Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

Determining the Location of the α-Synuclein Dimer Interface Using Native Top-Down Fragmentation and Isotope Depletion-Mass Spectrometry.

α-Synuclein (αSyn), a 140-residue intrinsically disordered protein, comprises the primary proteinaceous component of pathology-associated Lewy body inclusions in Parkinson's disease (PD). Due to its association with PD, αSyn is studied extensively; however, the endogenous structure and physiological roles of this protein are yet to be fully understood. Here, ion mobility-mass spectrometry and native top-down electron capture dissociation fragmentation have been used to elucidate the structural properties associated with a stable, naturally occurring dimeric species of αSyn. This stable dimer appears in both wild-type (WT) αSyn and the PD-associated variant A53E. Furthermore, we integrated a novel method for generating isotopically depleted protein into our native top-down workflow. Isotope depletion increases signal-to-noise ratio and reduces the spectral complexity of fragmentation data, enabling the monoisotopic peak of low abundant fragment ions to be observed. This enables the accurate and confident assignment of fragments unique to the αSyn dimer to be assigned and structural information about this species to be inferred. Using this approach, we were able to identify fragments unique to the dimer, which demonstrates a C-terminal to C-terminal interaction between the monomer subunits. The approach in this study holds promise for further investigation into the structural properties of endogenous multimeric species of αSyn.

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

Two-color coincidence single-molecule pulldown for the specific detection of disease-associated protein aggregates.

Protein misfolding and aggregation is a characteristic of many neurodegenerative disorders, including Alzheimer's and Parkinson's disease. The oligomers generated during aggregation are likely involved in disease pathogenesis and present promising biomarker candidates. However, owing to their small size and low concentration, specific tools to quantify and characterize aggregates in complex biological samples are still lacking. Here, we present single-molecule two-color aggregate pulldown (STAPull), which overcomes this challenge by probing immobilized proteins using orthogonally labeled detection antibodies. By analyzing colocalized signals, we can eliminate monomeric protein and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 picomolar. Furthermore, we show that STAPull can be used in a range of samples, including human biofluids. STAPull is applicable to protein aggregates from a variety of disorders and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases.

Probing TDP-43 condensation using an in silico designed aptamer.

Aptamers are artificial oligonucleotides binding to specific molecular targets. They have a promising role in therapeutics and diagnostics but are often difficult to design. Here, we exploited the catRAPID algorithm to generate aptamers targeting TAR DNA-binding protein 43 (TDP-43), whose aggregation is associated with Amyotrophic Lateral Sclerosis. On the pathway to forming insoluble inclusions, TDP-43 adopts a heterogeneous population of assemblies, many smaller than the diffraction-limit of light. We demonstrated that our aptamers bind TDP-43 and used the tightest interactor, Apt-1, as a probe to visualize TDP-43 condensates with super-resolution microscopy. At a resolution of 10 nanometers, we tracked TDP-43 oligomers undetectable by standard approaches. In cells, Apt-1 interacts with both diffuse and condensed forms of TDP-43, indicating that Apt-1 can be exploited to follow TDP-43 phase transition. The de novo generation of aptamers and their use for microscopy opens a new page to study protein condensation.

Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity.

Aggregation of alpha-synuclein (α-Syn) drives Parkinson's disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)-derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

Understanding protein-drug interactions using ion mobility-mass spectrometry.

Ion mobility-mass spectrometry (IM-MS) is an important addition to the analytical toolbox for the structural evaluation of proteins, and is enhancing many areas of biophysical analysis. Disease-associated proteins, including enzymes such as protein kinases, transcription factors exemplified by p53, and intrinsically disordered proteins, including those prone to aggregation, are all amenable to structural analysis by IM-MS. In this review we discuss how this powerful technique can be used to understand protein conformational dynamics and aggregation pathways, and in particular, the effect that small molecules, including clinically-relevant drugs, play in these processes. We also present examples of how IM-MS can be used as a relatively rapid screening strategy to evaluate the mechanisms and conformation-driven aspects of protein:ligand interactions.