Complex Effects of Salt on Small-Angle X-ray Scattering of BSA Originate from the Interplay of Ions and Hydration...

Extracting information on salt effects from small-angle X-ray scattering (SAXS) data of protein solutions remains challenging. Here, we combine the newly developed FMAPIq approach with explicit-solvent all-atom molecular dynamics simulations to quantitatively model the salt dependence of SAXS data for bovine serum albumin (BSA) and three other proteins. We find that buffer-background subtraction significantly contributes to this salt dependence because the buffer electron density varies with salt concentration. Furthermore, salt modulates the hydration-shell contrast, leading to a nonmonotonic dependence on salt concentration due to the complex interplay between ions and hydration water. Specifically, tightly bound ions displace hydration water, while loosely bound ions draw water into the shell, though this ability diminishes at high salt. At 1 M NaCl, the hydration-shell contrast strongly correlates with the protein surface charge density. This work provides an atomic-level framework for accurately interpreting salt-dependent protein SAXS profiles.