Liquid–Liquid Phase Separation Modifies the Dynamic Properties of Intrinsically Disordered Proteins

Abstract

Liquid−liquid phase separation of flexible biomolecules has been identified as a ubiquitous phenomenon underlying the formation of membraneless organelles that harbor a multitude of essential cellular processes. We use nuclear magnetic resonance (NMR) spectroscopy to compare the dynamic properties of an intrinsically disordered protein (measles virus N TAIL ) in the dilute and dense phases at atomic resolution. By measuring 15 N NMR relaxation at different magnetic field strengths, we are able to characterize the dynamics of the protein in dilute and crowded conditions and to compare the amplitude and timescale of the different motional modes to those present in the membraneless organelle. Although the local backbone conformational sampling appears to be largely retained, dynamics occurring on all detectable timescales, including librational, backbone dihedral angle dynamics and segmental, chainlike motions, are considerably slowed down. Their relative amplitudes are also drastically modified, with slower, chain-like motions dominating the dynamic profile. In order to provide additional mechanistic insight, we performed extensive molecular dynamics simulations of the protein under self-crowding conditions at concentrations comparable to those found in the dense liquid phase. Simulation broadly reproduces the impact of formation of the condensed phase on both the free energy landscape and the kinetic interconversion between states. In particular, the experimentally observed reduction in the amplitude of the fastest component of backbone dynamics correlates with higher levels of intermolecular contacts or entanglement observed in simulations, reducing the conformational space available to this mode under strongly self-crowding conditions.

Viscosity Prediction of High-Concentration Antibody Solutions with Atomistic Simulations

“…Molecular simulations have been used to investigate the structure and dynamics of the crowded cellular cytoplasm, using molecular-level Brownian dynamics (BD) simulations with implicit solvation models, , coarse-grained (CG) models, and also at the fully atomistic level , (see Ostrowska et al for a recent review). All-atom and coarse-grained MD simulations have also been used to characterize biomolecular condensates, as, for example, formed by liquid–liquid phase separation. − Simulations of dense antibody solutions are typically performed with colloidal hard-sphere models − or with super-CG models in which each individual domain of the mAb is represented by a single CG bead, resulting in the representation of the entire mAb by only a dozen CG beads. − Such models are computationally cheap and thus allow one to simulate large systems with a large copy number of mAbs in the simulation box and to screen a range of different conditions in the simulations such as mAb concentration, ionic strength of the solution, etc. However, the approximations inherent to the CG models can limit their accuracy and typically require extensive parametrization and calibration against experiments and/or higher-level computations, thus hampering the predictive capacity of such CG models.…”

Label-Free Techniques for Probing Biomolecular Condensates

“…Note that NMR spectroscopy can differentiate between dynamics of atomic bonds and of the whole molecule. Reprinted with permission under a Creative Commons Attribution 4.0 International License from ref . Copyright 2023 by the authors.…”

“…Harnessing spatially resolved NMR, Pantoja et al studied the inner composition of Tau protein condensates demonstrating how the composition of water, crowding agents and protein cofactors change within the condensates . NMR also has the ability to differentiate dynamics on both the atomic bond level and molecule level (Figure C) . Additionally, there is a whole host of X-ray scattering based works that have been reported in the field …”

Toward Accurate Simulation of Coupling between Protein Secondary Structure and Phase Separation

“…A critical limitation of Cα-only or MARTINI models, however, is their inability to accurately describe the interactions and secondary structures of the peptide backbone due to the single-bead representation of the entire residue (Cα-only models) or backbone (MARTINI). Yet, it is well recognized that anisotropic backbone interactions are important in driving IDP phase separations. ,,− Critically, IDPs are not simple polymers, and transient secondary and sometimes long-range structures are prevalent. , Recent studies have highlighted the strong interplay between transient secondary structures and phase separation behaviors. − For instance, IDPs and IDRs often adopt more expanded conformations within condensates and often with an increased propensity for β-structures. − It has also suggested phase separation can induce the folding of poly-(PR) peptides into helical conformations, , which is a characteristic feature of aging-related phase transition pathologies. Furthermore, recent studies have revealed that the C-terminal domain of TAR DNA-binding protein 43 (TDP-43) undergoes transient conformational changes during phase transitions, forming helices during phase separation and subsequently aging into amyloid structures .…”