X-ray scattering experiments with high-flux X-ray source coupled rapid mixing microchannel device and their potential...

Small-angle X-ray scattering provides global, shape-sensitive structural information about macromolecules in solution. Its extension to time dimension in the form of time-resolved SAXS investigations and combination with other time-resolved biophysical methods contributes immensely to the study of protein dynamics. TR-SAXS can also provide unique information about the global structures of transient intermediates during protein dynamics. An experimental set-up with low protein consumption is essential for an extensive use of TR-SAXS experiments on protein dynamics. In this direction, a newly developed 20-microchannel microfluidic continuous-flow mixer was combined with SAXS. With this set-up, we demonstrate ubiquitin unfolding dynamics after rapid mixing with the chaotropic agent Guanidinium-HCl within milliseconds using only ∼ 40 nanoliters of the protein sample per scattering image. It is suggested that, in the future, this new TR-SAXS platform will help to increase the use of time-resolved small-angle X-ray scattering, wide-angle X-ray scattering and neutron scattering experiments for studying protein dynamics in the early millisecond regime. The potential research field for this set-up includes protein folding, protein misfolding, aggregation in amyloidogenic diseases, function of intrinsically disordered proteins and various protein-ligand interactions.