Alexander K. Buell

Nanostructured films from hierarchical self-assembly of amyloidogenic proteins.

In nature, sophisticated functional materials are created through hierarchical self-assembly of simple nanoscale motifs. In the laboratory, much progress has been made in the controlled assembly of molecules into one-, two- and three-dimensional artificial nanostructures, but bridging from the nanoscale to the macroscale to create useful macroscopic materials remains a challenge. Here we show a scalable self-assembly approach to making free-standing films from amyloid protein fibrils. The films were well ordered and highly rigid, with a Youngs modulus of up to 5-7 GPa, which is comparable to the highest values for proteinaceous materials found in nature. We show that the self-organizing protein scaffolds can align otherwise unstructured components (such as fluorophores) within the macroscopic films. Multiscale self-assembly that relies on highly specific biomolecular interactions is an attractive path for realizing new multifunctional materials built from the bottom up.

Solution conditions determine the relative importance of nucleation and growth processes in alpha-synuclein aggregation

Significance The deposition of α-synuclein as insoluble amyloid fibrils and the spreading of such species in the brain are two hallmarks of Parkinson disease. It is therefore of great importance to understand in detail the process of aggregation of this protein. We show by a series of in vitro measurements that amyloid fibrils of α-synuclein can grow under a wide range of solution conditions but that they can multiply rapidly only under a much more select set of solution conditions, mimicking those in endosomes and other organelles. The quantitative characterization of α-synuclein aggregation described here provides new insights into the microscopic mechanisms underlying α-synuclein aggregation in the context of Parkinson disease. The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that α-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of α-synuclein aggregation and aggregate spreading in the context of Parkinson disease.

Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation

α-Synuclein (α-syn) is a 140-residue intrinsically disordered protein that is involved in neuronal and synaptic vesicle plasticity, but its aggregation to form amyloid fibrils is the hallmark of Parkinsons disease (PD). The interaction between α-syn and lipid surfaces is believed to be a key feature for mediation of its normal function, but under other circumstances it is able to modulate amyloid fibril formation. Using a combination of experimental and theoretical approaches, we identify the mechanism through which facile aggregation of α-syn is induced under conditions where it binds a lipid bilayer, and we show that the rate of primary nucleation can be enhanced by three orders of magnitude or more under such conditions. These results reveal the key role that membrane interactions can have in triggering conversion of α-syn from its soluble state to the aggregated state that is associated with neurodegeneration and to its associated disease states.

Protein Aggregation in Crowded Environments

The physicochemical parameters of biomolecules are the key determinants of the multitude of processes that govern the normal and aberrant behavior of living systems. A particularly important aspect of such behavior is the role it plays in the self-association of proteins to form organized aggregates such as the amyloid or amyloid-like fibrils that are associated with pathological conditions including Alzheimers disease and Type II diabetes. In this study we describe quantitative quartz crystal microbalance measurements of the kinetics of the growth of amyloid fibrils in a range of crowded environments and in conjunction with theoretical predictions demonstrate the existence of general relationships that link the propensities of protein molecules to aggregate with fundamental parameters that describe their specific structures and local environments.

Direct observation of heterogeneous amyloid fibril growth kinetics via two-color super-resolution microscopy.

The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases. In the present study, we show that specific events in the kinetics of the complex, multistep aggregation process of one such protein, α-synuclein, whose aggregation is a characteristic hallmark of Parkinson’s disease, can be followed at the molecular level using optical super-resolution microscopy. We have explored in particular the elongation of preformed α-synuclein fibrils; using two-color single-molecule localization microscopy we are able to provide conclusive evidence that the elongation proceeds from both ends of the fibril seeds. Furthermore, the technique reveals a large heterogeneity in the growth rates of individual fibrils; some fibrils exhibit no detectable growth, whereas others extend to more than ten times their original length within hours. These large variations in the growth kinetics can be attributed to fibril structural polymorphism. Our technique offers new capabilities in the study of amyloid growth dynamics at the molecular level and is readily translated to the study of the self-assembly of other nanostructures.

Ostwald’s rule of stages governs structural transitions and morphology of dipeptide supramolecular polymers

The self-assembly of molecular building blocks into nano- and micro-scale supramolecular architectures has opened up new frontiers in polymer science. Such supramolecular species not only possess a rich set of dynamic features as a consequence of the non-covalent nature of their core interactions, but also afford unique structural characteristics. Although much is now known about the manner in which such structures adopt their morphologies and size distributions in response to external stimuli, the kinetic and thermodynamic driving forces that lead to their transformation from soluble monomeric species into ordered supramolecular entities have remained elusive. Here we focus on Boc-diphenylalanine, an archetypical example of a peptide with a high propensity towards supramolecular self-organization, and describe the pathway through which it forms a range of nano-assemblies with different structural characteristics. Our results reveal that the nucleation process is multi-step in nature and proceeds by Ostwalds step rule through which coalescence of soluble monomers leads to the formation of nanospheres, which then undergo ripening and structural conversions to form the final supramolecular assemblies. We characterize the structures and thermodynamics of the different phases involved in this process and reveal the intricate nature of the transitions that can occur between discrete structural states of this class of supramolecular polymers.

Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein by Small Molecules as a Potential Therapeutic Strategy for Parkinson’s Disease

The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aβ peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson’s and Alzheimer’s diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson’s disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson’s disease and related conditions.

Expanding the Solvent Chemical Space for Self-Assembly of Dipeptide Nanostructures

Nanostructures composed of short, noncyclic peptides represent a growing field of research in nanotechnology due to their ease of production, often remarkable material properties, and biocompatibility. Such structures have so far been almost exclusively obtained through self-assembly from aqueous solution, and their morphologies are determined by the interactions between building blocks as well as interactions between building blocks and water. Using the diphenylalanine system, we demonstrate here that, in order to achieve structural and morphological control, a change in the solvent environment represents a simple and convenient alternative strategy to the chemical modification of the building blocks. Diphenylalanine (FF) is a dipeptide capable of self-assembly in aqueous solution into needle-like hollow micro- and nanocrystals with continuous nanoscale channels that possess advantageous properties such as high stiffness and piezoelectricity and have so emerged as attractive candidates for functional nanomaterials. We investigate systematically the solubility of diphenylalanine in a range of organic solvents and probe the role of the solvent in the kinetics of self-assembly and the structures of the final materials. Finally, we report the crystal structure of the FF peptide in microcrystalline form grown from MeOH solution at 1 Å resolution and discuss the structural changes relative to the conventional materials self-assembled in aqueous solution. These findings provide a significant expansion of the structures and morphologies that are accessible through FF self-assembly for existing and future nanotechnological applications of this peptide. Solvent mediation of molecular recognition and self-association processes represents an important route to the design of new supramolecular architectures deriving their functionality from the nanoscale ordering of their components.

A Label‐Free, Quantitative Assay of Amyloid Fibril Growth Based on Intrinsic Fluorescence

Kinetic assay of seeded growth: The graph shows the variation in intrinsic fluorescence intensity of amyloid fibrils. Fluorescence increases during the seeded aggregation of α-synuclein seeds with α-synuclein monomeric protein (blue curve) but not when α-synuclein seeds are incubated with β-synuclein monomeric protein (black curve), thus showing that no seeded growth occurred in this case.

Detailed Analysis of the Energy Barriers for Amyloid Fibril Growth

Solubility is a key requirement for the functioning of a protein within the complex network of cellular components. A class of highly debilitating disorders, including Alzheimer s and Parkinson s diseases, is related to the loss of solubility of peptides and proteins that is accompanied by their aggregation into ordered amyloid fibrils. It has been found that, under at least some physiological conditions, these aggregates are thermodynamically more stable than the native forms of biological polypeptides. This finding raises questions as to the factors governing the crucial ability of native proteins to remain soluble even under conditions where they do not necessarily correspond to global minima on free energy landscapes. In order to address this question, we have studied in detail the kinetics of elongation of amyloid fibrils formed by a wide range of polypeptides. The formation of amyloid fibrils from soluble protein molecules involves at least a primary nucleation step, an elongation step and, in general, a secondary nucleation process such as fibril fragmentation. In addition, multiple interconverting oligomeric intermediates can be involved. Measurements of amyloid growth in bulk solution often reflect all of these processes, and it can therefore be extremely challenging to determine accurately the concentrations of the different species and the rate constants for the individual elementary steps. In order to overcome these difficulties, surface-based sensing techniques, notably those based on quartz crystal microbalance (QCM) measurements, have been developed in recent years, by which the growth of a constant, surfacebound ensemble of fibrils can be monitored. These methods make use of the fact that in the presence of preexisting fibrils, aggregation can be highly accelerated through seeding. This seeding process corresponds to the elongation of existing fibrils and can be well described as diffusional motion over a single free energy barrier, involving no intermediate species between monomeric and fibrillar peptide. The elongation of the fibrils is monitored through the increase in hydrodynamic mass bound to the quartz crystal, as the rate of change of the resonant frequency is proportional to the average elongation rate of the fibrils. The opportunity to image the sensor surface enables an estimation of the surface density of fibrils, an important factor in the determination of the rate constants in this bimolecular reaction, the overall rate of which depends on both the concentration of soluble protein and the number of available fibril ends. In addition, the lengths of the fibrils before and after an experiment can be compared and therefore an independent measurement of the length increase can be made and used to calibrate the frequency response of the microbalance. The covalent irreversible attachment of the preformed fibrils to the sensor surface and the subsequent passivation of the remaining surface, as well as the short duration of individual experiments, ensure that only fibril elongation is measured, and that primary and secondary nucleation events can be neglected; this selectivity is confirmed by the high reproducibility of the data obtained from QCM measurements. The starting point of a systematic study of the energy barriers that separate the soluble from the fibrillar states of a protein is the measurement of the temperature dependence of the fibril elongation rate; such an approach allows for the determination of the enthalpy of activation from an Arrhenius plot. The temperature dependence of amyloid growth has already been measured for a range of amyloidogenic peptides and proteins, and where possible these literature data are included in the analysis described herein. These published data have been acquired with a range of different techniques, mainly involving small-molecule labels such as Thioflavin-T. In such experiments, the exclusive study of the elementary elongation reaction is challenging and therefore the published values on energy barriers may in some cases refer to a combination of different elementary steps. We have, however, used the QCM approach, which is particularly suited for such measurements, to increase substantially the size of the available dataset by studying peptides and proteins of very diverse sequence that form amyloid fibrils under varied solution conditions. Figure 1 shows, as an example, raw QCM data for the temperature dependence of PI3K-SH3 amyloid fibril elongation, as well as AFM images of the QCM sensor surface. Similar experiments were performed for a range of other peptides and proteins, and the resulting Arrhenius plots are shown in Figure 2. No pronounced curvature is apparent, unlike that sometimes observed for protein folding; this [*] A. K. Buell, M. E. Welland Nanoscience Centre, University of Cambridge 11 JJ Thomson Avenue, West Cambridge CB3 0FF (UK) E-mail: mew10@cam.ac.uk