Direct characterization of amyloidogenic oligomers by single-molecule fluorescence

Abstract

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-beta structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.

Fig. 1.

Principle of the TCCD method to detect oligomeric aggregates. (A) Detection of oligomer events in PI3–SH3 aggregation. The coincident fluorescent bursts on both channels show the presence of oligomers (marked as asterisks). (B) Expansion of fluorescence bursts in A. Comparison of the intensity of bursts from monomers and oligomers: The monomer events are not coincident and are much less intense than those due to oligomers.

Fig. 2.

Determination of aggregation kinetics. (A) Bulk aggregation kinetics of PI3–SH3 were measured by the increase in the fluorescence of Thioflavin T (squares) and the increase in the fraction of protein sedimentable by ultracentrifugation (circles). The ThT trace showed a lag phase of 18.4 ± 2.3 h, in contrast to the absence of an observable lag phase in the sedimentation data, indicating the formation of aggregated prefibrillar species in the early stages of the reaction that do not bind ThT. (B) Single-molecule TCCD measurement of blue (squares) and red (circles) monomer event rates (corrected for variations in dilution), proportional to monomer concentration, at different incubation times. The traces were globally fitted to a single exponential function, giving a decay time of 3.1 ± 0.4 h. (C) Association quotient variation with incubation time. The traces were fitted to a sum of exponential functions, showing a fast rise-time (1.9 ± 1.0 h) and a longer decay time (16.3 ± 1.3 h) related to the initial accumulation and subsequent growth of the oligomers.

Fig. 3.

Distribution of oligomer sizes. (A) A 2D contour plot of the number of red- and blue-labeled PI3–SH3 monomers per oligomer, from an aliquot taken after 8.9 h of incubation. (B) Histogram of the total number of monomers per oligomer, corrected for the different oligomer detection efficiency, from an aliquot taken after 8.9 h of incubation. (C) Median of the total number of monomers per oligomer as a function of incubation time.

Fig. 4.

Blue- and red-labeled monomer burst rates during TCCD measurements. (A) Aliquot taken after 2 h of incubation of PI3–SH3, showing an increase in the monomer burst rate due to oligomer dissociation. The traces were fitted to exponential functions with the same time constant, giving a rise-time value of 1,000 ± 56 s. (B) An aliquot taken after 72 h of incubation showing the constant rates of monomer events.

Fig. 5.

PI3–SH3 aggregation process as detected by TCCD. Single-molecule fluorescence two-color coincidence detection (TCCD) provides information concerning PI3–SH3 oligomerization prior to amyloid fibril formation and about the size of the oligomers, their growth, stability, and molecular rearrangement.