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.

The protein hydration shell is a key mediator of processes such as molecular recognition, protein folding, and proton transfer. How solvent-exposed amino acids shape the hydration shell structure is not well understood. We combine molecular dynamics simulations with explicit-solvent predictions of small-angle x-ray scattering (SAXS) curves to quantify the contributions of all 20 proteinogenic amino acids to the hydration shell of the globular GB3 domain and the intrinsically disordered protein (IDP) XAO. We focus on two quantities encoded by SAXS curves: the hydration shell effect on the radius of gyration and the electron density contrast between protein and solvent. We derive an amino acid-specific contrast score, revealing that acidic residues generate the strongest contrast with 1–1.5 excess water molecules relative to alanine, followed by cationic and polar residues. In contrast, apolar residues generate a water depletion layer. These trends are consistent across simulations with different water models. Around the XAO peptide, the hydration shell is generally far weaker compared with the globular GB3 domain, indicating unfavorable water-peptide packing at the IDP surface. The hydration shell effect on the radius of gyration of the IDP is strongly conformation-dependent. Together, the calculations show that the composition and spatial arrangement of solvent-exposed amino acids govern the hydration shell structure, with implications for a wide range of biological functions and for hydration-sensitive experimental techniques such as solution scattering.

Monkeypox virus (MPXV) is a poxvirus endemic to Central and West Africa with high epidemic potential. Poxviruses enter host cells via a conserved entry-fusion complex (EFC), which mediates viral fusion to the cell membrane. The EFC is a promising therapeutic target, but the absence of structural data has limited the development of fusion-inhibiting treatments. Here, we investigated A16/G9, a subcomplex of the EFC that controls fusion timing. Using cryo-electron microscopy, we showed how A16/G9 interacts with A56/K2, a viral fusion suppressor that prevents superinfection. Immunization with A16/G9 elicited a protective immune response in mice. Using X-ray crystallography, we characterized two neutralizing antibodies and engineered a chimeric antibody that cross-neutralizes several poxviruses more efficiently than 7D11, the most potent antibody targeting the EFC described to date. These findings highlight the potential of A16/G9 as a candidate for subunit vaccines and identify regions of the EFC as targets for antiviral development.