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Parkinson’s disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS–membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of OαS–membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.

Liquid–liquid phase separation underlies the formation of biological condensates. Physically, such systems are microemulsions that in general have a propensity to fuse and coalesce; however, many condensates persist as independent droplets in the test tube and inside cells. This stability is crucial for their function, but the physicochemical mechanisms that control the emulsion stability of condensates remain poorly understood. Here, by combining single-condensate zeta potential measurements, optical microscopy, tweezer experiments, and multiscale molecular modeling, we investigate how the nanoscale forces that sustain condensates impact their stability against fusion. By comparing peptide–RNA (PR25:PolyU) and proteinaceous (FUS) condensates, we show that a higher condensate surface charge correlates with a lower fusion propensity. Moreover, measurements of single condensate zeta potentials reveal that such systems can constitute classically stable emulsions. Taken together, these results highlight the role of passive stabilization mechanisms in protecting biomolecular condensates against coalescence.

Oligomers comprised of misfolded proteins are implicated as neurotoxins in the pathogenesis of protein misfolding conditions such as Parkinson’s and Alzheimer’s diseases. Structural, biophysical, and biochemical characterization of these nanoscale protein assemblies is key to understanding their pathology and the design of therapeutic interventions, yet it is challenging due to their heterogeneous, transient nature and low relative abundance in complex mixtures. Here, we demonstrate separation of heterogeneous populations of oligomeric α-synuclein, a protein central to the pathology of Parkinson’s disease, in solution using microfluidic free-flow electrophoresis. We characterize nanoscale structural heterogeneity of transient oligomers on a time scale of seconds, at least 2 orders of magnitude faster than conventional techniques. Furthermore, we utilize our platform to analyze oligomer ζ-potential and probe the immunochemistry of wild-type α-synuclein oligomers. Our findings contribute to an improved characterization of α-synuclein oligomers and demonstrate the application of microchip electrophoresis for the free-solution analysis of biological nanoparticle analytes.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with other cell components. In the case of outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because these proteins remain unfolded for extended periods of time during their biogenesis and rely on interactions with binding partners to support proper folding. Using a combination of ensemble and single-molecule spectroscopy, we have scrutinized the unfolded state of outer-membrane phospholipase A (OmpLA) to provide a detailed view of its structural dynamics on timescales from nanoseconds to milliseconds. We find that even under strongly denaturing conditions and in the absence of residual secondary structure, OmpLA populates an ensemble of slowly (>100 ms) interconverting and conformationally heterogeneous unfolded states that lack the fast chain-reconfiguration motions expected for an unstructured, fully unfolded chain. The drastically slowed sampling of potentially folding-competent states, as compared with a random-coil polypeptide, may contribute to the slow in vitro folding kinetics observed for many OMPs. In vivo, however, slow intramolecular long-range dynamics might be advantageous for entropically favored binding of unfolded OMPs to chaperones and, by facilitating conformational selection after release from chaperones, for preserving binding-competent conformations before insertion into the outer membrane.

An approach relying on nanocavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nanocavity diffusional sizing (NDS), measures particle residence times within nanofluidic cavities to determine their hydrodynamic radii. Using theoretical modeling and simulations, we show that the residence time of particles within nanocavities above a critical time scale depends on the diffusion coefficient of the particle, which allows the estimation of the particle’s size. We demonstrate this approach experimentally through the measurement of particle residence times within nanofluidic cavities using single-molecule confocal microscopy. Our data show that the residence times scale linearly with the sizes of nanoscale colloids, protein aggregates, and single DNA oligonucleotides. NDS thus constitutes a new single molecule optofluidic approach that allows rapid and quantitative sizing of nanoscale particles for potential applications in nanobiotechnology, biophysics, and clinical diagnostics.

Liquid–liquid phase separation (LLPS) of proteins into biomolecular condensates has emerged as a fundamental principle underpinning cellular function and malfunction. Indeed, many human pathologies, including protein misfolding diseases, are linked to aberrant liquid-to-solid phase transitions, and disease-associated protein aggregates often nucleate through phase separation. The molecular level determinants that promote pathological phase transitions remain, however, poorly understood. Here we study LLPS of the microtubule-associated protein Tau, whose aberrant aggregation is associated with a number of neurodegenerative diseases, including Alzheimer’s disease. Using single molecule spectroscopy, we probe directly the conformational changes that the protein undergoes as a result of LLPS. We perform single-molecule FRET and fluorescence correlation spectroscopy experiments to monitor the intra- and intermolecular changes and demonstrate that the N- and C-terminal regions of Tau become extended, thus exposing the microtubule-binding region. These changes facilitate intermolecular interactions and allow for the formation of nanoscale clusters of Tau. Our results suggest that these clusters can promote the fibrillization of Tau, which can be dramatically accelerated by disease-related mutations P301L and P301S. Our findings thus provide important molecular insights into the mechanism of protein phase separation and the conversion of protein condensates from functional liquid assemblies to pathological aggregates.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.