Altered dynamics may drift pathological fibrillization in membraneless organelles

Tüű-Szabó, Boldizsár; Hoffka, G.; Duro, Norbert; Fuxreiter, Mónika

doi:10.1016/j.bbapap.2019.04.005

Cited by 17 publications

(8 citation statements)

References 79 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…19 The linker flexibility can be a critical regulator of weak multivalent interactions in the liquid phase and can govern the material-state of biomolecular condensates. 51…”

Section: Resultsmentioning

confidence: 99%

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

Majumdar

Dogra

Maity

et al. 2019

Preprint

View full text Add to dashboard Cite

Liquid-liquid phase separation occurs via a multitude of transient, non-covalent, intermolecular interactions resulting in phase transition of intrinsically disordered proteins/regions (IDPs/IDRs) and other biopolymers into mesoscopic, dynamic, nonstoichiometric, supramolecular condensates. IDPs resemble associative polymers possessing stereospecific "stickers" and flexible "spacers" that govern the transient chain-chain interactions and fluidity in phase-separated liquid droplets. However, the fundamental molecular origin of phase separation remains elusive. Here we present a unique case to demonstrate that unusual conformational expansion events coupled with solvation and fluctuations drive phase separation of tau, an IDP associated with Alzheimer's disease. Using intramolecular excimer emission as a powerful proximity readout, we show the unraveling of polypeptide chains within the protein-rich interior environment that can promote critical interchain contacts. Using highly-sensitive picosecond time-resolved fluorescence depolarization measurements, we directly capture rapid large-amplitude torsional fluctuations in the extended chains that can control the relay of making-and-breaking of noncovalent intermolecular contacts maintaining the internal fluidity. Our observations, together with the existing polymer theories, suggest that such an orchestra of concerted molecular shapeshifting events involving chain expansion, solvation, and fluctuations can provide additional favorable free energies to overcome the entropy of mixing term during phase separation. The interplay of these key molecular parameters can also be of prime importance in modulating the mesoscale material property of liquid-like condensates and their maturation of into pathological gel-like and solid-like aggregates.

show abstract

“…19 The linker flexibility can be a critical regulator of weak multivalent interactions in the liquid phase and can govern the material-state of biomolecular condensates. 51…”

Section: Resultsmentioning

confidence: 99%

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

Majumdar

Dogra

Maity

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…19 The linker flexibility can be a critical regulator of weak multivalent interactions in the liquid phase and can govern the material-state of biomolecular condensates. 51 Our picosecond time-resolved fluorescence depolarization measurements allowed us to directly capture the unique dynamic signatures of the polypeptide chains within the phase-separated droplets. Rapid conformational fluctuations can provide a temporal control of the making and the breaking of stereospecific, intermolecular, noncovalent contacts between the "stickers" on a characteristic timescale that can further allow remaking of the new contacts with different polypeptide chain partners within the droplets.…”

Section: Discussionmentioning

confidence: 99%

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

Majumdar

Dogra

Maity

et al. 2019

J. Phys. Chem. Lett.

126

115

View full text Add to dashboard Cite

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

show abstract

“…Our results also support the hypothesis that pathological fibrils form within a biomolecular condensate when the conformational dynamics of the constituent IDP's are reduced. 56 LLPS of the α-synuclein, an IDP associated with Parkinson's disease, has been found to precede its aggregation into fibrils in vitro and in cells under oxidative stress. 57 In a healthy state, IDPs reversibly bind and retain strong local disorder, 20 as seen in our simulations.…”

Section: Discussionmentioning

confidence: 99%

“…60 If additional binding sites are thereby brought within range, the IDPs may progressively bind more tightly 9,61 and transform into rigid fibrils. 56 Experiments are now testing whether perturbing the conformational ensemble of a disease-prone IDP by adding a weakly-interacting ligand has therapeutic potential. 62…”

Section: Discussionmentioning

confidence: 99%

Structure of biomolecular condensates from dissipative particle dynamics simulations

Shillcock

Brochut

Chénais

et al. 2019

Preprint

View full text Add to dashboard Cite

Phase separation of immiscible fluids is a common phenomenon in polymer chemistry, and is recognized as an important mechanism by which cells compartmentalize their biochemical reactions. Biomolecular condensates are condensed fluid droplets in cells that form by liquid-liquid phase separation of intrinsically-disordered proteins. They have a wide range of functions and are associated with chronic neurodegenerative diseases in which they become pathologically rigid. Intrinsically-disordered proteins are conformationally flexible and possess multiple, distributed binding sites for each other or for RNA.However, it remains unclear how their material properties depend on the molecular structure of the proteins. Here we use coarse-grained simulations to explore the phase behavior and structure of a model biomolecular condensate composed of semi-flexible polymers with attractive end-caps in a good solvent.Although highly simplified, the model contains the minimal molecular features that are sufficient to observe liquid-liquid phase separation of soluble polymers.The polymers condense into a porous, three-dimensional network in which their end-caps reversibly bind at junctions. The spatial separation of connected junctions scales with the polymer backbone length as a self-avoiding random walk over a wide range of concentration with a weak affinity-dependent prefactor.By contrast, the average number of polymers that meet at the junctions depends strongly on the end-cap affinity but only weakly on the polymer length. The regularity and porosity of the condensed network suggests a mechanism for cells to regulate biomolecular condensates. Interaction sites along a protein may be turned on or off to modulate the condensate's porosity and tune the diffusion and interaction of additional proteins. ensemble of conformations, 14 and have little conserved sequence similarity. 15Instead of folding into a minimum-energy structure, IDPs sample many almost equi-energy conformational states in aqueous solution. 16,17 As many of these states are extended, their conformational flexibility allows them to bind to multiple proteins via distributed, multivalent, reversible binding sites even at low concentrations. 18 Their ability to explore interaction volumes whose size is comparable to the linear extent of the protein allows IDPs to phase separate from their surroundings into biomolecular condensates.A wide range of experimental techniques have revealed the structure of BCs to resemble a fluid, mesh-like network in which the constituent IDPs transiently bind to each other but are locally disordered. 19 Cryo-electron microscopy has shown that the condensed phase of FUS, an IDP associated with ALS, is amorphous, and lack internal ordered structures. 4 Solution NMR experiments of Burke et al. 20 show that FUS in its condensed phase is locally dynamic, making transient intermolecular contacts that are sufficiently short-lived that the proteins exhibit little or no secondary structure and remain disordered. At the same time, their fluores...

show abstract

Altered dynamics may drift pathological fibrillization in membraneless organelles

Cited by 17 publications

References 79 publications

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

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

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

Structure of biomolecular condensates from dissipative particle dynamics simulations

Contact Info

Product

Resources

About