Duncan A. White

Proliferation of amyloid-β42 aggregates occurs through a secondary nucleation mechanism

The generation of toxic oligomers during the aggregation of the amyloid-β (Aβ) peptide Aβ42 into amyloid fibrils and plaques has emerged as a central feature of the onset and progression of Alzheimer’s disease, but the molecular pathways that control pathological aggregation have proved challenging to identify. Here, we use a combination of kinetic studies, selective radiolabeling experiments, and cell viability assays to detect directly the rates of formation of both fibrils and oligomers and the resulting cytotoxic effects. Our results show that once a small but critical concentration of amyloid fibrils has accumulated, the toxic oligomeric species are predominantly formed from monomeric peptide molecules through a fibril-catalyzed secondary nucleation reaction, rather than through a classical mechanism of homogeneous primary nucleation. This catalytic mechanism couples together the growth of insoluble amyloid fibrils and the generation of diffusible oligomeric aggregates that are implicated as neurotoxic agents in Alzheimer’s disease. These results reveal that the aggregation of Aβ42 is promoted by a positive feedback loop that originates from the interactions between the monomeric and fibrillar forms of this peptide. Our findings bring together the main molecular species implicated in the Aβ aggregation cascade and suggest that perturbation of the secondary nucleation pathway identified in this study could be an effective strategy to control the proliferation of neurotoxic Aβ42 oligomers.

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.

Observation of spatial propagation of amyloid assembly from single nuclei

The crucial early stages of amyloid growth, in which normally soluble proteins are converted into fibrillar nanostructures, are challenging to study using conventional techniques yet are critical to the protein aggregation phenomena implicated in many common pathologies. As with all nucleation and growth phenomena, it is difficult to track individual nuclei in traditional macroscopic experiments, which probe the overall temporal evolution of the sample, but do not yield detailed information on the primary nucleation step as they mix independent stochastic events into an ensemble measurement. To overcome this limitation, we have developed microdroplet assays enabling us to detect single primary nucleation events and to monitor their subsequent spatial as well as temporal evolution, both of which we find to be determined by secondary nucleation phenomena. By deforming the droplets to high aspect ratio, we visualize in real-time propagating waves of protein assembly emanating from discrete primary nucleation sites. We show that, in contrast to classical gelation phenomena, the primary nucleation step is characterized by a striking dependence on system size, and the filamentous protein self-assembly process involves a highly nonuniform spatial distribution of aggregates. These findings deviate markedly from the current picture of amyloid growth and uncover a general driving force, originating from confinement, which, together with biological quality control mechanisms, helps proteins remain soluble and therefore functional in nature.

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

Biosensor‐based label‐free assays of amyloid growth

Uncontrolled fibrous protein aggregation is implicated in a range of aberrant biological phenomena. Much effort has consequently been directed towards establishing quantitative in vitro assays of this process with the aim of probing amyloid growth in molecular detail as well as elucidating the effect of additional species on this reaction. In this paper, we discuss some recent approaches based on label‐free technologies focussed on achieving these objectives. Several biosensor techniques have been developed to monitor biomolecular assembly without the requirement for fluorophore marker molecules; in particular quartz crystal microbalance and surface plasmon resonance measurements provide advantageous alternatives to traditional spectroscopic methods and are currently receiving increasing attention in the context of amyloid growth assays.

Surface attachment of protein fibrils via covalent modification strategies.

Chemical control of surface functionality and topography is an essential requirement for many technological purposes. In particular, the covalent attachment of monomeric proteins to surfaces has been the object of intense studies in recent years, for applications as varied as electrochemistry, immuno-sensing, and the production of biocompatible coatings. Little is known, however, about the characteristics and requirements underlying surface attachment of supramolecular protein nanostructures. Amyloid fibrils formed by the self-assembly of peptide and protein molecules represent one important class of such structures. These highly organized beta-sheet-rich assemblies are a hallmark of a range of neurodegenerative disorders, including Alzheimers disease and type II diabetes, but recent findings suggest that they have much broader significance, potentially representing the global free energy minima of the energy landscapes of proteins and having potential applications in material science. In this paper, we describe strategies for attaching amyloid fibrils formed from different proteins to gold surfaces under different solution conditions. Our methods involve the reaction of sulfur containing small molecules (cystamine and 2-iminothiolane) with the amyloid fibrils, enabling their covalent linkage to gold surfaces. We demonstrate that irreversible attachment using these approaches makes possible quantitative analysis of experiments using biosensor techniques, such as quartz crystal microbalance (QCM) assays that are revolutionizing our understanding of the mechanisms of amyloid growth and the factors that determine its kinetic behavior. Moreover, our results shed light on the nature and relative importance of covalent versus noncovalent forces acting on protein superstructures at metal surfaces.

Microfluidic diffusion analysis of the sizes and interactions of proteins under native solution conditions

Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space-time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures.

Dry-mass sensing for microfluidics

We present an approach for interfacing an electromechanical sensor with a microfluidic device for the accurate quantification of the dry mass of analytes within microchannels. We show that depositing solutes onto the active surface of a quartz crystal microbalance by means of an on-chip microfluidic spray nozzle and subsequent solvent removal provides the basis for the real-time determination of dry solute mass. Moreover, this detection scheme does not suffer from the decrease in the sensor quality factor and the viscous drag present if the measurement is performed in a liquid environment, yet allows solutions to be analysed. We demonstrate the sensitivity and reliability of our approach by controlled deposition of nanogram levels of salt and protein from a micrometer-sized channel.

Highly Non-linear Microfluidic Resistor Elements for Flow Rate-dependent Addressing of Microchannels

Abstract We have developed a microfluidic resistor element exhibiting a hydrodynamic resistance that is strongly dependent on the pressure at the inlet of the device. Such an element can be used to construct microfluidic circuits that possess a highly non-linear dependence between the pressure and the flow rate, in contrast to conventional microfluidic resistors. We show that this effect can be exploited to create valves which do not require an external control line, but are actuated directly through the pressure difference between the single inlet and outlet of the device. Furthermore, we show that the flux to each branch of the circuit controlled by the valve can be fixed for high or low flow rates by modifying the fixed resistances internal to the device.

Quantitative approaches for characterising fibrillar protein nanostructures

Polypeptide sequences have an inherent tendency to self-assemble into filamentous nanostructures commonly known as amyloid fibrils. Such self-assembly is used in nature to generate a variety of functional materials ranging from protective coatings in bacteria to catalytic scaffolds in mammals. The aberrant self-assembly of misfolded peptides and proteins is also, however, implicated in a range of disease states including neurodegenerative conditions such as Alzheimers and Parkinsons diseases. It is increasingly evident that the intrinsic material properties of these structures are crucial for understanding the thermodynamics and kinetics of the pathological deposition of proteins, particularly as the mechanical fragmentation of aggregates enhances the rate of protein deposition by exposing new fibril ends which can promote further growth. We discuss here recent advances in physical techniques that are able to characterise the hierarchical self-assembly of misfolded protein molecnles and define their properties.