Time-resolved crystallography of enzyme-catalyzed reactions

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I will focus exclusively on time-resolved crystallography (TRX) of irreversible enzyme reactions by pursuing 3 specific areas, each represented by a protein: 1) improving the time-resolution of TRX of fluoroacetate dehalogenase (FAcD), focusing on bond formation and breakage, 2) exploring new triggering mechanisms with glutathione reductase (GR), 3) investigating the photolysis of caged substrates with H-ras P21. All 3 proteins are active in crystals while preserving crystal integrity. We will use a chip that delivered many data sets in TRX experiments. Projects 1) and 2) need access to high-end synchrotron beamlines, project 3) requires an X-ray Free Electron Laser (XFEL). 18 TRX crystal structures, from 30 ms to 30 s, covering 4 full catalytic cycles of the irreversible FAcD reaction, provide the first visual proof of half-of-the-sites reactivity and reveal correlations between the catalytic steps and molecular breathing motions, the structure of a water network and small changes of the protein mold. The results also set rather narrow boundaries for when bond formation and breakage occur. We will investigate the processes of SN2 substitution and ester hydrolysis with much improved time resolution. The broad absorption spectra of flavoproteins forbid triggering reactions by photolysis of caged precursors. I propose 2 methods to start such TRX reactions: 1) use of an ultrasound-based mixing technique and 2) fast pH shifts caused by temperature (T) jumps. Changes in reaction rates based on accessible T shifts alone are too small, however, for some buffers, 20 C shifts change pH by 2 units, taking a dormant system to catalysis. T changes can both lower or increase the pH value. While compounds exist that lower pH quickly (caged protons') no caged bases' react sufficiently fast upon illumination. GR catalyzes two successive half-reactions, NADPH oxidation and glutathione reduction, which can also be stopped at intermediate states. Applying pL mixing and pH shifts, we will analyze GR's catalytic steps and correlate them to in-crystal VIS spectra and changes to the electron density maps. 3) The third sub-project is the most ambitious one, the structural investigation of the dark reaction following the photolysis of the 2-nitrobenzyl- and 1-(2-nitrophenyl)ethyl- caging groups. While the former has been used successfully in quite a number of TRX studies, e.g. attached to the ?-phosphate of GTP investigating GTP hydrolysis by H-ras P21, the exact mechanism of transformation of aci-nitro- and bicyclic intermediates to nitrosoketone and free compound are still discussed in the literature. TR-spectroscopy indicates time ranges of ?s to ms for the various steps for the dark reactions of these caging groups, making them accessible to TRX experiments. Earlier work has shown that a complex of H-ras P21 protein with caged GTP can be crystallized and the crystals will tolerate the decaging and GTP hydrolysis processes without disintegration.