Bio-SAXS of single-stranded DNA-binding proteins: radiation protection by the compatible solute ectoine

Biological small-angle X-ray scattering (bio-SAXS) is a versatile experimental technique to obtain structural information of biomolecules in solution. An inherent problem for bio-SAXS is radiation-induced damage to the analyte. Therefore, radiation damage is often the main limiting factor when it comes to maximum exposure time.

In this study, a new approach to extend the possible X-ray exposure time of the model protein Gene-V Protein (G5P) was investigated. For this purpose, the scavenger and compatible solute ectoine was utilized during bio-SAXS measurements of the DNA-binding protein. Ectoine was used to protect the G5P protein from radicals generated during radiation exposure and therefore allowing a longer radiation time. The radiation-induced changes in G5P were monitored during the bio-SAXS measurements and the resulting energy-damage relation was determined from microdosimetric calculations using Monte Carlo-based particle scattering simulations. In the presence of ectoine, a threefold increase in energy deposition was required to achieve the same damage level as in pure G5P solutions. This indicates that ectoine increases the potential exposure time by interacting with reactive species in aqueous solution before radiation damage to G5P is observed. In addition, the dominant mode of damage shifted from G5P aggregation in absence of ectoine towards G5P fragmentation for solutions containing ectoine as a cosolute. This could be related to the preferential exclusion of the cosolute from the protein surface.

Therefore, it was shown that ectoine offers a possibility to improve the structure determination of proteins via bio-SAXS in future studies and thus can be used as a radiation protection of DNA-binding proteins.