Liquid-liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is ...therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.
Single-molecule Förster resonance energy transfer (smFRET) has become a powerful nanoscopic tool in studies of biomolecular structures and nanoscale objects; however, conventional smFRET ...measurements are generally blind to distances above 10 nm thus impeding the study of long-distance phenomena. Here, we report the development of farFRET, a technique that extends the range in smFRET measurements beyond the 10 nm line by enhanced energy transfer using multiple acceptors. We demonstrate that farFRET can be readily employed to quantify FRET efficiencies and conformational dynamics using double-stranded DNA molecules, RecA-filament formation on single-stranded DNA and Holliday junction dynamics. farFRET allows quantitative measurements of large biomolecular complexes and nanostructures thus bridging the remaining gap to superresolution microscopy.
Intracellular phase separation of proteins into biomolecular condensates is increasingly recognized as a process with a key role in cellular compartmentalization and regulation. Different hypotheses ...about the parameters that determine the tendency of proteins to form condensates have been proposed, with some of them probed experimentally through the use of constructs generated by sequence alterations. To broaden the scope of these observations, we established an in silico strategy for understanding on a global level the associations between protein sequence and phase behavior and further constructed machine-learning models for predicting protein liquid-liquid phase separation (LLPS). Our analysis highlighted that LLPS-prone proteins are more disordered, less hydrophobic, and of lower Shannon entropy than sequences in the Protein Data Bank or the Swiss-Prot database and that they show a fine balance in their relative content of polar and hydrophobic residues. To further learn in a hypothesis-free manner the sequence features underpinning LLPS, we trained a neural network-based language model and found that a classifier constructed on such embeddings learned the underlying principles of phase behavior at a comparable accuracy to a classifier that used knowledge-based features. By combining knowledge-based features with unsupervised embeddings, we generated an integrated model that distinguished LLPS-prone sequences both from structured proteins and from unstructured proteins with a lower LLPS propensity and further identified such sequences from the human proteome at a high accuracy. These results provide a platform rooted in molecular principles for understanding protein phase behavior. The predictor, termed DeePhase, is accessible from https://deephase.ch.cam.ac.uk/.
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.
DNA origami represent powerful platforms for single-molecule investigations of biomolecular processes. The required structural integrity of the DNA origami may, however, pose significant limitations ...regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. Here, we therefore report a detailed study on the stability of 2D DNA origami triangles in the presence of the strong chaotropic denaturing agents urea and guanidinium chloride (GdmCl) and its dependence on concentration and temperature. At room temperature, the DNA origami triangles are stable up to at least 24 h in both denaturants at concentrations as high as 6 M. At elevated temperatures, however, structural stability is governed by variations in the melting temperature of the individual staple strands. Therefore, the global melting temperature of the DNA origami does not represent an accurate measure of their structural stability. Although GdmCl has a stronger effect on the global melting temperature, its attack results in less structural damage than observed for urea under equivalent conditions. This enhanced structural stability most likely originates from the ionic nature of GdmCl. By rational design of the arrangement and lengths of the individual staple strands used for the folding of a particular shape, however, the structural stability of DNA origami may be enhanced even further to meet individual experimental requirements. Overall, their high stability renders DNA origami promising platforms for biomolecular studies in the presence of chaotropic agents, including single-molecule protein folding or structural switching.
Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to ...follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion-like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs.
Molecular chaperones contribute to the maintenance of cellular protein homoeostasis through assisting de novo protein folding and preventing amyloid formation. Chaperones of the Hsp70 family can ...further disaggregate otherwise irreversible aggregate species such as α-synuclein fibrils, which accumulate in Parkinson's disease. However, the mechanisms and kinetics of this key functionality are only partially understood. Here, we combine microfluidic measurements with chemical kinetics to study α-synuclein disaggregation. We show that Hsc70 together with its co-chaperones DnaJB1 and Apg2 can completely reverse α-synuclein aggregation back to its soluble monomeric state. This reaction proceeds through first-order kinetics where monomer units are removed directly from the fibril ends with little contribution from intermediate fibril fragmentation steps. These findings extend our mechanistic understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and inform on possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies.
Abstract
Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from ...biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein–DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software.
The stability of DNA origami nanostructures under various environmental conditions constitutes an important issue in numerous applications, including drug delivery, molecular sensing, and ...single‐molecule biophysics. Here, the effect of Na+ and Mg2+ concentrations on DNA origami stability is investigated in the presence of urea and guanidinium chloride (GdmCl), two strong denaturants commonly employed in protein folding studies. While increasing concentrations of both cations stabilize the DNA origami nanostructures against urea denaturation, they are found to promote DNA origami denaturation by GdmCl. These inverse behaviors are rationalized by a salting‐out of Gdm+ to the hydrophobic DNA base stack. The effect of cation‐induced DNA origami denaturation by GdmCl deserves consideration in the design of single‐molecule studies and may potentially be exploited in future applications such as selective denaturation for purification purposes.
The effect of cations on DNA origami stability in the presence of the denaturing agents urea and guanidinium chloride (GdmCl) is investigated. While increasing Na+ and Mg2+ concentrations stabilize DNA origami nanostructures in urea, they promote DNA origami denaturation by GdmCl. This counterintuitive behavior is attributed to a salting‐out of Gdm+ to the hydrophobic DNA base stack.
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.
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