Extrinsic Amyloid-Binding Dyes for Detection of Individual Protein Aggregates in Solution.

Christopher G Taylor,Georg Meisl,David Klenerman,Mathew H Horrocks,Henrik Zetterberg,Tuomas P J Knowles

doi:10.1021/acs.analchem.8b02226

Abstract

Protein aggregation is a key molecular feature underlying a wide array of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. To understand protein aggregation in molecular detail, it is crucial to be able to characterize the array of heterogeneous aggregates that are formed during the aggregation process. We present here a high-throughput method to detect single protein aggregates, in solution, from a label-free aggregation reaction, and we demonstrate the approach with the protein associated with Parkinson’s disease, α-synuclein. The method combines single-molecule confocal microscopy with a range of amyloid-binding extrinsic dyes, including thioflavin T and pentameric formylthiophene acetic acid, and we show that we can observe aggregates at low picomolar concentrations. The detection of individual aggregates allows us to quantify their numbers. Furthermore, we show that this approach also allows us to gain structural insights from the emission intensity of the extrinsic dyes that are bound to aggregates. By analyzing the time evolution of the aggregate populations on a single-molecule level, we then estimate the fragmentation rate of aggregates, a key process that underlies the multiplication of pathological aggregates. We additionally demonstrate that the method permits the detection of these aggregates in biological samples. The capability to detect individual protein aggregates in solution opens up a range of new applications, including exploiting the potential of this method for high-throughput screening of human biofluids for disease diagnosis and early detection.

Highlights

S elf-assembly of amyloidogenic proteins underlies a multitude of neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease (PD)
A single-molecule total internal reflection fluorescence microscopy (TIRFM) method has recently been developed to detect surface-immobilized individual aggregates of αS, amyloid β, and tau without covalently attached labels. This was used to investigate the number of αS aggregates in cerebrospinal fluid (CSF) samples from PD patients in comparison with controls.[16]. This technique does not require a dye to be conjugated to the protein of interest but instead relies upon the utilization of the benzothiazole salt dye thioflavin T (ThT), which binds to the β-sheet structure of protein aggregates.[17,18]
The obtained Kd values of 1.37 ± 0.25 μM for ThT (2.3 μM with insulin),22 48.7 ± 8.4 nM for diThT (34 nM with insulin),[22] and 15.7 ± 3.4 nM for pentameric formylthiophene acetic acid (pFTAA) (142 nM with tau)[24] correspond well with literature values

Summary

■ RESULTS AND DISCUSSION

To confirm that the single-molecule confocal technique is able to sensitively and reliably detect aggregates down to picomolar concentrations, we optimized each extrinsic dye concentration using the same fibrillar sample of αS aggregates. The data show that ThT and diThT are similar in both the number of αS aggregate events they track and the evolution of average intensity of monitored species over time This can be expected since these two dyes are so closely related structurally and are likely detecting the same aggregate species. There is no clear trend in either the number of events observed or the magnitude of their average intensity between the beginning and end of the aggregation time course for this extrinsic dye This suggests that PicoGreen is not a suitable choice for such experiments, where greater sensitivity of detection for aggregates is pivotal. The number of dye-active species was monitored (Figure 5A) by ThT and pFTAA (selected since the two dyes had shown different behaviors) over the course of aggregation, and the average intensity was calculated and normalized for each dye independently with respect to the maximum value for that dye (Figure 5B). This experiment shows the potential of the confocal technique to make measurements in complex biofluids such as cerebrospinal fluid

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES