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.

The bacterial nucleoid-associated proteins, HU and Dps, condense DNA into context-dependent biphasic or multiphasic complex coacervates.

The bacterial chromosome, known as its nucleoid, is an amorphous assemblage of globular nucleoprotein domains. It exists in a state of phase separation from the cell's cytoplasm, as an irregularly-shaped, membrane-less, intracellular compartment. This state (the nature of which remains largely unknown) is maintained through bacterial generations ad infinitum. Here, we show that HU and Dps, two of the most abundant nucleoid-associated proteins (NAPs) of Escherichia coli, undergo spontaneous complex coacervation with different forms of DNA/RNA, both individually and in each other's presence, to cause accretion and compaction of DNA/RNA into liquid-liquid phase separated condensates in vitro. Upon mixing with nucleic acids, HU-A and HU-B form (a) biphasic heterotypic mixed condensates in which HU-B helps to lower the Csat of HU-A and also (b) multiphasic heterotypic condensates, with Dps, in which demixed domains display different contents of HU and Dps. We believe that these modes of complex coacervation that are seen in vitro can serve as models for the in vivo relationships among NAPs in nucleoids, involving local and global variations in the relative abundances of the different NAPs, especially in demixed subdomains that are characterized by differing grades of phase separation. Our results clearly demonstrate some quantitative, and some qualitative, differences in the coacervating abilities of different NAPs with DNA, potentially explaining (i) why E. coli has two isoforms of HU, and (ii) why changes in the abundances of HU and Dps facilitate the lag, logarithmic, and stationary phases of E. coli growth.

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.

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.

Biophysics of biomolecular condensates.

The formation of biomolecular condensates has emerged as a new biophysical principle for subcellular compartmentalization within cells to facilitate the spatiotemporal regulation of a multitude of complex biomolecular reactions. In this Research Highlight, we summarize the findings that were published in Biophysical Journal during the past two years (2021 and 2022). These papers provided biophysical insights into the formation of biomolecular condensates via phase separation of proteins with or without nucleic acids.

Substoichiometric Hsp104 regulates the genesis and persistence of self-replicable amyloid seeds of Sup35 prion domain.

Prion-like self-perpetuating conformational conversion of proteins is involved in both transmissible neurodegenerative diseases in mammals and non-Mendelian inheritance in yeast. The transmissibility of amyloid-like aggregates is dependent on the stoichiometry of chaperones such as heat shock proteins (Hsps), including disaggregases. To provide the mechanistic underpinnings of the formation and persistence of prefibrillar amyloid seeds, we investigated the role of substoichiometric Hsp104 on the in vitro amyloid aggregation of the prion domain (NM-domain) of Saccharomyces cerevisiae Sup35. At low substoichiometric concentrations, we show Hsp104 exhibits a dual role: it considerably accelerates the formation of prefibrillar species by shortening the lag phase but also prolongs their persistence by introducing unusual kinetic halts and delaying their conversion into mature amyloid fibers. Additionally, Hsp104-modulated amyloid species displayed a better seeding capability compared to NM-only amyloids. Using biochemical and biophysical tools coupled with site-specific dynamic readouts, we characterized the distinct structural and dynamical signatures of these amyloids. We reveal that Hsp104-remodeled amyloidogenic species are compositionally diverse in prefibrillar aggregates and are packed in a more ordered fashion compared to NM-only amyloids. Finally, we show these Hsp104-remodeled, conformationally distinct NM aggregates display an enhanced autocatalytic self-templating ability that might be crucial for phenotypic outcomes. Taken together, our results demonstrate that substoichiometric Hsp104 promotes compositional diversity and conformational modulations during amyloid formation, yielding effective prefibrillar seeds that are capable of driving prion-like Sup35 propagation. Our findings underscore the key functional and pathological roles of substoichiometric chaperones in prion-like propagation.

Molecular Origin of Internal Friction in Intrinsically Disordered Proteins.

Protein folding and dynamics are controlled by an interplay of thermal and viscosity effects. The effect of viscous drag through the solvent molecules is described by the classic Kramers theory in the high friction limit, which considers the dampening of the reactant molecules in the solution and quantifies the dependence of the reaction rate on the frictional drag. In addition to the external energy dissipation originating from the surrounding solvent molecules, there is an additional mode of internal energy dissipative force operative within the polypeptide chain reflecting the internal resistance of the chain to its conformational alterations. This dry, solvent-independent intrinsic frictional drag is termed internal friction. In the case of natively folded proteins, the physical origin of internal friction is primarily attributed to the intrachain interactions or other nonnative interactions in their compact states. However, the molecular origin of internal friction in intrinsically disordered proteins (IDPs) remains elusive.In this Account, we address this fundamental issue: what are the principal drivers of viscosity-independent (dry) friction in highly solvated, expanded, conformationally flexible, rapidly fluctuating IDPs that do not possess persistent intrachain interactions? IDPs exhibit diffusive conformational dynamics that is predominantly dominated by the segmental motion of the backbone arising due to the dihedral rotations in the Ramachandran Φ-Ψ space. The physical origin of friction in a complex biopolymeric system such as IDPs can be described by classic polymer models, namely, Rouse/Zimm models with internal friction. These one-dimensional models do not invoke torsional fluctuation components. Kuhn's classic description includes the connection between internal friction and microscopic dihedral hopping. Based on our time-resolved fluorescence anisotropy results, we describe that the sequence-dependent, collective, short-range backbone dihedral rotations govern localized internal friction in an archetypal IDP, namely, α-synuclein. The highly sensitive, residue-specific fluorescence depolarization kinetics offers a potent methodology to characterize and quantify the directional decorrelation engendered due to the short-range dihedral relaxation of the polypeptide backbone in the dihedral space. We utilized this characteristic relaxation time scale as our dynamic readout to quantify the site-specific frictional component. Our linear viscosity-dependent model of torsional relaxation time scale furnished a finite nonzero time constant at the zero solvent viscosity representing the solvent-independent internal friction. These results unveil the effect of the degree of dihedral restraining parameter on the internal friction component by showing that a restrained proline residue imparts higher torsional stiffness in the chain segments and, therefore, exhibits higher internal friction. This Account sheds light on the molecular underpinning of the sequence-specific internal friction in IDPs and will be of interest to unmask the role of internal friction in a diverse range of biomolecular processes involving binding-induced folding, allosteric interaction, protein misfolding and aggregation, and biomolecular condensation via phase separation.

Short-Range Backbone Dihedral Rotations Modulate Internal Friction in Intrinsically Disordered Proteins.

Protein folding and dynamics are governed by an intricate interplay of thermal and viscosity-mediated effects. The solvent viscosity contributes to the frictional drag in protein dynamics. In addition to this viscosity-dependent effect, there is also an intriguing viscosity-independent component that represents the intrinsic resistance of the polypeptide chain to changing its conformation. This solvent-independent component is termed internal friction. A longstanding question is what is the fundamental molecular origin of internal friction in highly solvated and rapidly fluctuating intrinsically disordered proteins (IDPs) devoid of any persistent intrachain interactions? Here, we present a unique case to directly demonstrate that sequence-specific backbone dihedral barriers control local internal friction in an archetypal IDP, namely, α-synuclein. We performed site-directed fluorescence depolarization kinetics using picosecond time-resolved fluorescence anisotropy measurements to directly observe the directional decorrelation arising due to short-range backbone torsional fluctuations in the dihedral space. A linear viscosity-dependent model of the dihedral relaxation time yielded a finite zero-viscosity intercept that corresponds to internal friction. Our site-specific dynamic readouts were able to detect localized sequence-specific frictional components that are otherwise skewed in viscosity-dependent long-range chain fluctuations. Our results revealed the presence of low internal friction in nonproline sequence segments. In contrast, a proline introduces torsional stiffness in the segment exhibiting high internal friction that can be compensated by a conformationally flexible glycine. Such an intriguing interplay of local dihedral dynamics can modulate sequence-dependent internal friction in a wide range of IDPs involved in a myriad of important events including folding, binding, assembly, and phase transitions.

Distinct types of amyloid-ß oligomers displaying diverse neurotoxicity mechanisms in Alzheimer's disease.

Soluble oligomers of amyloid-ß (Aß) are recognized as key pernicious species in Alzheimer's disease (AD) that cause synaptic dysfunction and memory impairments. Numerous studies have identified various types of Aß oligomers having heterogeneous peptide length, size distribution, structure, appearance, and toxicity. Here, we review the characteristics of soluble Aß oligomers based on their morphology, size, and structural reactivity toward the conformation-specific antibodies and then describe their formation, localization, and cellular effects in AD brains, in vivo and in vitro. We also summarize the mechanistic pathways by which these soluble Aß oligomers cause proteasomal impairment, calcium dyshomeostasis, inhibition of long-term potentiation, apoptosis, mitochondrial damage, and cognitive decline. These cellular events include three distinct molecular mechanisms: (i) high-affinity binding with the receptors for Aß oligomers such as N-methyl- d-aspartate receptors, cellular prion protein, nerve growth factor, insulin receptors, and frizzled receptors; (ii) the interaction of Aß oligomers with the lipid membranes; (iii) intraneuronal accumulation of Aß by α7-nicotinic acetylcholine receptors, apolipoprotein E, and receptor for advanced glycation end products. These studies indicate that there is a pressing need to carefully examine the role of size, appearance, and the conformation of oligomers in identifying the specific mechanism of neurotoxicity that may uncover potential targets for designing AD therapeutics.

Conformation-specific perturbation of membrane dynamics by structurally distinct oligomers of Alzheimer's amyloid-ß peptide.

The accumulation of toxic soluble oligomers of the amyloid-ß peptide (Aß) is a key step in the pathogenesis of Alzheimer's disease. There are mainly two conformationally distinct oligomers, namely, prefibrillar and fibrillar oligomers, that are recognized by conformation-specific antibodies, anti-amyloid oligomer antibody (A11) and anti-amyloid fibrillar antibody (OC), respectively. Previous studies have shown that the interaction of Aß oligomers with the lipid membrane is one of the key mechanisms of toxicity produced by Aß oligomers. However, the mechanism by which structurally distinct Aß oligomers interact with the lipid membrane remains elusive. In this work, we dissect the molecular mechanism underlying the interaction of structurally distinct Aß42 oligomers with the lipid membrane derived from the brain total lipid extract. Using picosecond time-resolved fluorescence spectroscopy, we show that the A11-positive Aß42 oligomers undergo a membrane-induced conformational change that promotes the deeper immersion of these oligomers into the lipid hydrocarbon region and results in an increase in the membrane micro-viscosity. In sharp contrast, OC-positive Aß42 oligomers interact with the lipid membrane via electrostatic interactions between the negatively-charged lipid headgroup and positively-charged residues of Aß42 without perturbing the membrane dynamics. We show that the two structurally distinct Aß42 oligomers demonstrating different interaction mechanisms with the lipid membrane eventually lead to the formation of typical amyloid fibrils. Our findings provide the mechanistic underpinning of the perturbation of lipid membranes by two conformationally distinct Aß42 oligomers and can be of prime importance in designing anti-Alzheimer's therapeutic agents targeting Aß-membrane interactions.

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.

Intermolecular Charge-Transfer Modulates Liquid-Liquid Phase Separation and Liquid-to-Solid Maturation of an Intrinsically Disordered pH-Responsive Domain.

Liquid-liquid phase separation of intrinsically disordered proteins into mesoscopic, dynamic, liquid-like supramolecular condensates is thought to govern critical cellular functions. These condensates can mature from a functional liquid-like state to a pathological gel-like or solid-like state. Here, we present a unique case to demonstrate that an unusual cascade of intermolecular charge-transfer coupled with a multitude of transient noncovalent interactions and conformational fluctuations can promote liquid phase condensation of a pH-responsive, intrinsically disordered, oligopeptide repeat domain of a melanosomal protein. At neutral cytosolic pH, the repeat domain forms highly dynamic, mesoscopic, permeable, liquid-like droplets possessing rapid internal diffusion and torsional fluctuations. These liquid condensates mature via pervasive intermolecular charge-transfer and persistent backbone interactions driving the liquid-to-solid phase transition into heterogeneous solid-like aggregates that are structurally and morphologically distinct from typical amyloids formed at mildly acidic melanosomal pH. Our findings reveal the regulatory role of the repeat domain as a specific pH-sensor that critically controls the phase transition and self-assembly processes akin to prion-like low-complexity domains modulating intracellular phase separation.

Femtosecond Hydration Map of Intrinsically Disordered α-Synuclein.

Protein hydration water plays a fundamentally important role in protein folding, binding, assembly, and function. Little is known about the hydration water in intrinsically disordered proteins that challenge the conventional sequence-structure-function paradigm. Here, by combining experiments and simulations, we show the existence of dynamical heterogeneity of hydration water in an intrinsically disordered presynaptic protein, namely α-synuclein, implicated in Parkinson's disease. We took advantage of nonoccurrence of cysteine in the sequence and incorporated a number of cysteine residues at the N-terminal segment, the central amyloidogenic nonamyloid-ß component (NAC) domain, and the C-terminal end of α-synuclein. We then labeled these cysteine variants using environment-sensitive thiol-active fluorophore and monitored the solvation dynamics using femtosecond time-resolved fluorescence. The site-specific femtosecond time-resolved experiments allowed us to construct the hydration map of α-synuclein. Our results show the presence of three dynamically distinct types of water: bulk, hydration, and confined water. The amyloidogenic NAC domain contains dynamically restrained water molecules that are strikingly different from the water molecules present in the other two domains. Atomistic molecular dynamics simulations revealed longer residence times for water molecules near the NAC domain and supported our experimental observations. Additionally, our simulations allowed us to decipher the molecular origin of the dynamical heterogeneity of water in α-synuclein. These simulations captured the quasi-bound water molecules within the NAC domain originating from a complex interplay between the local chain compaction and the sequence composition. Our findings from this synergistic experimental simulation approach suggest longer trapping of interfacial water molecules near the amyloidogenic hotspot that triggers the pathological conversion into amyloids via chain sequestration, chain desolvation, and entropic liberation of ordered water molecules.

Formation of Heterotypic Amyloids: α-Synuclein in Co-Aggregation.

Protein misfolding resulting in the formation of ordered amyloid aggregates is associated with a number of devastating human diseases. Intrinsically disordered proteins (IDPs) do not autonomously fold up into a unique stable conformation and remain as an ensemble of rapidly fluctuating conformers. Many IDPs are prone to convert into the ß-rich amyloid state. One such amyloidogenic IDP is α-synuclein that is involved in Parkinson's disease. Recent studies have indicated that other neuronal proteins, especially IDPs, can co-aggregate with α-synuclein in many pathological ailments. This article describes several such observations highlighting the role of heterotypic protein-protein interactions in the formation of hetero-amyloids. It is believed that the characterizations of molecular cross talks between amyloidogenic proteins as well as the mechanistic studies of heterotypic protein aggregation will allow us to decipher the role of the interacting proteins in amyloid proteomics.

Human Fibrinogen Inhibits Amyloid Assembly of Biofilm-Forming CsgA.

Curli is a biofilm-forming amyloid that is expressed on the surface of Gram-negative enteric bacteria such as Escherichia coli and Salmonella spp. Curli is primarily composed of the major structural subunit, CsgA, and interacts with a wide range of human proteins that contribute to bacterial virulence. The adsorption of curli onto the contact-phase proteins and fibrinogen results in a hypocoagulatory state. Using an array of biochemical and biophysical tools, we elucidated the molecular mechanism of interaction between human fibrinogen and CsgA. Our results revealed that a substoichiometric concentration of fibrinogen delays the onset of CsgA aggregation by inhibiting the early events of CsgA assembly. The presence of fibrinogen prevents the maturation of CsgA into fibrils and maintains the soluble state of CsgA. We also demonstrate that fibrinogen interacts more effectively with the disordered conformational state of CsgA than with the ordered ß-rich state. Our study suggested that fibrinogen is an anti-curli protein and that the interplay of CsgA and fibrinogen might be a host defense mechanism against curli biogenesis, biofilm formation, bacterial colonization, and infection.

Electrostatic lipid-protein interactions sequester the curli amyloid fold on the lipopolysaccharide membrane surface.

Curli is a functional amyloid protein in the extracellular matrix of enteric Gram-negative bacteria. Curli is assembled at the cell surface and consists of CsgA, the major subunit of curli, and a membrane-associated nucleator protein, CsgB. Oligomeric intermediates that accumulate during the lag phase of amyloidogenesis are generally toxic, but the underlying mechanism by which bacterial cells overcome this toxicity during curli assembly at the surface remains elusive. Here, we elucidated the mechanism of curli amyloidogenesis and provide molecular insights into the strategy by which bacteria can potentially bypass the detrimental consequences of toxic amyloid intermediates. Using a diverse range of biochemical and biophysical tools involving circular dichroism, fluorescence, Raman spectroscopy, and atomic force microscopy imaging, we characterized the molecular basis of the interaction of CsgB with a membrane-mimetic anionic surfactant as well as with lipopolysaccharide (LPS) constituting the outer leaflet of Gram-negative bacteria. Aggregation studies revealed that the electrostatic interaction of the positively charged C-terminal region of the protein with a negatively charged head group of surfactant/LPS promotes a protein-protein interaction that results in facile amyloid formation without a detectable lag phase. We also show that CsgB, in the presence of surfactant/LPS, accelerates the fibrillation rate of CsgA by circumventing the lag phase during nucleation. Our findings suggest that the electrostatic interactions between lipid and protein molecules play a pivotal role in efficiently sequestering the amyloid fold of curli on the membrane surface without significant accumulation of toxic oligomeric intermediates.

Stepwise unfolding of human ß2-microglobulin into a disordered amyloidogenic precursor at low pH.

Amyloid fibril formation by human ß2-microglobulin (ß2m) is associated with dialysis-related amyloidosis. In order to understand the mechanism of protein misfolding, it is important to characterize the nature and properties of various intermediates formed during protein unfolding. In this work, we studied the effect of pH change on the unfolding of ß2m using a range of spectroscopic readouts. In order to investigate the local structural changes, we created single tryptophan (W60 and W95) mutants of ß2m. The equilibrium results suggested that in the acid-unfolded state of ß2m at pH 2.5, the W60 residue attains non-native local structure whereas the W95 residue becomes more exposed. Our stopped-flow kinetic data revealed that ß2m undergoes unfolding in a stepwise manner. Initial unfolding of ß2m involves non-uniform protein expansion with the unpacking of tertiary structure and significant core solvation while maintaining a native-like structure around residue W95. The resolved-phase of unfolding exhibits a timescale of ~500 ms that describes the transition from the native-like swollen intermediate to an acid-induced disordered state. Taken together, our results demonstrate that ß2m has a complex pH-induced unfolding mechanism yielding a disordered amyloidogenic precursor comprising both exposed and buried segments.