Umeå University's logo

Time-resolved X-ray solution scattering (TR-XSS) is a synchrotron-based methodology that enables real-time structural characterization under near-native conditions to provide insight into dynamic and transient structural changes inaccessible to static high-resolution methods such as cryo-electron microscopy (cryo-EM) or X-ray crystallography. Here, we present a workflow for light-triggered TR-XSS experiments that spans data collection, data processing, kinetic analysis, and structural refinement, with accompanying Python scripts. A calcium-transporting P-type ATPase membrane protein (LMCA1) is used as an illustrative example, but the protocol is broadly applicable to diverse protein systems. This workflow offers a practical framework for collecting TR-XSS synchrotron data and subsequent data analysis and interpretation.

Proteins are dynamic molecules whose function depends on structural changes that occur over a broad range of timescales. Protein motions can be linked to important functionalities such as ligand binding, catalysis, transport and regulation. To understand such processes, it is necessary to go beyond determination of static structures and follow protein conformational changes in time. The work presented in this thesis focuses on ATP-dependent protein dynamics in solution using time-resolved X-ray solution scattering (TR-XSS), combined with molecular dynamics-based structural refinement and ensemble analysis. 

A detector readout-based TR-XSS setup was established using adenylate kinase (AdK) as a model system (Paper I). AdK is a soluble protein that carries out the interconversion of ATP, AMP and ADP, helping the cell balance adenine nucleotide levels. Using laser-induced release of caged-ATP, we collected time-resolved scattering data at a general-purpose synchrotron beamline, and the radiation damage, and data quality was evaluated. Although the temporal resolution was lower than what can be achieved at dedicated time-resolved beamlines, the setup enabled detection of structural changes on the millisecond timescale and provided a promising and accessible workflow for TR-XSS measurements.

Then we proceeded to investigate conformational heterogeneity and ATP-induced domain motions in AdK (Paper III). Ensemble based refinement was applied to time-resolved difference scattering data, showing that AdK exists as a heterogeneous conformational ensemble in solution. This ensemble shifts towards catalytically active conformations after ATP release. In later work (Paper IV), an application of an improved ATP releasing strategy, combined with TR-XSS and metadynamics-derived structure pools enabled us to resolve conformational changes in the microsecond range. These results showed that the substrate binding domains of AdK do not close simultaneously but follow a defined sequence of events and reach the closed state of the enzyme on a sub-millisecond timescale.

The final part of the work focused on a bacterial Ca²⁺ ATPase (LMCA1). We used TR-XSS combined with targeted molecular dynamics to investigate ATP-dependent structural changes in LMCA1 (Paper II). The results identified that phosphorylation is the rate-limiting step of the Ca²⁺ transport cycle.

Overall, this thesis demonstrates that TR-XSS, when combined with simulation-based structural refinement, is a powerful methodology for the study of structural dynamics in solution and makes a contribution to the characterization of ATP-driven protein function.

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.

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 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.

CoSAXS is a state-of-the-art SAXS/WAXS beamline exploiting the high brilliance of the MAX IV 3 GeV synchrotron. By coupling advances in sample environment control with fast X-ray detectors, millisecond time-resolved scattering methods can follow structural dynamics of proteins in solution. In the present work, four sample environments are discussed. A sample environment for combined SAXS with UV–vis and fluorescence spectroscopy (SUrF) enables a comprehensive understanding of the time evolution of conformation in a model protein upon acid-driven denaturation. The use of microfluidic chips with SAXS allows the mapping of concentration with very small sample volumes. For highly reproducible sequences of mixing of components, it is possible using stopped-flow and SAXS to access the initial effects of mixing at 2 millisecond timescales with good signal to noise to allow structural interpretation. The intermediate structures in a protein are explored under light and temperature perturbations by using lasers to "pump" the protein and SAXS as the "probe". The methods described demonstrate that features at low q, corresponding to cooperative motions of the atoms in a protein, could be extracted at millisecond timescales, which results from CoSAXS being a highly-stable, low background, dedicated SAXS beamline.

One of the fundamental aspects of chemistry learning is to visualize chemical structures. Through the application of Alex Johnstone's (1991) multilevel thought, the submicroscopic level is often a challenge for students, especially the shift between 2D and 3D, i.e., spatial thinking or spatial ability (Harle & Towns, 2011). With small molecules, plastic ball-and-stick models are commonly used, but on university level, the structures are often larger. By applying digital tools and techniques, as Virtual Reality (VR), there are less limitations in size to represent molecules, and even large structures and reaction mechanisms can be explored (Won et al., 2019). In a five-year design-based research project (Anderson & Shattuck, 2012), a collaboration between university chemistry teachers and a chemistry education researcher, has had an aim to develop university chemistry students' spatial thinking.

Students and teachers have, in workshops and tutorials, applied VR with both simple and more advanced tools, see figures 1 and 2. Empirical data has been collected using surveys, interviews, and observations. Standard ethical considerations have been considered throughout the whole project.

In this presentation, students' cognitive and affective learning related to spatial thinking will be discussed, as well as students', teachers', and researcher’s perspectives from the application of VR to visualize chemistry will be elaborated further. Implications for chemistry teaching at all levels will also be explored.