Biophys J. 2015 Apr 21;108(8):2038-47.
Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis.
- 1Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
- 2Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.
- 3Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York; Tri-Institutional Training Program in Chemical Biology, Weill Cornell Medical College, and Rockefeller University, and Memorial Sloan-Kettering Cancer Center, New York, New York.
- 4Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska. Electronic address: email@example.com.
The aggregation of α-synuclein (α-Syn) is linked to Parkinson’s disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of α-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free α-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) α-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of α-Syn dimers could lead to different aggregation pathways.
The self-assembly of proteins and peptides into nano-aggregates of various sizes and morphologies is a widespread phenomenon relevant to human health. Aggregation is accompanied with the protein conformational change termed misfolding. Misfolding and aggregation of proteins contribute to a wide range of human pathologies termed protein misfolding and deposition neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. More than 40 human protein misfolding diseases are associated with the aggregation of a single protein or peptide. Understanding the mechanisms underlying self-assembly into nano-aggregates could facilitate the development of efficient therapeutic and diagnostic tools for these devastating diseases. However, due to a lack of such knowledge, little progress has been made in curing these diseases.
The early onset of Parkinson’s disease (PD) is associated with the misfolding and aggregation of a-synuclein (AS). Our long-term goal is to understand the mechanism by which AS misfolds and self-assembles into nano-aggregates. Our ultimate goal is to translate this knowledge into preventive and therapeutic strategies. Our objective is to characterize the oligomers formed at early stages of the self-assembly process and identify the species critical for the assembly of aggregates. We hypothesize that oligomer stability is a key characteristic that promotes the self-assembly of disease-prone species. Oligomers are formed transiently during the aggregation process, and their characterization requires special approaches capable of probing transient species. One approach termed a tethered approach for probing intermolecular interactions (TAPIN) was described in reference (1) and is schematically depicted in Fig. 1. In this approach, the unlabeled AS monomers are covalently immobilized onto the substrate while the labeled AS monomers are allowed to freely diffuse in the solution. When a labeled monomer comes close to the surface and makes a dimer complex with an unlabeled monomer, a fluorescence burst can be observed that persists until the dimer dissociates. The duration of the fluorescence burst can be measured and is considered the lifetime of a dimer. A typical time trajectory is shown in Fig. 2. The fluorescence burst is clearly seen, showing that the dimer persists for 1.7 seconds. By analyzing hundreds of such events, we show that the dimer is a very stable complex. This conclusion is supported by comparing the dimer lifetime with the characteristic time of monomer dynamics that occur in the microsecond time scale. Therefore, we conclude that AS dimerization is a key initial step that leads toward protein self-assembly for disease prone aggregates. There are a number of AS mutants that are associated with early PD development. We analyzed the A30P, E46K and A53T familial mutants and discovered that they all assemble into more stable dimers than the wild type (WT) variant of AS.
Figure 1. Schematic for the tethered approach for probing intermolecular interactions (TAPIN) used to directly measure the lifetime of AS dimers. Total internal reflection fluorescence microscopy was used to detect the dwell time of a dimer complex formed within the evanescent field. 1 nM of labeled AS monomers solution was used to ensure the single-molecule observation of dimer complexes.
Figure 2. A representative time trajectory shows a fluorescence burst with duration time of 1.7 s. The sudden increase in fluorescence indicates the formation of a dimer. The abrupt drop of fluorescence is due to the dissociation of the dimer. The duration is considered the lifetime of the dimer.
It is known that AS aggregation and PD development depend on environmental conditions. Specifically, AS aggregation is very sensitive to pH and acidic conditions accelerate AS aggregation. We recently used the TAPIN approach to test how dimer stability depends on pH by comparing the lifetimes of AS dimers at pH 7 and pH 5 (2). The results assembled as histograms in Fig. 3 show unambiguously that dimers are much more stable at pH 5 than at pH 7. Importantly, this trend is observed not only for WT protein, but also for the two familial mutations E46K and A53T.
Figure 3. Comparison of lifetimes of all AS dimers at pH 7 and pH 5.
In conclusion, the TAPIN approach allowed us to probe the dimeric transient states of AS. We demonstrate that the high stability of dimers, and possibly higher order oligomers, contributes to the AS self-assembly process. Given the fact that small oligomers are neurotoxic and dimers have a high stability, approaches that prevent AS dimerization should be considered as potential PD therapeutic and preventative strategies. This would require specific techniques that could probe transient species. Our TAPIN methodology could be utilized as a test system for the qualitative characterization of candidate drugs and the selection of those that are most efficacious. The discovery of the impact of acidic pH on dimer stability advances our knowledge of the molecular mechanisms leading to aggregation and may help identify the most efficient therapeutic targets.
- Lv Z, Krasnoslobodtsev AV, Zhang Y, Ysselstein D, Rochet JC, Blanchard SC, Lyubchenko YL. Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis. Biophys J. 2015, 108(8):2038-2047.
- Lv Z, Krasnoslobodtsev AV, Zhang Y, Ysselstein D, Rochet JC, Blanchard SC, Lyubchenko YL. Effect of acidic pH on the stability of α-Synuclein dimers. 2016, Biopolymers, in press, doi: 10.1002/bip.22874.
The work was supported by grants GM096039 (NIH) and MCB 1515346 (NSF) to YLL, grant GM098859 to SCB, and a grant from the Branfman Family Foundation to JCR. The authors declare no conflict of interest.
Yuri L. Lyubchenko, PhD, DSc, Professor
Department of Pharmaceutical Sciences
College of Pharmacy, University of Nebraska Medical Center
Joseph & Milliee Williams Science Hall
986025 Nebraska Medical Center
Omaha, NE 68198-6025