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Protein dynamics are essential to biological function, and methods to determine such structural rearrangements constitute a frontier in structural biology. Synchrotron radiation can track real-time protein dynamics, but accessibility to dedicated high-flux single X-ray pulse time-resolved beamlines is scarce and protein targets amendable to such characterization are limited. These limitations can be alleviated by triggering the reaction by laser-induced activation of a caged compound and probing the structural dynamics by fast-readout detectors. In this work, we established time-resolved X-ray solution scattering (TR-XSS) at the CoSAXS beamline at the MAX IV Laboratory synchrotron. Laser-induced activation of caged ATP initiated phosphoryl transfer in the adenylate kinase (AdK) enzyme, and the reaction was monitored up to 50 ms with a 2-ms temporal resolution achieved by the detector readout. The time-resolved structural signal of the protein showed minimal radiation damage effects and excellent agreement to data collected by a single X-ray pulse approach.

Calcium (Ca²⁺) signaling is fundamental to cellular processes in both eukaryotic and prokaryotic organisms. While the mechanisms underlying eukaryotic Ca²⁺ transport are well documented, an understanding of prokaryotic transport remains nascent. LMCA1, a Ca²⁺ adenosine triphosphatase (ATPase) from Listeria monocytogenes, has emerged as a prototype for elucidating structure and dynamics in prokaryotic Ca²⁺ transport. Here, we used a multidisciplinary approach integrating kinetics, structure, and dynamics to unravel the intricacies of LMCA1 function. A cryo–electron microscopy (cryo-EM) structure of a Ca²⁺-bound E1 state showed ion coordination by Asp⁷²⁰, Asn⁷¹⁶, and Glu²⁹². Time-resolved x-ray solution scattering experiments identified phosphorylation as the rate-determining step. A cryo-EM E2P state structure exhibited remarkable similarities to a SERCA1a E2-P* state, which highlights the essential role of the unique P-A domain interface in enhancing dephosphorylation rates and reconciles earlier proposed mechanisms. Our study underscores the distinctiveness between eukaryotic and prokaryotic Ca²⁺ ATPase transport systems and positions LMCA1 as a promising drug target for developing antimicrobial strategies.

Protein molecules typically carry out their biological function by adopting multiple, transient conformations, which complicates their structural characterization. Synchrotron-based time-resolved X-ray solution scattering (TR-XSS) combined with triggering by caged compounds enables real-time monitoring of protein structural transitions in a wide range of protein targets. However, non-instantaneous release of photosensitive cages and undefined equilibrium states complicate data interpretation. In this work, we addressed these challenges with the Escherichia coli adenylate kinase (AdK) enzyme as a model system. To account for, and visualize, heterogeneity resulting from overlap between the ATP release kinetics and protein catalytic motions, we based the structural refinement on ensembles from a pool of putative target structures generated by molecular dynamics (MD) simulations. Under equilibrium conditions, protein conformations preferentially occupied intermediate states in which the ATP- and AMP-binding domains were never fully opened or closed. Upon ATP availability, ensembles successively shifted toward fully closed and open conformations accompanying partial unfolding, which is consistent with a cracking model for triggering the enzymatic reaction. The findings demonstrate that non-instantaneous substrate release can significantly impact protein transition kinetics but can be tackled with the use of ensemble-based structural refinement. Hence, this work establishes a framework for dissecting rapid protein conformational changes in solution induced by caged compounds.