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Structural And Functional Characterization Of A Bacterial Photosensing Light-Oxygen-Voltage (LOV) Protein Domain From Rhizobium Leguminosarum


Light-Oxygen-Voltage (LOV) domains are an important family of sensors that can be found in bacteria, archaea and eukaryotes. This thesis focuses on the blue light-sensing LOV domains and their signal transduction pathways (STPs) in bacteria. Specifically, a LOV domain from Rhizobium leguminosarum (Rle) is characterized. Biophysical characterization of the protein’s cyclic light response (photocycle) and structure were addressed in order to further our understanding of this important signalling pathway.

Chapter 1 introduces the fundamental characteristics of light sensing with a specific emphasis on bacterial LOV domains. After introducing these sensors and their associated signalling pathways, Chapter 2 focuses on a LOV-histidine ki- nase (HK) from Rle. The importance of Rhizobium-plant symbiosis and previous research on the in vivo characterization of the Rh-LOV-HK STP are summarized. Then, the cloning, protein expression and purification methods that were used throughout this thesis research are described. In the results section, various optical spectroscopy experiments that were used to characterize the photocycle properties of Rh-LOV-HK and the Rh-LOV domain in isolation are presented. Experiments included time-resolved flow-flash absorption measurements to measure early changes in the photocycle, and absorption and fluorescence spectroscopy to characterize the average light-activated (adduct) state lifetime. Chapter 2 con- cludes with circular dichroism (CD) experiments, which can provide insights into the conformational changes that lead to the active signalling state.

In Chapter 3, the experimental procedures that were used to crystallize and solve the structure of Rh-LOV at 1.89 A resolution are described. Analysis of this high-resolution structure includes a close look at interactions with FMN in the active pocket, the dimerization interface and the position of the C-terminal region. For comparison, the structure of Rh-LOV is contrasted with previously crystallized bacterial LOV domain structures. These homologous LOV domains can have distinctly different temporal photocycle characteristics, which may arise from small differences in structure. Finally, we discuss the experimental procedures that were carried out with the intention of resolving the light-activated crystal structure of Rh-LOV.

In Chapter 4, the structural-functional characterization of Rh-LOV using electron paramagnetic resonance (EPR) techniques is presented. At various positions in the Rh-LOV structure, we show that changes in the continuous wave (CW) EPR spectrum of a protein-bound paramagnetic label corresponds to the localized protein environment. Using the pulsed EPR technique, double electron-electron resonance (DEER), distance probability distributions were determined between various positions in the Rh-LOV solution structure. We show that it is a dimer and compare the solution and crystal structure dimer orientations. The results from DEER experiments that were undertaken to resolve conformational dynamics of the light-activated signalling state of Rh-LOV are also discussed.

Chapter 5 summarizes the results of this research and considers what we have learned about Rh-LOV-HK and its STP. We will also return to research obstacles that were encountered in this project and propose an alternative approach for isolating full-length Rh-LOV-HK. In conclusion, we consider the obstacles in producing an accurate model of full-length Rh-LOV-HK structure and discuss the trajectory of future LOV domain studies.

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