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
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.
Interactions and subsequent structural organization of fibronectin (FibN) (major component of an extracellular matrix) molecules in contact with chemically modified surfaces form an essential basis to the understanding of the regulation of matrix assembly in pathological states and to the development of new biomedical devices. Here, using poly-L-lysine (PLL)-coated liquid crystal (LC) droplets, we present a simple but fruitful advance of an experimental system that can report label-free imaging of adsorption of FibN at the LC−aqueous interface. We observed that the interfacial intermolecular interactions of anionic FibN molecules and cationic PLL residues is accompanied by a director configuration transition of the LC droplets from radial to bipolar/preradial, giving a distinct optical output. Specifically, it is found that the ordering transitions of LC can be effectively tuned by blocking the anionic sites of FibN with divalent cations (Ca 2+ ). Combination of fluorescence measurements, circular dichroism, and atomic force microscopy further revealed that PLL induces significant conformational changes in FibN at the aqueous−LC interface. This report provides a simple method based on LC droplets to understand polymer−protein interactions at membrane interfaces which would have potential applications in biomedical and interfacial systems.
significantly faster in vivo than in vitro, so the passenger domain likely folds along a faster, vectorial pathway as its being secreted through the outer membrane. Experimental results show that the relative stability at the N-and C-terminus of the passenger domain largely affects the secretion rate of these proteins, suggesting that the folding of the b-helix structure plays an important role in efficient secretion. In this study, we used three different simulation methods to investigate the folding mechanism and kinetics of the passenger domain of Pertactin, an autotransporter from Bordetella pertussis. Multidimensional replica-exchange umbrella sampling simulations reveal how cooperative folding beginning at the C-terminus enhances the kinetics of folding, while steered molecular dynamics simulations show differences in mechanical responses at the N-and C-terminus. Lastly, we use Markov state modelling to find intermediate folding states as well as possible misfolded structures, which may act as kinetic traps.
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