A Combined Methodology for Radiation Damage Mitigation and Enzyme Intermediate Study in Macromolecular Crystals

Due to radiation damage, the majority of metalloproteins structures are incorrect. Radiation damage in X-ray crystallography manifests itself either globally or at specific radiation sensitive sites. Global damage can be monitored from data processing statistics, whereas specific damage is more clandestine and presents as structural changes within the electron density. Serial crystallography using an X-ray free-electron laser promises a pseudo zero dose structure, however, the paucity of beamlines means beamtime is highly competitive. Method development, therefore, is required to collect low-dose structures using synchrotron X-ray crystallography. One dose-reducing phenomenon is photoelectron escape, where the generated photoelectrons escape the crystal volume before depositing their energy.

This thesis conducted the first serial crystallography experiment at VMXm (Diamond Light Source, UK), where photoelectron escape is significant for the targeted microcrystal sizes. An oxidised iron intermediate in myoglobin, Compound II, was tested as FeIV-oxo “ferryl” intermediates, which are known to be particularly susceptible to radiation damage. Despite not being a formal heme peroxidase, myoglobin is an excellent model for testing dose-limiting techniques. An NADP+-specific glyceraldehyde 3’-phosphate dehydrogenase from the enteric pathogen Helicobacter pylori was also investigated. NADP+-specificity is unusual amongst GAPDHs and are therefore poised for therapeutic targets. The kinetics of GAPDHA were investigated, and amongst the first structures of an NADP+-specific GAPDH outside of photosynthetic organisms are reported. An underreported form of radiation damage was observed. Therefore, a transition to microcrystals for a prospective dose-series and time-resolved investigation was performed.

A Mix and Quench Microcrystal Reactor was developed to initiate a reaction within microcrystals with rapid mixing and to trap intermediates by quenching in liquid ethane. Current systems exist for time-resolved crystallography or time-resolved cryoEM; however, a system was developed to react and spray microcrystals onto a TEM grid for use on the specific goniometry at VMXm.