Intrinsically disordered proteins are ubiquitous throughout all known proteomes, playing essential roles in all aspects of cellular and extracellular biochemistry. To understand their function, it is necessary to determine their structural and dynamic behavior and to describe the physical chemistry of their interaction trajectories. Nuclear magnetic resonance is perfectly adapted to this task, providing ensemble averaged structural and dynamic parameters that report on each assigned resonance in the molecule, unveiling otherwise inaccessible insight into the reaction kinetics and thermodynamics that are essential for function. In this review, we describe recent applications of NMR-based approaches to understanding the conformational energy landscape, the nature and time scales of local and long-range dynamics and how they depend on the environment, even in the cell. Finally, we illustrate the ability of NMR to uncover the mechanistic basis of functional disordered molecular assemblies that are important for human health.

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.