Breaking the Cage: Transformative Time-resolved Crystallography using Fixed Targets at Synchrotrons and XFELs

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X-ray crystallography has been the leading method to understand structure and mechanisms of proteins for decades. However, the structures obtained generally represent averaged or static states and cannot fully represent dynamic proteins. Obtaining time resolved, high-resolution structures of proteins as they carry out their functions is a grand challenge for life science. Time resolved x-ray crystallography has largely been limited to reversible, naturally photoactivatable e.g. rhodopsins (a tiny fraction of all proteins), with more recent developments allowing access to non-reversible processes such as enzyme reactions. We will use laser activated photocages and silicon fixed targets to initiate reactions in microcrystals of proteins of fundamental and biotechnological importance. These include enzymes relevant to biofuel production (lytic polysaccharide monooxygenases) and gas sensor proteins (cytochromes c') and cytochrome P450Nor. Our approach using time-dependent x-ray serial crystallography will allow access to a very wide range of timescales, from milliseconds to minutes, allowing on-pathway intermediates to be characterised. E.g. catalytic intermediates arising from oxygen activation by 2 mononuclear copper centres will be identified along with capturing the elusive distal intermediate/structural reorganisation associated with binding in a range of haem gas sensor proteins. Time-dependent crystallographic data will be measured with reaction progress and identity of intermediates monitored by complimentary spectroscopies providing essential validation of structures and allowing maximum biological information to be obtained. Data will be measured using microfocus synchrotron and x-ray free electron laser facilities. The results obtained will provide important time-dependent and dynamic mechanistic information. Our research program will also establish a general method, addressing the grand challenge of acquiring time resolved structures of enzymes in action