New Generation Stopped-Flow Time-Resolved BioSAXS Experiments at the P12 Beamline.

Abstract Time-resolved small-angle X-ray scattering enables studying dynamic structural changes in biological macromolecules. Developments in fast mixing devices, such as the significant reduction in sample volume requirements, now allow the technique to be applied to a wider range of biologically relevant macromolecules, often available in limited quantities. Advances in synchrotron radiation sources and detector technology have dramatically increased the time resolution, allowing real-time monitoring of reaction kinetics and folding processes at millisecond and sub-millisecond timescales. Here, we present recent updates to the P12 SAXS beamline at EMBL Hamburg, including integrating the new Micro Stopped-Flow Mixer (µSFM) generation. We demonstrated the kinetics of pH-induced conformational dynamics in two case studies of oxygen-carrying proteins, capturing rapid reorganization of the multimeric state on a millisecond time scale. Based on the experimental findings, the new system significantly improves time resolution and sample consumption, facilitating more efficient TR-SAXS experiments.