Biology

Prof. Arwen Pearson
Universitat Hamburg

One of the major aims of structural biology is to obtain a molecular level mechanistic understanding of biological processes. Ideally this means imaging the reaction or process of interest as it occurs and with near-atomic resolution. One option is the, now well established, use of mechanistic and cryo-trapping approaches that accumulate specific meta-stable intermediates in the crystal or solution long enough for them to be structurally interrogated. However, it can be difficult to find conditions to trap all intermediates in a reaction pathway, especially those that are very short-lived. For these reasons a true time-resolved experiment has long been the dream of many structural enzymologists.
However, such an experiment is not technically easy to achieve. Although 100 ps time-resolved experiments have been possible for decades on Laue beamlines and more recently fs time-resolved experiments have hit the headlines as the new ultra-bright free electron X-ray laser sources have come on line, sub-ms time-resolved structural studies have still only been carried out for a handful of proteins. The major bottle-neck is the ability to quickly and uniformly initiate the reaction throughout the sample. Our collaborative research team at the Hamburg Centre for Ultrafast Imaging is addressing this challenge in two ways. The first is the development of microfluidic rapid mixing devices. These are suitable for slower reactions that occur on the order of 100s µs - ms. For faster reactions initiation with a laser flash is required. However, current photocages are not ideal for time-resolved structural experiments for a number of reasons. We are actively developing both new photocages with improved properties and alternate caging approaches to decouple the decaging and reaction chemistries. I will present an overview of the possible approaches to reaction initiation as well as some of our recent advances. I will also present a variation on the standard pump-probe data collection scheme for time-resolved experiments that enables fast experiments to be carried out on standard monochromatic X-ray sources and with slower detectors.