Single-molecule FRET unmasks structural subpopulations and crucial molecular events during FUS low-complexity domain phase separation.

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to be associated with a wide range of cellular functions and dysfunctions. We dissect critical molecular events associated with phase separation of an intrinsically disordered prion-like low-complexity domain of Fused in Sarcoma by performing single-molecule studies permitting us to access the wealth of molecular information that is skewed in conventional ensemble experiments. Our single-molecule FRET experiments reveal the coexistence of two conformationally distinct subpopulations in the monomeric form. Single-droplet single-molecule FRET studies coupled with fluorescence correlation spectroscopy, picosecond time-resolved fluorescence anisotropy, and vibrational Raman spectroscopy indicate that structural unwinding switches intramolecular interactions into intermolecular contacts allowing the formation of a dynamic network within condensates. A disease-related mutation introduces enhanced structural plasticity engendering greater interchain interactions that can accelerate pathological aggregation. Our findings provide key mechanistic underpinnings of sequence-encoded dynamically-controlled structural unzipping resulting in biological phase separation.

Hydrogen-Deuterium Exchange Vibrational Raman Spectroscopy Distinguishes Distinct Amyloid Polymorphs Comprising Altered Core Architecture.

Amyloid fibrils are ordered protein aggregates comprising a hydrogen-bonded central cross-ß core displaying a structural diversity in their supramolecular packing arrangements within the core. Such an altered packing results in amyloid polymorphism that gives rise to morphological and biological strain diversities. Here, we show that vibrational Raman spectroscopy coupled with hydrogen/deuterium (H/D) exchange discerns the key structural features that are responsible for yielding diverse amyloid polymorphs. Such a noninvasive and label-free methodology allows us to structurally distinguish distinct amyloid polymorphs displaying altered hydrogen bonding and supramolecular packing within the cross-ß structural motif. By using quantitative molecular fingerprinting and multivariate statistical analysis, we analyze key Raman bands for the protein backbone and side chains that allow us to capture the conformational heterogeneity and structural distributions within distinct amyloid polymorphs. Our results delineate the key molecular factors governing the structural diversity in amyloid polymorphs and can potentially simplify studying amyloid remodeling by small molecules.

ATP modulates self-perpetuating conformational conversion generating structurally distinct yeast prion amyloids that limit autocatalytic amplification.

Prion-like self-perpetuating conformational conversion of proteins into amyloid aggregates is associated with both transmissible neurodegenerative diseases and non-Mendelian inheritance. The cellular energy currency ATP is known to indirectly regulate the formation, dissolution, or transmission of amyloid-like aggregates by providing energy to the molecular chaperones that maintain protein homeostasis. In this work, we demonstrate that ATP molecules, independent of any chaperones, modulate the formation and dissolution of amyloids from a yeast prion domain (NM domain of Saccharomyces cerevisiae Sup35) and restricts autocatalytic amplification by controlling the amount of fragmentable and seeding-competent aggregates. ATP, at (high) physiological concentrations in the presence of Mg2+, kinetically accelerates NM aggregation. Interestingly, ATP also promotes phase separation-mediated aggregation of a human protein harboring a yeast prion-like domain. We also show that ATP disaggregates preformed NM fibrils in a dose-independent manner. Our results indicate that ATP-mediated disaggregation, unlike the disaggregation by the disaggregase Hsp104, yields no oligomers that are considered one of the critical species for amyloid transmission. Furthermore, high concentrations of ATP delimited the number of seeds by giving rise to compact ATP-bound NM fibrils that exhibited nominal fragmentation by either free ATP or Hsp104 disaggregase to generate lower molecular weight amyloids. In addition, (low) pathologically relevant ATP concentrations restricted autocatalytic amplification by forming structurally distinct amyloids that are found seeding inefficient because of their reduced ß-content. Our results provide key mechanistic underpinnings of concentration-dependent chemical chaperoning by ATP against prion-like transmissions of amyloids.

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates.

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.

Single-droplet surface-enhanced Raman scattering decodes the molecular determinants of liquid-liquid phase separation.

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.

Spatiotemporal modulations in heterotypic condensates of prion and α-synuclein control phase transitions and amyloid conversion.

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.

An intrinsically disordered pathological prion variant Y145Stop converts into self-seeding amyloids via liquid-liquid phase separation.

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, ß-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.

Ultrasensitive Characterization of the Prion Protein by Surface-Enhanced Raman Scattering: Selective Enhancement via Electrostatic Tethering of the Intrinsically Disordered Domain with Functionalized Silver Nanoparticles.

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.

Excitation Energy Migration Unveils Fuzzy Interfaces within the Amyloid Architecture.

Amyloid fibrils are highly ordered nanoscopic protein aggregates comprising a cross-ß amyloid core and are associated with deadly human diseases. Structural studies have revealed the supramolecular architecture of a variety of disease-associated 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-Förster resonance energy transfer can decipher the architecture of amyloid fibrils of human α-synuclein. Site-specific homo-Förster resonance energy transfer efficiencies measured by fluorescence depolarization allowed us to construct a two-dimensional proximity correlation map that defines the supramolecular packing of α-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.

Intrinsically disordered proteins in the formation of functional amyloids from bacteria to humans.

Amyloids are nanoscopic ordered self-assemblies of misfolded proteins that are formed via aggregation of partially unfolded or intrinsically disordered proteins (IDPs) and are commonly linked to devastating human diseases. An enlarging body of recent research has demonstrated that certain amyloids can be beneficial and participate in a wide range of physiological functions from bacteria to humans. These amyloids are termed as functional amyloids. Like disease-associated amyloids, a vast majority of functional amyloids are derived from a range of IDPs or hybrid proteins containing ordered domains and intrinsically disordered regions (IDRs). In this chapter, we describe an account of recent studies on the aggregation behavior of IDPs resulting in the formation of functional amyloids in a diverse range of organisms from bacteria to human. We also discuss the strategies that are used by these organisms to regulate the spatiotemporal amyloid assembly in their physiological functions.