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 in 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 remains less understood due to the lack of hydration shell-sensitive
structural probes with high spatial resolution. We combined temperature-ramp
small-angle X-ray scattering (T-ramp SAXS) from 255
to 335 K with molecular simulations to demonstrate that the hydration
shells of the IgG-binding domain of Protein G (GB3) and the villin
headpiece are remarkably temperature-sensitive. For proteins in the
folded state, T-ramp SAXS data and explicit-solvent
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 caused not merely 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 of the protein hydration
shell, which underlies temperature-dependent processes such as cold
denaturation, thermophoresis, or biomolecular phase separation.