Direct Characterization of Amyloidogenic Oligomers by Single-Molecule Fluorescence
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Direct characterization of amyloidogenic oligomers by single-molecule fluorescence Angel Orte*, Neil R. Birkett*, Richard W. Clarke*, Glyn L. Devlin†, Christopher M. Dobson†, and David Klenerman† Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom Edited by William E. Moerner, Stanford University, Stanford, CA, and approved August 1, 2008 (received for review March 28, 2008) A key issue in understanding the pathogenic conditions associated hydrogen/deuterium exchange kinetics and controlled proteol- with the aberrant aggregation of misfolded proteins is the iden- ysis (16–18). Before fibril proliferation, however, PI3–SH3 forms tification and characterization of species formed during the ag- smaller, granular aggregates that have been found to be as toxic gregation process. Probing the nature of such species has, how- as aggregates of the A peptide associated with Alzheimer’s ever, proved to be extremely challenging to conventional disease (9, 19). These observations show that such toxicity is not techniques because of their transient and heterogeneous charac- simply restricted to those aggregates formed by disease-related ter. We describe here the application of a two-color single-mole- peptides and proteins but, like the ability to form the fibrils cule fluorescence technique to examine the assembly of oligomeric themselves, could be a generic property of polypeptides (2, 9). species formed during the aggregation of the SH3 domain of PI3 Studies of PI3–SH3 can therefore provide valuable information kinase. The single-molecule experiments show that the species not only on the nature of amyloid fibril formation, but also on formed at the stage of the reaction where aggregates have the population of the cytotoxic oligomers that populate the previously been found to be maximally cytotoxic are a heteroge- assembly pathway. neous ensemble of oligomers with a median size of 38 ؎ 10 molecules. This number is remarkably similar to estimates from Results and Discussion bulk measurements of the critical size of species observed to seed Bulk Experiments. Initially, we performed bulk kinetic studies of ordered fibril formation and of the most infective form of prion the formation of amyloid fibrils by wild-type PI3–SH3 at room particles. Moreover, although the size distribution of the SH3 temperature and pH 2.0 by monitoring the fluorescence of the oligomers remains virtually constant as the time of aggregation amyloidophilic dye, thioflavin T (ThT). These data yield a increases, their stability increases substantially. These findings sigmoidal kinetic profile that could be well fitted by using a together provide direct evidence for a general mechanism of simple logistic model, where the overall aggregation rate de- amyloid aggregation in which the stable cross- structure emerges creases as soluble precursors are consumed and that has been via internal reorganization of disordered oligomers formed during used previously to describe PI3–SH3 aggregation kinetics (see the lag phase of the self-assembly reaction. Fig. 2A) (20). Based on this fit, the initial lag phase for fibril proliferation (assigned here as the time until the ThT fluores- amyloid aggregation ͉ amyloid oligomers ͉ two-color coincidence cence has increased by 5% of its maximal increase) of 18.4 Ϯ spectroscopy ͉ PI3-SH3 domain ͉ neurodegenerative diseases 2.3 h is followed by an exponential fibril growth phase with a rate constant of 0.16 Ϯ 0.02 hϪ1. The presence of a lag phase is typical he tissue deposition of the -sheet-rich, filamentous protein of the conversion of soluble proteins into amyloid fibrils and is Taggregates, amyloid fibrils, represents the common patho- generally considered to represent the nucleation phase for logical hallmark of a range of degenerative disorders including ordered aggregation (8). Characteristically, this early stage of Alzheimer’s and Parkinson’s diseases. However, the observation fibril formation also corresponds to the highest population of that many proteins unrelated to disease can also form amyloid toxic prefibrillar aggregates (9–13). Indeed, aggregates were fibrils suggests that the structural motif common to these shown to form during this stage of the reaction by determination aggregates is broadly accessible by polypeptide chains (1, 2). As of the fraction of PI3–SH3 that remained soluble subsequent such, developing an understanding of the mechanisms by which to ultracentrifugation of the solution at various time points (see Fig. 2A). soluble protein molecules assemble into these fibrils is of fun- In contrast to the ThT data, the fraction of soluble protein damental and biomedical importance (3–7). Amyloid fibrils have decreased without a lag phase and with a single exponential rate been demonstrated to assemble typically via nucleation and constant of 0.033 Ϯ 0.006 hϪ1 (see Fig. 2A); Ϸ40% of the protein growth kinetics, characterized by an initial lag phase before fibril became sedimentable during the ThT-determined lag phase for elongation (8). A wealth of data indicates that species formed fibril proliferation. From the centrifugation parameters used in during this phase of the reaction are cytotoxic, and, furthermore, this experiment, particles with a sedimentation coefficient of that soluble, oligomeric precursors to amyloid fibrils likely Ͼ38 S are expected to have been cleared from these solutions. represent the critical pathological species in some amyloid Although the sedimentation coefficient of an oligomeric assem- disorders (7, 9–13). Probing the initial stages of the assembly bly is highly dependent on particle shape and its degree of process is, however, challenging because of the low populations of heterogeneous, unstable oligomeric species. We have studied the early stages of amyloid fibril formation Author contributions: A.O., N.R.B., R.W.C., G.L.D., C.M.D., and D.K. designed research; A.O., by the SH3 domain from bovine phosphatidylinositol-3Ј-kinase N.R.B., R.W.C., and G.L.D. performed research; A.O., N.R.B., R.W.C., and G.L.D. analyzed (PI3–SH3) using an approach involving both bulk and single- data; and A.O., N.R.B., R.W.C., G.L.D., C.M.D., and D.K. wrote the paper. molecule techniques. PI3–SH3 has been shown to form highly The authors declare no conflict of interest. ordered structures upon incubation at low pH and low ionic This article is a PNAS Direct Submission. strength that have the key characteristics of amyloid fibrils *A.O., N.R.B., and R.W.C. contributed equally to this work. associated with human disease (14, 15). The most well charac- †To whom correspondence may be addressed. E-mail: [email protected], terized example of the amyloid fibrils formed by PI3–SH3 [email protected], or [email protected]. comprises a double helix of protofilament pairs, in which the This article contains supporting information online at www.pnas.org/cgi/content/full/ great majority of the 84 amino acid-residue protein is contained 0803086105/DCSupplemental. within the fibril structure, as shown from measurements of © 2008 by The National Academy of Sciences of the USA 14424–14429 ͉ PNAS ͉ September 23, 2008 ͉ vol. 105 ͉ no. 38 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803086105 Downloaded by guest on September 23, 2021 hydration, by simple comparison, 40 S particles correspond approximately to biomolecular complexes with diameters in the range of 10–20 nm (21). This value falls within the broad range of aggregate sizes observed by TEM in the early stages of the reaction [see supporting information (SI) Text and Figs. S1–S4] for this and other systems (9, 22–25). Taken together, the marked differences in the kinetic profiles of the aggregation reaction acquired from the ThT and sedimentation data strongly suggest the existence of multiple processes on the reaction pathway. To resolve the individual species present during the reaction and, hence, to probe the nature of these processes, we have applied a single-molecule fluorescence technique that employs simulta- neous two-color detection (26). Single-Molecule Experiments. To enable these single-molecule experiments, which were otherwise performed under identical conditions to the bulk studies, an N-terminal cysteine mutation (M1C) was introduced into the polypeptide sequence of PI3– SH3 to allow site-specific labeling. Batches of protein were labeled separately with either Alexa Fluor 488 or Alexa Fluor 647 fluorophores. Fibrils produced from a mixture of these labeled proteins were found by TEM to be morphologically similar to those formed by unmodified PI3–SH3 under the same conditions (see SI Text and Fig. S4). For the single-molecule experiments, equimolar mixtures of Alexa Fluor 488- and Alexa Fluor 647-labeled PI3–SH3 were incubated at room temperature for 4 d, corresponding to the lag and growth phases of aggre- gation detected by ThT fluorescence, during which time aliquots were taken at different time points for analysis. The aliquots Fig. 1. Principle of the TCCD method to detect oligomeric aggregates. (A) were then rapidly diluted by a factor of (1–2) ϫ 105 to enable the Detection of oligomer events in PI3–SH3 aggregation. The coincident fluores- detection of single-molecule fluorescence bursts and were sub- cent bursts on both channels show the presence of oligomers (marked as sequently subjected to single-molecule analysis using the two- asterisks). (B) Expansion of fluorescence bursts in A. Comparison of the inten- color coincidence detection (TCCD) technique. sity of bursts from monomers and oligomers: The monomer events are not The principles of oligomer detection by TCCD are shown in coincident and are much less intense than those due to oligomers. Fig. 1. TCCD is capable of detecting oligomeric complexes even in the presence of monomers, based on a molecule-by-molecule of the reaction to bind ThT strongly suggests that they do not analysis of the species as they diffuse through a confocal volume possess a well defined cross- structural motif common to mature excited by overlapped red and blue lasers (26, 27).