scholarly journals Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Emil G. P. Stender ◽  
Soumik Ray ◽  
Rasmus K. Norrild ◽  
Jacob Aunstrup Larsen ◽  
Daniel Petersen ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Amyloid Fibrils ◽  
Capillary Flow ◽  
Solid Phase ◽  
Liquid Phase Separation ◽  
Dead Box ◽  
Phase Concentration ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP3. Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

scholarly journals G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

2021 ◽  
Author(s):  
Jun Gao ◽  
Zhaofeng Gao ◽  
Andrea A. Putnam ◽  
Alicia K. Byrd ◽  
Sarah L. Venus ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Dead Box ◽  
G4 Dna ◽  
G Quadruplex ◽  
The Dead ◽  
Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

scholarly journals Prion-Like Proteins in Phase Separation and Their Link to Disease

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1014
Author(s):  
Macy L. Sprunger ◽  
Meredith E. Jackrel
Keyword(s):  
Protein Folding ◽  
Phase Transitions ◽  
Phase Separation ◽  
Liquid Phase ◽  
Protein Misfolding ◽  
Solid Phase ◽  
Liquid Phase Separation ◽  
Therapeutic Approaches ◽  
Important Physiological Process ◽  
Liquid Liquid Phase Separation

Aberrant protein folding underpins many neurodegenerative diseases as well as certain myopathies and cancers. Protein misfolding can be driven by the presence of distinctive prion and prion-like regions within certain proteins. These prion and prion-like regions have also been found to drive liquid-liquid phase separation. Liquid-liquid phase separation is thought to be an important physiological process, but one that is prone to malfunction. Thus, aberrant liquid-to-solid phase transitions may drive protein aggregation and fibrillization, which could give rise to pathological inclusions. Here, we review prions and prion-like proteins, their roles in phase separation and disease, as well as potential therapeutic approaches to counter aberrant phase transitions.

scholarly journals Microfluidic characterization of macromolecular liquid–liquid phase separation

Lab on a Chip ◽  
2020 ◽  
Vol 20 (22) ◽  
pp. 4225-4234
Author(s):  
Anne Bremer ◽  
Tanja Mittag ◽  
Michael Heymann
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Precise Determination ◽  
Phase Morphology ◽  
Liquid Phase Separation ◽  
Dense Phase ◽  
Liquid Liquid Phase Separation

The microfluidic phase chip allows precise determination of the saturation concentrations of biomolecules that undergo liquid–liquid phase separation while also monitoring the dense-phase morphology.

scholarly journals Liquid-liquid phase separation of full-length prion protein initiates conformational conversion in vitro

2020 ◽  
Author(s):  
Hiroya Tange ◽  
Daisuke Ishibashi ◽  
Takehiro Nakagaki ◽  
Yuzuru Taguchi ◽  
Yuji O. Kamatari ◽  
...  
Keyword(s):  
Phase Transition ◽  
Phase Separation ◽  
Liquid Phase ◽  
Prion Protein ◽  
Solid Phase ◽  
Terminal Region ◽  
Liquid Phase Separation ◽  
Solid Phase Transition ◽  
Liquid Liquid Phase Separation

AbstractPrion diseases are characterized by accumulation of amyloid fibrils. The causative agent is an infectious amyloid that is comprised solely of misfolded prion protein (PrPSc). Prions can convert PrPC to proteinase-resistant PrP (PrP-res) in vitro; however, the intermediate steps involved in the spontaneous conversion remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation in the absence of PrPSc. We found that rPrP underwent liquid-liquid phase separation at the interface of the aqueous two-phase system (ATPS) of polyethylene glycol (PEG) and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired proteinase-resistant amyloid accompanied by β-sheet conversion, as confirmed by western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23-89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote liquid-liquid phase separation. Thus, structural conversion via liquid–liquid phase separation and liquid–solid phase transition are intermediate steps in the conversion of prions.

scholarly journals Spider silk self-assembly via modular liquid-liquid phase separation and nanofibrillation

2020 ◽  
Vol 6 (45) ◽  
pp. eabb6030
Author(s):  
Ali D. Malay ◽  
Takehiro Suzuki ◽  
Takuya Katashima ◽  
Nobuaki Kono ◽  
Kazuharu Arakawa ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Self Assembly ◽  
Spider Silk ◽  
Solid Phase ◽  
Silk Gland ◽  
Liquid Phase Separation ◽  
Hierarchical Organization ◽  
Amino Terminal ◽  
Liquid Liquid Phase Separation

Spider silk fiber rapidly assembles from spidroin protein in soluble state via an incompletely understood mechanism. Here, we present an integrated model for silk formation that incorporates the effects of multiple chemical and physical gradients on the different spidroin functional domains. Central to the process is liquid-liquid phase separation (LLPS) that occurs in response to multivalent anions such as phosphate, mediated by the carboxyl-terminal and repetitive domains. Acidification coupled with LLPS triggers the swift self-assembly of nanofibril networks, facilitated by dimerization of the amino-terminal domain, and leads to a liquid-to-solid phase transition. Mechanical stress applied to the fibril structures yields macroscopic fibers with hierarchical organization and enriched for β-sheet conformations. Studies using native silk gland material corroborate our findings on spidroin phase separation. Our results suggest an intriguing parallel between silk assembly and other LLPS-mediated mechanisms, such as found in intracellular membraneless organelles and protein aggregation disorders.

scholarly journals Tau conformers in FTLD-MAPT undergo liquid-liquid phase separation and perturb the nuclear envelope

2020 ◽  
Author(s):  
Sang-Gyun Kang ◽  
Zhuang Zhuang Han ◽  
Nathalie Daude ◽  
Emily McNamara ◽  
Serene Wohlgemuth ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nuclear Envelope ◽  
Solid Phase ◽  
Critical Mass ◽  
Liquid Phase Separation ◽  
Liquid Droplets ◽  
Cytoplasmic Transport ◽  
End Stage ◽  
Liquid Liquid Phase Separation

AbstractRecent studies show that a single MAPT gene mutation can promote alternative tau misfolding pathways engendering divergent forms of frontotemporal dementia and that under conditions of molecular crowding, the repertoire of tau forms can include liquid-liquid phase separation (LLPS). We show here that following pathogenic seeding, tau condenses on the nuclear envelope (NE) and disrupts nuclear-cytoplasmic transport (NCT). Interestingly, NE fluorescent tau signals and small fluorescent inclusions behaved as demixed liquid droplets in living cells. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 μm2, indicating that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions. Our findings indicate that tau undergoing LLPS is more toxic amongst a spectrum of alternative conformers; LLPS droplets on the NE that disrupt NCT serve to trigger cell death and can act as nurseries for fibrillar structures abundantly detected in end-stage disease.

scholarly journals A generic approach to study the kinetics of liquid–liquid phase separation under near-native conditions

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Joris Van Lindt ◽  
Anna Bratek-Skicki ◽  
Phuong N. Nguyen ◽  
Donya Pakravan ◽  
Luis F. Durán-Armenta ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Stress Protein ◽  
Low Complexity ◽  
Liquid Phase Separation ◽  
Phase Separation Behavior ◽  
Liquid Liquid Phase Separation

AbstractUnderstanding the kinetics, thermodynamics, and molecular mechanisms of liquid–liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.

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