abstract: Photosystem II (PSII) is a large protein-cofactor complex. The first step in photosynthesis involves the harvesting of light energy from the sun by the antenna (made of pigments) of the PSII trans-membrane complex. The harvested excitation energy is transferred from the antenna complex to the reaction center of the PSII, which leads to a light-driven charge separation event, from water to plastoquinone. This phenomenal process has been producing the oxygen that maintains the oxygenic environment of our planet for the past 2.5 billion years. The oxygen molecule formation involves the light-driven extraction of 4 electrons and protons from two water molecules through a multistep reaction, in which the Oxygen Evolving Center (OEC) of PSII cycles through 5 different oxidation states, S0 to S4. Unraveling the water-splitting mechanism remains as a grant challenge in the field of photosynthesis research. This requires the development of an entirely new capability, the ability to produce molecular movies. This dissertation advances a novel technique, Serial Femtosecond X-ray crystallography (SFX), into a new realm whereby such time-resolved molecular movies may be attained. The ultimate goal is to make a “molecular movie” that reveals the dynamics of the water splitting mechanism using time-resolved SFX (TRSFX) experiments and the uniquely enabling features of X-ray Free-Electron Laser (XFEL) for the study of biological processes. This thesis presents the development of SFX techniques, including development of new methods to analyze millions of diffraction patterns (~100 terabytes of data per XFEL experiment) with the goal of solving the X-ray structures in different transition states. ii The research comprises significant advancements to XFEL software packages (e.g., Cheetah and CrystFEL). Initially these programs could evaluate only 8-10% of all the data acquired successfully. This research demonstrates that with manual optimizations, the evaluation success rate was enhanced to 40-50%. These improvements have enabled TR-SFX, for the first time, to examine the double excited state (S3) of PSII at 5.5-Å. This breakthrough demonstrated the first indication of conformational changes between the ground (S1) and the double-excited (S3) states, a result fully consistent with theoretical predictions. The power of the TR-SFX technique was further demonstrated with proof-of principle experiments on Photoactive Yellow Protein (PYP) micro-crystals that high temporal (10-ns) and spatial (1.5-Å) resolution structures could be achieved. In summary, this dissertation research heralds the development of the TR-SFX technique, protocols, and associated data analysis methods that will usher into practice a new era in structural biology for the recording of ‘molecular movies’ of any biomolecular process. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

abstract: This thesis focuses on serial crystallography studies with X-ray free electron lasers (XFEL) with a special emphasis on data analysis to investigate important processes in bioenergy conversion and medicinal applications. First, the work on photosynthesis focuses on time-resolved femtosecond crystallography studies of Photosystem II (PSII). The structural-dynamic studies of the water splitting reaction centering on PSII is a current hot topic of interest in the field, the goal of which is to capture snapshots of the structural changes during the Kok cycle. This thesis presents results from time-resolved serial femtosecond (fs) crystallography experiments (TR-SFX) where data sets are collected at room temperature from a stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an XFEL with the goal to obtain structural information from the transient state (S4) state of the cycle where the O=O bond is formed, and oxygen is released. The most current techniques available in SFX/TR-SFX to handle hundreds of millions of raw diffraction patterns are discussed, including selection of the best diffraction patterns, allowing for their indexing and further data processing. The results include two 4.0 Å resolution structures of the ground S1 state and triple excited S4 transient state. Second, this thesis reports on the first international XFEL user experiments in South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this new XFEL in a proof-of-principle experiment for the study of microcrystals of human taspase1 (an important cancer target) by SFX has been tested. The descriptions of experiments and discussions of specific data evaluation challenges of this project in light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å, are included in this report In summary, this thesis examines current techniques that are available in the SFX/TR-SFX domain to study crystal structures from microcrystals damage-free, with the future potential of making movies of biological processes. / Dissertation/Thesis / Masters Thesis Chemistry 2020

<p>HMG-CoA reductase from Pseudomonas mevalonii (<i>Pm</i>HMGR) catalyzes the oxidation of mevalonate and mevaldyl-CoA to form HMG-CoA using CoA-SH and two NAD+ cofactors. While the enzyme has been used extensively as a drug target in humans to treat hypercholesterolemia, its pathway has also been found to be critical for the survival of antibiotic resistant gram-positive bacteria. Structural studies using non-productive and slow-substrate binary complexes as well as biochemical studies using half and full reactions led to the proposal that the conversion of mevalonate to HMG-CoA occurs through the generation of two intermediates, mevaldehyde and mevaldyl-CoA (Shown in Fig 1.1). However, several intermediary changes along the <i>Pm</i>HMGR reaction pathway remain unclear. By gathering information about the enzyme’s intermediate states via structural studies, we could identify potential allosteric sites that further the reaction mechanism. Using this knowledge, we could design enzyme inhibitors that act as novel antibacterials. The application of time-resolved crystallographic methods would provide structural information about transitory states in the PmHMGR reaction mechanism. The <i>Pm</i>HMGR crystal has been shown to be suitable for time-resolved crystallographic measurements for the reaction steps resulting in mevaldyl-CoA formation. However, our structural investigations of the mevalonate, CoA and NAD+ complex that are expected to result in the formation of mevaldehyde (Fig 1.1) do not show any changes corresponding to a turnover in the crystal environment. <br></p><p><br></p><p>To investigate the factors limiting enzymatic activity in the crystal, we investigated the effects of pH and specific ions in the crystallization environment. Kinetic studies indicated a strong <i>Pm</i>HMHGR inhibition in the crystallization buffer that is dependent on the concentration of the crystallization precipitant ammonium sulfate. These studies also indicated an increase in enzyme turnover with increasing pH. Utilizing the ionic concentration and pH-dependent properties of the enzyme in the crystallization environment, we have developed a reaction triggering approach using pH changes for <i>Pm</i>HMGR crystals.<br></p><p><br></p><p>We have demonstrated our application of this ‘pH-jump’ method by observing changes in <i>Pm</i>HMGR crystals after reaction initiation. Changes in the density of mevalonate, CoA and NAD+have indicated mevaldehyde and mevaldyl-CoA formation. Additionally, the appearance of a unique NADH absorbance peak after the pH-change has also highlighted the initiation of the <i>Pm</i>HMGR reaction and the occurrence of a hydride transfer step. Our analysis of the movements using time-resolved structures post reaction-initiation have also highlighted structural changes and inter-domain contacts in the small and flap domain that would allow cofactor exchange and product release. The pH-jump method can hence be utilized as a novel approach for triggering the <i>Pm</i>HMGR reaction in crystals and further studying transitory states along its reaction pathway.<br></p>