Addressing Structural Heterogeneity in Real-time Tracking of Protein Dynamics Triggered by Caged Compounds

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.