Molecular Motion of Viscous Solvents from Solid-State NMR.

Liquid-liquid phase separation (LLPS) is a ubiquitous phenomenon in biological systems and involves molecular motions across multiple time scales, especially in viscous, crowded environments. These dynamics include not only biomacromolecules but also small solutes, ions, and water. To establish an NMR framework for characterizing such heterogeneous dynamic regimes, we employ two homogeneous viscous model systems, glycerol and PEG400, which reproduce the slowed molecular motions characteristic of condensed biomolecular environments while avoiding the structural complexity of phase-separated systems. Using solid-state NMR relaxometry, we investigate multi-time scale dynamics in these model solvents and examine water behavior under crowded conditions. Nanosecond motions are characterized by heteronuclear Overhauser effects and 13C T1 relaxation, while microsecond dynamics are probed via T2 and T1ρ relaxation; and translational self-diffusion is quantified using diffusion-ordered spectroscopy (DOSY). Glycerol and PEG400 exhibit similar fast nanosecond rotational motions (∼ 0.7-1.0 ns), yet their distinct viscosities are associated with differences in slower rotational dynamics and translational self-diffusion. Water exhibits constrained nanosecond motion in both systems, indicating partially immobilized populations shaped by variations in free volume. This work establishes an NMR-based framework for resolving multi-time scale dynamics in viscous systems, providing insights into LLPS and hydration behavior.