Nano-assembly of amyloid ß peptide: role of the hairpin fold.

Structural investigations have revealed that ß hairpin structures are common features in amyloid fibrils, suggesting that these motifs play an important role in amyloid assembly. To test this hypothesis, we characterized the effect of the hairpin fold on the aggregation process using a model ß hairpin structure, consisting of two Aß(14-23) monomers connected by a turn forming YNGK peptide. AFM studies of the assembled aggregates revealed that the hairpin forms spherical structures whereas linear Aß(14-23) monomers form fibrils. Additionally, an equimolar mixture of the monomer and the hairpin assembles into non-fibrillar aggregates, demonstrating that the hairpin fold dramatically changes the morphology of assembled amyloid aggregates. To understand the molecular mechanism underlying the role of the hairpin fold on amyloid assembly, we performed single-molecule probing experiments to measure interactions between hairpin and monomer and two hairpin complexes. The studies reveal that the stability of hairpin-monomer complexes is much higher than hairpin-hairpin complexes. Molecular dynamics simulations revealed a novel intercalated complex for the hairpin and monomer and Monte Carlo modeling further demonstrated that such nano-assemblies have elevated stability compared with stability of the dimer formed by Aß(14-23) hairpin. The role of such folding on the amyloid assembly is also discussed.

Thermodynamics of amyloid formation and the role of intersheet interactions.

The self-assembly of proteins into ß-sheet-rich amyloid fibrils has been observed to occur with sigmoidal kinetics, indicating that the system initially is trapped in a metastable state. Here, we use a minimal lattice-based model to explore the thermodynamic forces driving amyloid formation in a finite canonical (NVT) system. By means of generalized-ensemble Monte Carlo techniques and a semi-analytical method, the thermodynamic properties of this model are investigated for different sets of intersheet interaction parameters. When the interactions support lateral growth into multi-layered fibrillar structures, an evaporation/condensation transition is observed, between a supersaturated solution state and a thermodynamically distinct state where small and large fibril-like species exist in equilibrium. Intermediate-size aggregates are statistically suppressed. These properties do not hold if aggregate growth is one-dimensional.

Shedding Light on the Dock-Lock Mechanism in Amyloid Fibril Growth Using Markov State Models.

We investigate how the molecular mechanism of monomer addition to a growing amyloid fibril of the transthyretin TTR105-115 peptide is affected by pH. Using Markov state models to extract equilibrium and dynamical information from extensive all atom simulations allowed us to characterize both productive pathways in monomer addition as well as several off-pathway trapped states. We found that multiple pathways result in successful addition. All productive pathways are driven by the central hydrophobic residues in the peptide. Furthermore, we show that the slowest transitions in the system involve trapped configurations, that is, long-lived metastable states. These traps dominate the rate of fibril growth. Changing the pH essentially reweights the system, leading to clear differences in the relative importance of both productive paths and traps, yet retains the core mechanism.

Assessment of the effect of macromolecular crowding on aggregation behaviour of a model amyloidogenic Peptide.

Accumulation of ordered protein aggregates (or amyloids) represents a hallmark of many diseases (e.g., Alzheimer's disease, type II diabetes, Parkinson's diseases etc.), results from intermolecular association of partially unfolded proteins/ peptides. Such associations usually take place in highly crowded conditions. The aggregates, which are formed under in vitro and in vivo conditions exhibit substantial variations in their structure and function. Such heterogeneities in amyloids might arise due to macromolecular crowding that is usually omitted under in vitro conditions. The current study is an attempt to assess the effects of macromolecular crowding on amyloid formation using a model amyloidogenic peptide. The sequence of the peptide was derived from C-terminal region (RATQIPSYKKLIMY) of PAP(248-286), which naturally occurs in human semen as amyloid aggregates and is known for boosting HIV infectivity. This model peptide forms sedimentable and fibrillar aggregates in aqueous buffer and shows the characteristic features of amyloids. In the presence of macromolecular crowders the morphological features of the amyloids are significantly altered and resulted in the formation of shorter amyloid aggregates. The current study assumes the hypothesis that macromolecular crowding in the biological system favours formation of heterogeneous classes of aggregates and each of them might differ in their biophysical and biological properties.

Dynamical phase transitions reveal amyloid-like states on protein folding landscapes.

Developing an understanding of protein misfolding processes presents a crucial challenge for unlocking the mysteries of human disease. In this article, we present our observations of ß-sheet-rich misfolded states on a number of protein dynamical landscapes investigated through molecular dynamics simulation and Markov state models. We employ a nonequilibrium statistical mechanical theory to identify the glassy states in a protein's dynamics, and we discuss the nonnative, ß-sheet-rich states that play a distinct role in the slowest dynamics within seven protein folding systems. We highlight the fundamental similarity between these states and the amyloid structures responsible for many neurodegenerative diseases, and we discuss potential consequences for mechanisms of protein aggregation and intermolecular amyloid formation.

ß-hairpin-mediated formation of structurally distinct multimers of neurotoxic prion peptides.

Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into ß-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109-122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109-122 peptide has a preference for α-helical conformations, but that this peptide can also form ß-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109-122 peptide (A117V, associated with familial prion disease) increases the prevalence of ß-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106-126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a ß-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel ß-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-ß structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both ß-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for ß-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of ß-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies.

Aggregate geometry in amyloid fibril nucleation.

We present and study a minimal structure-based model for the self-assembly of peptides into ordered ß-sheet-rich fibrils. The peptides are represented by unit-length sticks on a cubic lattice and interact by hydrogen bonding and hydrophobicity forces. Using Monte Carlo simulations with >10(5) peptides, we show that fibril formation occurs with sigmoidal kinetics in the model. To determine the mechanism of fibril nucleation, we compute the joint distribution in length and width of the aggregates at equilibrium, using an efficient cluster move and flat-histogram techniques. This analysis, based on simulations with 256 peptides in which aggregates form and dissolve reversibly, shows that the main free-energy barriers that a nascent fibril has to overcome are associated with changes in width.