Visualizing structural dynamics: from small molecules to membrane proteins

To fully understand the mechanism of a chemical reaction, it is important to characterize the rapid structural dynamics of transient chemical species as the reaction propagates. Since chemical processes are heavily influenced by the solvent in which they occur, the interaction with the surrounding solution also needs to be elucidated. The emerging technique of time-resolved X-ray scattering provides a way to directly visualize these structural dynamics in solution. The chemical reactions studied in this work were all initiated by means of photo-activation. Before mapping the structural events of a propagating chemical reaction in solution, the structural dynamics of the solvent itself was characterized. Indeed, the thermal expansion of neat CH2Cl2 was captured at high time resolution and showed excellent agreement with the theoretical framework. Solvent-dependent effects were assessed for a transient structural intermediate, specifically the structural isomer CH2I-I. Following the photo-dissociation of CH2I2, the transient intermediate was shown to display slightly different geometries in non-polar and polar solvents. The lifetime of this transient species was also shown to be strongly dependent on the polarity of the solvent. Moreover, this study proved the methodology of time-resolved X-ray scattering to be powerful enough for resolving subtle solvent-dependent structural perturbations. To extend the technique of time-resolved X-ray scattering from visualizing simple structural dynamics in solution to incorporate complex biological systems, a virtual experiment was performed by means of molecular dynamics simulations. It was shown that artificially introduced heavy atoms could aid the visualisation of local structural rearrangements of the halide ion pump, halorhodopsin. Furthermore, as the large conformational changes of the protein could, in principle, be resolved, the relative timing of substrate translocation could be measured in such an experiment. Finally, the structural dynamics of two membrane proton pumps, bacteriorhodopsin and proteorhodopsin, were recorded using time-resolved X-ray scattering. It was found that significant α-helical rearrangements occurred already within 2 μs and that deprotonation of the Schiff base in bacteriorhodopsin preceded the transition to the late structural changes, characterized by significantly larger movements. Instead of being divided in an extracellular and a cytoplasmic movement, the structural rearrangements throughout the bacteriorhodopsin photocycle proved to follow the same basic motion. Moreover, the photocycle of proteorhodopsin was shown to be dynamically related to that of bacteriorhodopsin