An ancient endosymbiosis involving early eukaryotic cells and cyanobacteriaresulted in the modern chloroplast organelle of the Archaeplastida clade, including all land plants. Throughout chloroplast evolution, many genes from the chloroplast genome were transferred to the nuclear genome, including the genetic pathway for synthesis of the bacterial polymer peptidoglycan (PG). In the ~1 billion years since the acquisition of the chloroplast, seed plants can now be grouped into species that retain ten PG genes (“full-PG”) or only four of the ten (“4-PG”). In Arabidopsis thaliana, a 4-PG flowering plant, knockout mutants for the PG gene murE show a proplastid phenotype, where chloroplast development is completely halted. In this report, A. thaliana mutants for the remaining three PG genes (mraY, murG, and ddl) were phenotyped to test for their functions. Two T-DNA insertion lines for each gene were selected from the Arabidopsis Biological Resource Center, and either confirmed as homozygotes or selfed to produce homozygous lines. These lines were then grown for at least two generations to confirm homozygous status and the location of the T-DNA insertion was identified using Sanger sequencing. All mutant lines had an insertion in either an exon or 5’ UTR of target genes. All lines were then grown on soil in conjunction with wild type plants for comparison of germination, morphology, and transcriptomics. No differences were observed in time to germination or morphology compared to the wild type control. Transcriptomics showed that target PG genes were differentially expressed in each line, but usually at a non-significant value. Genes related to flowering and reproduction were significantly differentially expressed in the mraY lines. Protoplasts were produced to image chloroplasts and identify any morphological or developmental effects. Chloroplast morphology was comparable to wild type; additionally, measurements of photosynthetic efficiency were not found to be significantly different than wild type. Together these data show that A. thaliana mutants for three PG genes do not display a phenotype like those for mutants of the fourth gene, murE. The results suggest that PG genes in A. thaliana are no longer linked to PG production and have diverged from the ancestral bacterial pathway.

The thylakoid membranes of oxygenic photosynthetic organisms contain four large membrane complexes vital for photosynthesis: photosystem II and photosystem I (PSII and PSI, respectively), the cytochrome b6f complex and ATP synthase. Two of these complexes, PSII and PSI, utilize solar energy to carry out the primary reaction of photosynthesis, light induced charge separation. In vivo, both photosystems associate with multiple antennae to increase their light absorption cross section. The antennae, Iron Stress Induced A (IsiA), is expressed in cyanobacteria as part of general stress response and forms a ring system around PSI. IsiA is a member of a large and relatively unexplored antennae family prevalent in cyanobacteria. The structure of the PSI-IsiA super-complex from the cyanobacteria Synechocystis sp. PCC 6803 was resolved to high resolution, revealing how IsiA interacts with PSI as well as the chlorophyll organization within this antennae system. Despite these structural insights, the basis for the binding between 18 IsiA subits and PSI is not fully resolved. Several IsiA mutants were constructed using insights from the atomic structure of PSI-IsiA, revealing the role of the C-terminus of IsiA in its interaction with PSI.

The complex network of the immune system defends the human body against infection, providing protection from pathogens. This work aims to improve preparation and structural knowledge of two proteins on opposite sides of the immune system spectrum. The first protein, secreted autotransporter toxin (Sat) is a class I serine protease autotransporter of Enterobacteriaceae (SPATE) that has cytotoxic and immunomodulatory effects on the host. Previous studies on Sat show its ability to aid in bacterial colonization and evasion of the immune system. This work improves the stability of Sat by making mutations to the active serine protease motif (GDSGS) while inhibiting remaining activity with reversible and irreversible serine protease inhibitors. Characterization of Sat by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and size-exclusion chromatography led to the first structural studies of Sat by x-ray crystallography and cryo-EM. Human leukocyte antigen class I proteins play an important role in the adaptive immune system by presenting endogenous viral peptides at the cell surface for CD8+ T cell recognition. In vitro production of HLA-I proteins is a difficult task without endoplasmic reticulum chaperones as present in vivo. Disulfide bond formation, folded light chain and a peptide bound are all key to refolding the HLA-I heavy chain for complex formation. The work presented in this dissertation represents systematic studies aimed at improving the production of HLA-I proteins in vitro in bacterial expression systems. Optimization of every step of the preparation was investigated providing higher expression yields, quality of inclusion bodies, and refolding improvements. With further improvements in the future, this work forms the basis for a more efficient small and large-scale production of HLA-I molecules for functional and structural studies.

The growing global energy demand coupled with the need for a low-carbon economy requires innovative solutions. Microalgal oxygenic photosynthesis provides a sustainable platform for efficient capture of sunlight and storage of some of the energy in the form of reduced carbon derivatives. Under certain conditions, the photosynthetic reductant can be shunted to molecular hydrogen production, yet the efficiency and longevity of such processes are insufficient. In this work, re-engineering of the heterodimeric type I reaction center, also known as photosystem I (PSI), in the green microalga Chlamydomonas reinhardtii was shown to dramatically change algal metabolism and improve photobiological hydrogen production in vivo. First, an internal fusion of the small PsaC subunit of PSI harboring the terminal photosynthetic electron transport chain cofactors with the endogenous algal hydrogenase 2 (HydA2) was demonstrated to assemble on the PSI core in vivo, albeit at ~15% the level of normal PSI accumulation, and make molecular hydrogen from water oxidation. Second, the more physiologically active algal endogenous hydrogenase 1 (HydA1) was fused to PsaC in a similar fashion, resulting in improved levels of accumulation (~75%). Both algal hydrogenases chimeras remained extremely oxygen sensitive and benefited from oxygen removal methods. On the example of PSI-HydA1 chimera, it was demonstrated that the active site of hydrogenase can be reactivated in vivo after complete inactivation by oxygen without the need for new polypeptide synthesis. Third, the hydrogenase domain of Megasphaera elsdenii bacterial hydrogenase (MeHydA) was also fused with psaC, resulting in expression of a PSI-hydrogenase chimera at ~25% the normal level. The heterologous hydrogenase chimera could be activated with the algal maturation system, despite only 32 % sequence identity (43 % similarity). All constructs demonstrated diminished ability to reduce PSI electron acceptors (ferredoxin and flavodoxin) in vitro and indirect evidence indicated that this was true in vivo as well. Finally, chimeric design considerations are discussed in light of the models generated by Alphafold2 and how could they be used to further optimize stability of the PSI-hydrogenase chimeric complexes.

First evolving in cyanobacteria, the light reactions of oxygenic photosynthesis are carried out by the membrane proteins, photosystem II and photosystem I, located in the thylakoid membrane. Both utilize light captured by their core antenna systems to catalyze a charge separation event at their respective reaction centers and energizes electrons to be transferred energetically uphill, eventually to be stored as a high energy chemical bond. These protein complexes are highly conserved throughout different photosynthetic lineages and understanding the variations across species is vital for a complete understanding of how photosynthetic organisms can adapt to vastly different environmental conditions. Most knowledge about photosynthesis comes from only a handful of model organisms grown under laboratory conditions. Studying model organisms has facilitated major breakthroughs in understanding photosynthesis, however, due to the vast global diversity of environments where photosynthetic organisms are found, certain aspects of this process may be overlooked or missed by focusing on a select group of organisms optimized for studying in laboratory conditions. This dissertation describes the isolation of a new extremophile cyanobacteria, Cyanobacterium aponinum 0216, from the Arizona Sonoran Desert and its innate ability to grow in light intensities that exceed other model organisms. A structure guided approach was taken to investigate how the structure of photosystem I can influence the spectroscopic properties of chlorophylls, with a particular focus on long wavelength chlorophylls, in an attempt to uncover if photosystem I is responsible for high light tolerance in Cyanobacterium aponinum 0216. To accomplish this, the structure of photosystem I was solved by cryogenic electron microscopy to 2.7-anstrom resolution. By comparing the structure and protein sequences of Cyanobacterium aponinum to other model organisms, specific variations were identified and explored by constructing chimeric PSIs in the model organism Synechocystis sp. PCC 6803 to determine the effects that each specific variation causes. The results of this dissertation describe how the protein structure and composition affect the spectroscopic properties of chlorophyll molecules and the oligomeric structure of photosystem I, possibly providing an evolutionary advantage in the high light conditions observed in the Arizona Sonoran Desert.

The understanding of protein functions in vivo is very important since the protein is the building block of a cell. Cryogenic electron microscopy (cryo-EM) is capable of visualizing protein samples in their near-native states in high-resolution details. Cryo-EM enables the visualization of biomolecular structures at multiscale ranging from a cellular structure to an atomic structure of protein subunit.Neurodegenerative diseases, like Alzheimer’s disease and frontotemporal dementia, have multiple dysregulated signaling pathways. In my doctoral studies, I investigated two protein complexes relevant to these disorders: one is the proNGF- p75 neurotrophin receptor (p75NTR)- sortilin neurotrophin complex and the other is the p97R155H mutant complex. The neurotrophins are a family of soluble basic growth factors involved in the development, maintenance, and proliferation of neurons in the central nervous system (CNS) and peripheral nervous system (PNS). The ligand for the neuronal receptors dictates the fate of the neuronal cells. My studies focused on understanding the binding interfaces between the proteins in the proNGF-p75NTR-sortilin neuronal apoptotic complex. I have performed the biochemical characterization of the complex to understand how the complex formation occurs. Single amino-acid mutation of R155H on the N-domain of p97 is known to be the prevalent mutation in 40% patients suffering from neurodegenerative disease. The p97R155H mutant exhibits abnormal ATPase activity and cofactor dysregulation. I pursued biochemical characterization in combination with single-particle cryo-EM to explore the interaction of p97R155H mutant with its cofactor p47 and determined the full-length structures of the p97R155H-p47 assemblies for the first time. About 40% p97R155H organizes into higher order dodecamers, which lacks nucleotide binding, does not bind to p47, and closely resembles the structure of p97 bound with an adenosine triphosphate (ATP)-competitive inhibitor, CB-5083, suggesting an inactive state of the p97R155H mutant. The structures also revealed conformational changes of the arginine fingers which might contribute to the elevated p97R155H ATPase activity. Because the D1-D2 domain communication is important in regulating the ATPase function, I further studied the functions of the conserved L464 residue on the D1-D2 linker using mutagenesis and single-particle cryo-EM. The biochemical and structural results suggested the torsional constraint of the D1-D2 linker likely modulates the D2 ATPase activity. Our studies thus contributed to develop deeper knowledge of the intricate cellular mechanisms and the proteins affected in disease pathways.

G protein coupled receptors (GPCRs) mediate various of physiologicalactivities which makes them significant drug targets. Determination of atomic level structure of GPCRs facilitates the structure-based drug design. The most widely used method currently for solving GPCR structure is still protein crystallography especially lipidic cubic phase (LCP) crystallization. LCP could mimic the native environment of membrane protein which stable the membrane proteins. Traditional synchrotron source requires large size large size protein crystals (>30 micron) due to the radiation damage during data collection. However, acquiring large sized protein crystals is challenging and not guaranteed practically. In this study, a novel method was developed which combined LCP technology and micro-electron diffraction (MicroED) technology. LCP-MicroED technology was able to collect complete diffraction data sets from serval submicron protein crystals and deliver high resolution protein structures. This technology was first confirmed with soluble protein crystals, proteinase K and small molecule crystals, cholesterol. Furthermore, this novel method was applied to a human GPCR target, Î22- adrenergic receptor (Î22AR). The structure model was successfully built which proved the feasibility of applying LCP-MicroED method to GPCRs and other membrane proteins. Besides, in this research, a novel human GPCR target, human histamine 4 receptor(H4R) was studied. Different constructs were expressed, purified, and characterized. Some key residuals that affect ligand binding were confirmed.

Cryogenic Electron Microscopy (Cryo-EM) is a method that can be used for studying the structure of biological systems. Biological samples are frozen to cryogenic temperatures and embedded in a vitreous ice when they are imaged by electrons. Due to its ability to preserve biological specimens in near-native conditions, cryo-EM has a significant contribution to the field of structural biology.Single-particle cryo-EM technique was utilized to investigate the dynamical characteristics of various protein complexes such as the Nogo receptor complex, polymerase ζ (Polζ) in yeast and human integrin ⍺vβ8-pro-TGFβ1-GARP complex. Furthermore, I proposed a new method that can potentially improve the sample preparation for cryo-EM. The Nogo receptor complex was expressed using baculovirus expression system in sf9 insect cells and isolated for structural studies. Nogo receptor complex was found to have various stoichiometries and interactions between individual proteins. A structural investigation of the yeast apo polymerase ζ holoenzyme was also carried out. The apo Polζ displays a concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. Furthermore, a lysine residue that obstructs the DNA-binding channel in apo Polζ was found and suggested a gating mechanism. In addition, cryo-EM studies of the human integrin ⍺vβ8-pro-TGFβ1-GARP complex was conducted to assess its dynamic interactions. The 2D classifications showed the ⍺vβ8-pro-TGFβ1-GARP complex is highly flexible and required several sample preparation techniques such as crosslinking and graphene oxide coating to improve protein homogeneity on the EM grid. To overcome challenges within the cryo-EM technique such as particle adsorption on air-water interface, I have documented a collaborative work on the development and application of lipid monolayer sandwich on cryo-EM grid. Cryogenic electron tomography (cryo-ET) along with cryo-EM were used to study the characteristics of lipid monolayer sandwich as a potential protective layer for EM grid. The cryo-ET results demonstrated that the thickness of lipid monolayer is adequate for single-particle cryo-EM processing. Furthermore, there was no appearance of preferred orientations in cryo-EM and cryo-ET images. To establish that this method is actually beneficial, more data must be collected, and high-resolution structures of protein samples must be obtained using this methodology.