Liquid–Liquid Phase Separation Is Driven by Large-Scale Conformational Unwinding and Fluctuations of Intrinsically Disordered Protein Molecules

Abstract

MaterialsAll of the chemicals used for preparing buffer solutions, such as, sodium phosphate monobasic dihydrate, Tris (2-carboxyethyl) phosphine hydrochloride (TCEP), MES (2-(N-Morpholino)ethanesulfonic acid hydrate, EDTA (Ethylenediaminetetraacetic acid), Magnesium chloride hexahydrate, DTT (DL-Dithiothreitol) were of highest purity grade obtained from Sigma Aldrich (St. Louis, MO). The fluorescent probes, namely, fluorescein-5maleimide, N-(1-pyrene) maleimide, Acrylodan (6-Acryloyl-2-Dimethylaminonaphthalene), AlexaFluor 488 C5-maleimide, and AlexaFluor 594 C5-maleimide were purchased from Molecular Probes, Invitrogen. The free fluorescein dye was purchased from Fluka Analytical. SP Sepharose resin used for protein purification and PD-10 columns were purchased from GE Healthcare Life Sciences (USA). The protein concentrators and filters were procured from Merck Millipore. A Metrohm 827 lab pH meter was used to adjust the final pH ( 0.01) of all the buffer solutions prepared in Milli-Q water and filtered before use. Expression and Purification of tau K18Tau K18 was expressed in Escherichia coli BL21(DE3) and purified using the procedure described previously (51). Briefly, using a lysis buffer of pH 8, (50 mM Tris, 150 mM NaCl, 10 mM EDTA), the cells were lysed by boiling it for half an hour at a 100 ˚C. The lysate was then centrifuged at 11,500 rpm at 4 ˚C for 30 min, following which the supernatant was treated with 136 µL/mL of 10% streptomycin sulfate and 228 µL/mL of glacial acetic acid for the precipitation of DNA. After the removal of DNA by further centrifugation at 11,500

Relevance of Electrostatic Charges in Compactness, Aggregation, and Phase Separation of Intrinsically Disordered Proteins

“…The relentless tug of war between electrostatics and the hydrophobic effect is also a determinant for the link between aggregation and LLPS. To cite an instance, a connection between aggregation and LLPS of tau protein has for a long time been suggested [151][152][153] and recently ruled out [154]. For tau protein, LLPS is driven by complex coacervation mechanisms, dominated by long-range multivalent electrostatic attractions [155].…”

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

“…Phase transition into demixed liquid state of tau has been reported mainly using in vitro cell-free systems with purified recombinant proteins under conditions of molecular crowding (Ambadipudi et al, 2017; Boyko et al, 2019; Hernandez-Vega et al, 2017; Majumdar, Dogra, Maity, & Mukhopadhyay, 2019; Singh et al, 2020; Vega et al, 2019; Wegmann et al, 2018; X. Zhang et al, 2017).…”

Common Functions of Disordered Proteins across Evolutionary Distant Organisms

“…Under these conditions, intermolecular interactions between IDPs stabilize the condensed phase overcoming intramolecular and solvent interactions [59,60]. The level of anisotropy remarkably increases in phase separated droplets, indicating an expanded state of IDPs in comparison to a more compacted state in solution [59,60] ( Figure 1D). The resulting MCs are highly concentrated in proteinous components and can also rapidly dissociate once the concentration falls below the critical point or protein interactions are altered (e.g., by PTMs) [61][62][63][64][65].…”

“…To achieve LLPS and formation of MCs, their contents have to reach a critical concentration to enable a de-mixing effect and form non-miscible phases [58]. Under these conditions, intermolecular interactions between IDPs stabilize the condensed phase overcoming intramolecular and solvent interactions [59,60]. The level of anisotropy remarkably increases in phase separated droplets, indicating an expanded state of IDPs in comparison to a more compacted state in solution [59,60] ( Figure 1D).…”