Enhancing biological SAXS by solvent contrast variation: Effect on structural stability and enzymatic activity.

Small-angle x-ray scattering (SAXS) has evolved over the past decades into an essential structural biology technique for the study of conformational states of solubilized biomacromolecules and assemblies. It complements a multitude of other methods, including the atomic-resolution techniques NMR, crystallography, and cryo-EM. In the vast majority of applications, the biological systems of interest are measured by SAXS in aqueous buffers. This approach, although quick and straightforward, limits the information content of the internal topology of biomacromolecular assemblies of variable electron density, such as protein-RNA/DNA complexes, and detergent/lipid-solubilized membrane proteins. These shortcomings can be partially overcome by altering the solvent electron density by the addition of small, electron-rich molecules such as sugar, glycerol, salt, and heavy-atom compounds, so-called contrast agents. A crucial issue in these contrast-variation SAXS experiments is the question whether the conformational state and the enzymatic activity of biomolecules are maintained after long incubation times with contrast agents, which are often required at molar concentrations. Here, we present a structural SAXS study of two proteins, a bacterial endo-β-1,4-mannanase and a malate dehydrogenase, in the presence of high concentrations of sucrose and a lanthanide compound and evaluate the residual enzymatic activity as a function of the incubation time. Our results indicate that the global conformation and the oligomeric state of both proteins are preserved over the entire concentration range of both contrast agents, and that their enzymatic activities are unaltered after long incubation times (up to 24 h). Taken together, our results encourage a more routine use, as well as further developments, of contrast variation SAXS experiments for structural studies of a variety of important and challenging biomacromolecular assemblies.