The nature of trehalose-protein interactions in aqueous solutions revealed by neutron scattering

Trehalose is widely recognized for stabilising proteins under conditions that promote dehydration, denaturation, or loss of function, yet its underlying mechanisms remain elusive. We examine myoglobin in trehalose solutions of two concentrations to evaluate whether trehalose forms a protective sugar shell beyond the hydration layer-a concept suggested by molecular dynamics simulations but not experimentally verified. Using neutron diffraction, we demonstrate that myoglobin remains almost fully hydrated in both systems, even at high trehalose concentrations. To probe myoglobin-trehalose interactions, we constructed two starting models: (i) trehalose molecules dispersed randomly in the solvent and (ii) a pre-assembled trehalose shell positioned adjacent to the protein surface. After refinement, both models converged with experimental data in the Q range (1-30 Å^-1), despite resulting in markedly different final configurations, although the protein was preferentially hydrated by water in both cases. However, the shell model yields substantially higher low-Q intensities (0.03-0.5 Å^-1), inconsistent with the experimental data, demonstrating that trehalose does not form a pronounced layer outside the hydration shell. Residue-resolved analysis further confirms that there are almost no direct trehalose-protein interactions for hydrophilic or hydrophobic amino acids. In addition, quasielastic neutron scattering reveals slower dynamics for all components in the concentrated system. Furthermore, the protein motions are considerably slower in the three-component systems compared to those in a trehalose-free binary solution. This finding shows the significance of the dynamically stabilising effect of trehalose on proteins in cryoprotective and pharmaceutical applications.