Crowding by Poly(ethylene glycol) Destabilizes Chemotaxis Protein Y (CheY).

The prevailing understanding of various aspects of biochemical processes, including folding, stability, intermolecular interactions, and the binding of metals, substrates, and inhibitors, is derived from studies carried out under dilute and homogeneous conditions devoid of a crowding-related environment. The effect of crowding-induced modulation on the structure and stability of native and magnesium-dependent Chemotaxis Y (CheY), a bacterial signaling protein, was probed in the presence and absence of poly(ethylene glycol) (PEG). A combined analysis from circular dichroism, intrinsic and extrinsic fluorescence, and tryptophan fluorescence lifetime changes indicates that PEG perturbs the structure but leaves the thermal stability largely unchanged. Intriguingly, while the stability of the protein is enhanced in the presence of magnesium under dilute buffer conditions, PEG-induced crowding leads to reduced thermal stability in the presence of magnesium. Nuclear magnetic resonance (NMR) chemical shift perturbations and resonance broadening for a subset of residues indicate that PEG interacts specifically with a subset of hydrophilic and hydrophobic residues found predominantly in α helices, ß strands, and in the vicinity of the metal-binding region. Thus, PEG prompted conformational perturbation, presumably provides a different situation for magnesium interaction, thereby perturbing the magnesium-prompted stability. In summary, our results highlight the dominance of enthalpic contributions between PEG and CheY via both hydrophilic and hydrophobic interactions, which can subtly affect the conformation, modulating the metal-protein interaction and stability, implying that in the context of cellular situation, structure, stability, and magnesium binding thermodynamics of CheY may be different from those measured in dilute solution.

Residue-specific identification of phase separation hot spots of Alzheimer's-related protein tau.

Liquid-liquid phase separation (LLPS) of proteins enables the formation of non-membrane-bound organelles in cells and is associated with cancer and neurodegeneration. Little is known however about the structure and dynamics of proteins in LLPS conditions, because of the polymorphic nature of liquid-like protein droplets. Using carbon-detected NMR experiments we here show that the conversion of the aggregation-prone repeat region of the Alzheimer's-related protein tau from the dispersed monomeric state to phase-separated liquid-like droplets involves tau's aggregation-prone hexapeptides and regulatory KXGS motifs. Droplet dissolution in presence of 1,6-hexanediol revealed that chemical shift perturbations in the hexapeptide motifs are temperature driven, while those in KXGS motifs report on phase separation. Residue-specific secondary structure analysis further indicated that tau's repeat region exists in extended conformation in the dispersed state and attains transient ß-hairpin propensity upon LLPS. Taken together our work shows that NMR spectroscopy can provide high-resolution insights into LLPS-induced changes in intrinsically disordered proteins.

Simultaneous determination of fast and slow dynamics in molecules using extreme CPMG relaxation dispersion experiments.

Molecular dynamics play a significant role in how molecules perform their function. A critical method that provides information on dynamics, at the atomic level, is NMR-based relaxation dispersion (RD) experiments. RD experiments have been utilized for understanding multiple biological processes occurring at micro-to-millisecond time, such as enzyme catalysis, molecular recognition, ligand binding and protein folding. Here, we applied the recently developed high-power RD concept to the Carr-Purcell-Meiboom-Gill sequence (extreme CPMG; E-CPMG) for the simultaneous detection of fast and slow dynamics. Using a fast folding protein, gpW, we have shown that previously inaccessible kinetics can be accessed with the improved precision and efficiency of the measurement by using this experiment.