SummaryHere, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.

In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a novel risk factor in Alzheimer's disease (AD). We have examined the interactions between clusterin and the AD-associated amyloid-β 1-40 peptide (Aβ 1-40 ) which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single molecule fluorescence methods, we have found that Aβ 1-40 forms a heterogeneous distribution of small oligomers (from dimers to 50mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence strongly both the aggregation and disaggregation of Aβ 1-40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and AD.

A key issue in understanding the pathogenic conditions associated with the aberrant aggregation of misfolded proteins is the identification and characterization of species formed during the aggregation process. Probing the nature of such species has, however, proved to be extremely challenging to conventional techniques because of their transient and heterogeneous character. We describe here the application of a two-color single-molecule fluorescence technique to examine the assembly of oligomeric species formed during the aggregation of the SH3 domain of PI3 kinase. The single-molecule experiments show that the species formed at the stage of the reaction where aggregates have previously been found to be maximally cytotoxic are a heterogeneous ensemble of oligomers with a median size of 38 ؎ 10 molecules. This number is remarkably similar to estimates from bulk measurements of the critical size of species observed to seed ordered fibril formation and of the most infective form of prion particles. Moreover, although the size distribution of the SH3 oligomers remains virtually constant as the time of aggregation increases, their stability increases substantially. These findings together provide direct evidence for a general mechanism of amyloid aggregation in which the stable cross-␤ structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.amyloid aggregation ͉ amyloid oligomers ͉ two-color coincidence spectroscopy ͉ PI3-SH3 domain ͉ neurodegenerative diseases

Measurements of the static permittivity of pure samples of eight polar liquids—acetone, butan-1-ol, dimethyl sulphoxide, ethanediol, ethanol, methanol, propan-1-ol and water—and two non-polar liquids—cyclohexane and silicone oil—were measured in a shielded micrometer-driven, parallel-plate admittance cell at temperatures from 5 to 50 °C. The experimental method, which requires measurements of capacitance using a four-terminal-pair impedance analyser, is described in detail. The full measurement results have been tabulated separately in a National Physical Laboratory report. The present paper provides a comparative study of these latest results with previously published data and also describes the details of the uncertainty analyses employed. Admittance cell measurement techniques are also briefly reviewed to put this work into context.

We have extended the method of single-molecule fluorescence, two-color coincidence detection (TCCD) to detect coincident events due to a low fraction of a complex against a background of chance coincident events, due to monomers. We developed two complementary methods to determine the number of chance coincident events using the experimental data and without the need for additional experiments. We show that the subtraction of the chance coincidence level is essential for accurate quantification of the relative number of complexes and their stoichiometry. By performing experiments on model samples made from fluorophore-labeled duplex DNA and free dye, a linear dependence on the fraction of duplex DNA was found, independent of the level or ratio of free dye, with quantification down to a level of 0.5% and 500 fM duplex DNA. The method was then used to measure the equilibrium dissociation constant and offrate of a 9-mer duplex DNA, demonstrating the application of this method to systems with nanomolar dissociation constants. These improvements to the method of TCCD analysis significantly extend the application of TCCD to weakly bound complexes and large multicomponent biomolecular systems.

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Coaxial sensors are used in applications that require accurate and traceable measurements of complex permittivity. For example, coaxial sensors are often used for measurement of the complex permittivity of tissue equivalent materials (TEMs) used in specific absorption rate (SAR) measurements of exposure to RF fields. It is therefore important that well-founded metrological techniques for their use are developed and published. Although there are many published papers on coaxial sensors, few discuss the experimental techniques required to obtain the most accurate results. In this paper experimental approaches for obtaining the most accurate measurements are described. Common pitfalls with the technique are discussed. A Monte Carlo modelling (MCM) technique is used to provide estimates of uncertainty which are compared to those of measurements on reference liquids made with 3.5 mm, 7 mm and 15.1 mm diameter sensors. The MCM technique allows uncertainties to be estimated when measuring dielectrics for which there are no reference materials that have comparable properties, for example for TEMs at frequencies below 100 MHz.