Real-time Structural Tracking of Slow P-type ATPase Dynamics.

Time-resolved X-ray solution scattering (TR-XSS) provides direct access to protein structural dynamics but has largely been restricted from the microsecond range up to approximately 100 milliseconds. As a result, slower enzymatic systems, including many P-type ATPases, remain difficult to probe. Here, we extend the temporal reach of TR-XSS by sequentially positioning radiation damage-free acquisition windows to enable capturing structural evolution across sub-second to second timescales. Implemented at the CoSAXS beamline at MAX IV Laboratory, this strategy enables continuous tracking of slow protein dynamics while preserving structural sensitivity. Using adenylate kinase (AdK) as a benchmark, we observed a single conformational transition accompanied by signal amplitude decay. In contrast, application to the prokaryotic P-type ATPase LMCA1 revealed clear evolution in scattering profiles, consistent with sequential conformational transitions. Kinetic analysis identified two transitions on the 140 ms and 660 ms timescales, which correspond monitoring rise and decay of a rate-limiting step which can symbolize intermediate dynamics in a slow transport cycle. The results demonstrate that extended-time TR-XSS can resolve multi-step reaction pathways in slow membrane proteins. The approach broadens the accessible timescale of TR-XSS and establishes a general framework for studying slow conformational dynamics in P-type ATPases and related systems.