Although many proteins possess a distinct folded structure lying at a minimum in a funneled free energy landscape, thermal energy causes any protein to continuously access lowly populated excited states. The existence of excited states is an integral part of biological function. Although transitions into the excited states may lead to protein misfolding and aggregation, little structural information is currently available for them. Here, we show how NMR spectroscopy, coupled with pressure perturbation, brings these elusive species to light. As pressure acts to favor states with lower partial molar volume, NMR follows the ensuing change in the equilibrium spectroscopically, with residue-specific resolution. For T4 lysozyme L99A, relaxation dispersion NMR was used to follow the increase in population of a previously identified “invisible” folded state with pressure, as this is driven by the reduction in cavity volume by the flipping-in of a surface aromatic group. Furthermore, multiple partly disordered excited states were detected at equilibrium using pressure-dependent H/D exchange NMR spectroscopy. Here, unfolding reduced partial molar volume by the removal of empty internal cavities and packing imperfections through subglobal and global unfolding. A close correspondence was found for the distinct pressure sensitivities of various parts of the protein and the amount of internal cavity volume that was lost in each unfolding event. The free energies and populations of excited states allowed us to determine the energetic penalty of empty internal protein cavities to be 36 cal⋅Å⁻³. Significance Proteins exist as ensembles of microstates governed by a free energy landscape, with multiple “excited” states coexisting with the minimum energy structure. These alternate folded and partially disordered states are continuously being accessed through protein dynamics and are key elements required for a comprehensive understanding of protein function and stability. Unfortunately, their low abundance makes these “invisible” states hard to characterize experimentally. A unique view of the hierarchy of unfolding states on the protein energy landscape was obtained here using pressure perturbation. Furthermore, pressure perturbation can directly identify empty protein cavities and determine the energetic penalty of filling these with water.

The RNA-binding protein fused in sarcoma (FUS) undergoes liquid–liquid phase separation (LLPS) both in vivo and in vitro. Self-assembled liquid droplets of FUS transform into reversible hydrogels and into more irreversible and toxic aggregates. Although LLPS can be a precursor of irreversible aggregates, a generic method to study kinetics of the formation of LLPS has not been developed. Here, we demonstrated the pressure-jump kinetics of phase transition between the 1-phase state and FUS-LLPS states observed at low pressure (2 kbar, HP-LLPS) using high-pressure UV/vis spectroscopy. Absorbance (turbidity) changes were reproduced repeatedly using pressure cycles. The Johnson–Mehl–Avrami–Kolmogorov theory was used to understand droplet formation occurring via nucleation and growth. The Avrami exponent n, representing the dimensionality of growing droplets, and the reaction rate constant k were calculated. The HP-LLPS formation rate was ∼2-fold slower than that of LP-LLPS. The Avrami exponent obtained for both LLPS states could be explained by diffusion-limited growth. Nucleation and growth rates decreased during LP-LLPS formation (n = 0.51), and the nucleation rate decreased with a constant growth rate in HP-LLPS formation (n = 1.4). The HP-LLPS vanishing rate was ∼20-fold slower than that of LP-LLPS. This difference in vanishing rates indicates a stronger intermolecular interaction in HP-LLPS than in LP-LLPS, which might promote transformation into irreversible aggregates in the droplets. Further, direct transition from HP-LLPS to LP-LLPS was observed. This indicates that interconversion between LP-LLPS and HP-LLPS occurs in equilibrium. Formation of reversible liquid droplets, followed by phase transition into another liquid phase, could thus be part of the physiological maturation process of FUS-LLPS.

Internal cavities are important elements in protein structure, dynamics, stability and function. Here we use NMR spectroscopy to investigate the binding of molecular oxygen (O₂) to cavities in a well-studied model for ligand binding, the L99A mutant of T4 lysozyme. On increasing the O₂ concentration to 8.9 mM, changes in ¹H, ¹⁵N and ¹³C chemical shifts and signal broadening were observed specifically for backbone amide and side chain methyl groups located around the two hydrophobic cavities of the protein. O₂-induced longitudinal relaxation enhancements for amide and methyl protons could be adequately accounted for by paramagnetic dipolar relaxation. These data provide the first experimental demonstration that O₂ binds specifically to the hydrophobic and not the hydrophilic cavities, in a protein. Molecular dynamics simulations visualized the rotational and translational motions of O₂ in the cavities, as well as the binding and egress of O₂, suggesting that the channel consisting of helices D, E, G, H and J could be the potential gateway for ligand binding to the protein. Due to strong paramagnetic relaxation effects, O₂ gas-pressure NMR measurements can detect hydrophobic cavities when populated to as little as 1% and thereby provide a general and highly sensitive method for detecting oxygen binding in proteins.