A frontier challenge for many structural biologists is to determine time-resolved crystal structures directly from systems engaged in catalysis at physiological temperature and pressure. The goal is to ‘unambiguously’ describe as many steps as possible around a proposed reaction cycle, including those of reactive intermediates. Our deepest understanding of function is most often obtained through a multidisciplinary research strategy applied to samples at physiological temperatures and/or conditions.
This philosophy leads to a desire to collect structural, spectroscopic, and if possible, kinetic data too, from the same crystalline samples. However, a set of general tools to fully correlate enzyme kinetics with time-resolved crystallography are not yet readily available. To address this grand challenge, we are working to extended enzyme kinetics strategies to crystalline samples, and to couple these methods to room-temperature, time-resolved, serial crystallographic methods with correlations to single-crystal spectroscopy.
Serial (femtosecond) crystallography (SFX) is a rapidly developing field with the potential to produce time-resolved, atomic resolution movies of macromolecules engaged in catalysis. It uses a large number of several µm3 or smaller crystals, from each of which only a small fraction of data is collected, which is then merged together. Our guiding hypothesis is that many enzyme microcrystals (on the order 2 μm3 and smaller) will equilibrate with substrate(s) many times faster than their overall reaction cycle. We will use several µm3-sized crystals at synchrotrons to probe time domains in the ~ms and longer regime. X-ray free electron lasers (XFEL) sources enable us to collect diffraction and spectroscopic data simultaneously from the same sample, on the fs and longer time-scale, and with smaller crystals.
The project will include the design and testing of rapid mixing jets, acoustic conveyor belt, and/or microfluidics systems incorporated with microspectroscopy and particle traps (optical and/or acoustic) to assay enzyme microcrystals and compare the results with enzyme in solution. Slurries of well-characterized micro-crystals may include protocatechuate 3,4-dioxygenase, isopenicillin N synthase, 2-oxoglutarate-dependent oxygneases, and several clinically relevant b-lactamases which are drug targets. Assays will include spectroscopic methods that follow concentration changes of substrate(s) or product(s), or signals associated with catalytic metal centre(s). These results will serve as the basis for serial time-resolved experiments conducted at Diamond Light Source and/or one or more XFEL facility located in either the USA, Japan, Germany, Korea, or Switzerland.
This studentship will be linked with the EPSRC and MRC Systems Approaches to Biomedical Science, Centre for Doctoral Training (SABS-CDT
Applications to this studentship will open in early 2018.
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