Serial and time-resolved crystallography of metalloproteins

X-ray crystallography is an important biological research technique that has provided most of the structures in the Protein Data Bank. An inherent challenge of crystallography is that the X-rays which produce diffraction images also cause damage to the crystal, resulting in loss of diffraction quality as the absorbed dose increases. To minimise this effect, serial crystallography techniques have been developed to spread dose over many crystals, by collecting a small amount of data, usually only one diffraction image, from each. This is especially beneficial for metalloproteins since metal centres are particularly susceptible to site-specific radiation damage and are important to the function of many proteins. The benefits of serial crystallography include the ability to perform experiments at room temperature, where proteins are more likely to be in physiologically relevant conformations, rather than at the cryogenic temperatures usually used to limit radiation damage. Room temperature also allows increased diffusion and molecular motions, so reactions can take place within protein crystals. These can be investigated with time-resolved serial experiments, capturing structures of short-lived intermediate states of a protein undergoing a dynamic process. An important part of any time-resolved experiment is reaction initiation, including ensuring the reaction begins in the whole sample at the same time. This thesis describes the development of a method for time-resolved serial crystallography using the fixed-target chip system available on beamline I24 at Diamond Light Source, which is also applicable to serial femtosecond crystallography at X-ray free electron laser (XFEL) sources. These experiments explore the structures of highly radiation-sensitive metalloproteins, including time-resolved studies of nitric oxide binding in the haem site using a UV laser pump-probe technique and a photocaged substrate molecule.