Ultrasensitive Detection of Lipid-Induced Misfolding of the Prion Protein at the Aqueous-Liquid Crystal Interface.

The misfolding of the α-helical cellular prion protein into a self-propagating ß-rich aggregated form is a key pathogenic event in fatal and transmissible neurodegenerative diseases collectively known as prion diseases. Herein, we utilize the interfacial properties of liquid crystals (LCs) to monitor the lipid-membrane-induced conformational switching of prion protein (PrP) into ß-rich amyloid fibrils. The lipid-induced conformational switching resulting in aggregation occurs at the nanomolar protein concentration and is primarily mediated by electrostatic interactions between PrP and lipid headgroups. Our LC-based methodology offers a potent and sensitive tool to detect and delineate molecular mechanisms of PrP misfolding mediated by lipid-protein interactions at the aqueous interface under physiological conditions.

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.

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.

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.

Fluorescence Depolarization Kinetics Captures Short-Range Backbone Dihedral Rotations and Long-Range Correlated Dynamics of an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) do not autonomously fold into well-defined three-dimensional structures and are best described as a heterogeneous ensemble of rapidly interconverting conformers. It is challenging to elucidate their complex dynamic signatures using a single technique. In this study, we employed sensitive fluorescence depolarization kinetics by following picosecond time-resolved fluorescence anisotropy decays to directly capture the essential dynamical features of intrinsically disordered α-synuclein (α-syn) site-specifically labeled with thiol-active fluorophores. By utilizing a long-lifetime (≥10 ns) anisotropic label, we were able to discern three distinct rotational components of α-syn. The subnanosecond component represents the local wobbling-in-cone motion of the fluorophore, whereas the slower (∼1.4 ns) component corresponds to the short-range backbone dynamics governed by collective torsional fluctuations in the Ramachandran Φ-Ψ dihedral space. This backbone dihedral rotational time scale is sensitive to the local chain stiffness and slows down in the presence of an adjacent proline residue. We also observed a small-amplitude (≤10%) slower rotational correlation time (6-10 ns) that represents the long-range correlated dynamics involving a much longer segment of the polypeptide chain. These intrinsic dynamic signatures of IDPs will provide critical mechanistic underpinnings in a mosaic of biophysical phenomena involving internal friction, allosteric interactions, and phase separation.