Biomolecular condensation via liquid–liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, β-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.

Biomolecular condensation via liquid-liquid phase separation of proteins and nucleic acids is associated with a range of critical cellular functions and neurodegenerative diseases. Here, we demonstrate that complex coacervation of the prion protein and α-synuclein within narrow stoichiometry results in the formation of highly dynamic, reversible, thermo-responsive liquid droplets via domain-specific electrostatic interactions between the positively-charged intrinsically disordered N-terminal segment of prion and the acidic C-terminal tail of α-synuclein. The addition of RNA to these coacervates yields multiphasic, vesicle-like, hollow condensates. Picosecond time-resolved measurements revealed the presence of transient electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making of interactions on slower timescales giving rise to a liquid-like behavior in the mesoscopic regime. The liquid-to-solid transition drives a rapid conversion of complex coacervates into heterotypic amyloids. Our results suggest that synergistic prion-α-synuclein interactions within condensates provide mechanistic underpinnings of their physiological role and overlapping neuropathological features.

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) are involved in a myriad of critical cellular functions and debilitating neurodegenerative diseases. Elucidating the role of intrinsic disorder and conformational heterogeneity of intrinsically disordered proteins/regions (IDPs/IDRs) in these phase-separated membrane-less organelles is crucial to understanding the mechanism of formation and regulation of biomolecular condensates. Here we introduce a unique single-droplet surface-enhanced Raman scattering (SERS) methodology that utilizes surface-engineered, plasmonic, metal nanoparticles to unveil the inner workings of mesoscopic liquid droplets of Fused in Sarcoma (FUS) in the absence and presence of RNA. These highly sensitive measurements offer unprecedented sensitivity to capture the crucial interactions, conformational heterogeneity, and structural distributions within the condensed phase in a droplet-by-droplet manner. Such an ultra-sensitive single-droplet vibrational methodology can serve as a potent tool to decipher the key molecular drivers of biological phase transitions of a wide range of biomolecular condensates involved in physiology and disease.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins, namely tau and prion, undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing symphony of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, and ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau:PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman results in conjunction with atomic force microscopy and multi-color fluorescence imaging reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader biological role of complex phase transitions in physiology and disease.

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Biomolecular condensates formed via liquid-liquid phase separation (LLPS) of proteins and nucleic acids are thought to govern critical cellular functions. These multicomponent assemblies provide dynamic hubs for competitive homotypic and heterotypic interactions. Here, we demonstrate that the complex coacervation between the prion protein (PrP) and α-synuclein (α-Syn) within a narrow stoichiometry regime results in the formation of highly dynamic liquid droplets. Domain-specific electrostatic interactions between the positively charged intrinsically disordered N-terminal segment of PrP and the negatively charged C-terminal domain of α-Syn drive the formation of these highly tunable, reversible, thermo-responsive condensates. Picosecond time-resolved measurements revealed the existence of relatively ordered electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making on a much slower timescale giving rise to the liquid-like behavior on the second timescale and mesoscopic length-scale. The addition of RNA to these preformed coacervates yields multiphasic, anisotropic, vesicle-like, hollow condensates. LLPS promotes liquid-to-solid maturation of α-Syn-PrP condensates resulting in the rapid conversion into heterotypic amyloids. Our results suggest that synergistic interactions between PrP and α-Syn in liquid condensates can offer mechanistic underpinnings of their physiological role and overlapping neuropathological features.

Amyloid fibrils are highly ordered nanoscopic protein aggregates comprising a cross-b amyloid core and are associated with deadly human diseases. Structural studies have revealed the supramolecular architecture of a variety of diseaseassociated amyloids. However, the critical role of transient intermolecular interactions between the disordered polypeptide segments of protofilaments in directing the supramolecular structure and nanoscale morphology remains elusive. Here, we present a unique case to demonstrate that interchain excitation energy migration via intermolecular homo-Fö rster resonance energy transfer can decipher the architecture of amyloid fibrils of human a-synuclein. Site-specific homo-Fö rster resonance energy transfer efficiencies measured by fluorescence depolarization allowed us to construct a two-dimensional proximity correlation map that defines the supramolecular packing of a-synuclein within the fibrils. These studies captured unique heteroterminal cross talks between the fuzzy interprotofilament interfaces of the parallel-in-register amyloid spines. Our results will find applications in discerning the broader role of protein disorder and fuzziness in steering the distinct polymorphic amyloids that exhibit strain-specific disease phenotypes.

Surface-enhanced Raman scattering (SERS) circumvents the inherent insensitivity of Raman spectroscopy and offers a powerful tool for the ultrasensitive detection and characterization of biomolecules at low concentrations. Here we show that SERS via electrostatic tethering between surface-modified negatively charged silver nanoparticles and highly positively charged intrinsically disordered N-terminal domain of the prion protein allows highly sensitive and reproducible protein detection and characterization at as low as hundreds of nanomolar protein concentrations. These measurements preferentially illuminate a selective part of the protein due to a sharp dependence of the near-field intensity on the distance between the nanoparticle surface and the protein. We also demonstrate that by shortening the length of the disordered tail it is possible to achieve a domain-selective Raman enhancement to study the C-terminal globular domain. Our tether-length-dependent SERS methodology will serve as a potent, noninvasive, and label-free strategy to detect and characterize a wide range of proteins possessing disordered segments.