Anfinrud, P. | bioRxiv Science

bioRxiv2025

Depletion of the protein hydration shell with increasing temperature observed by small-angle X-ray scattering and molecular simulations

The hydration shell is an integral part of proteins since it plays key roles for conformational transitions, molecular recognition, and enzymatic activity. While the dynamics of the hydration shell have been described by spectroscopic techniques, the structure of the hydration shell remain less understood due to the lack of hydration shell-sensitive structural probes with high spatial resolution. We combined temperature-ramp smallangle X-ray scattering (T -ramp SAXS) from 255-335 K with molecular simulations to show that the hydration shells of the GB3 domain and villin headpiece are remarkably temperature-sensitive. For proteins in the folded state, T -ramp SAXS data and explicitsolvent SAXS predictions consistently demonstrate decays of protein contrasts and radii of gyration with increasing temperature, which are shown to reflect predominantly temperature-sensitive depleting hydration shells. The depletion is not merely caused by enhanced disorder within the hydration shells but also by partial displacements of surface-coordinated water molecules. Together, T -ramp SAXS and explicit-solvent SAXS calculations provide a novel structural view on the protein hydration shell, which underlies temperature-dependent processes such as cold denaturation, thermophoresis, or biomolecular phase separation.

biophysics↗

bioRxiv2018

Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme

Correlated motions of proteins and their bound solvent molecules are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved methods hold promise for addressing this challenge but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature-jumps (T-jumps), through thermal excitation of the solvent, have been implemented to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform T-jump small- and wide-angle X-ray scattering (SAXS/WAXS) measurements on a dynamic enzyme, cyclophilin A (CypA), demonstrating that these experiments are able to capture functional intramolecular protein dynamics. We show that CypA displays rich dynamics following a T-jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes in the enzyme. Two relaxation processes are resolved, which can be characterized by Arrhenius behavior. We also used mutations that have distinct functional effects to disentangle the relationship of the observed relaxation processes. A fast process is related to surface loop motions important for substrate specificity, whereas a slower process is related to motions in the core of the protein that are critical for catalytic turnover. These results demonstrate the power of time-resolved X-ray scattering experiments for characterizing protein and solvent dynamics on the s-ms timescale. We expect the T-jump methodology presented here will be useful for understanding kinetic correlations between local conformational changes of proteins and their bound solvent molecules, which are poorly explained by the results of traditional, static measurements of molecular structure.

biophysics↗