abstract: The need for a renewable and sustainable light-driven energy source is the motivation for this work, which utilizes a challenging, yet practical and attainable bio-inspired approach to develop an artificial oxygen evolving complex, which builds upon the principles of the natural water splitting mechanism in oxygenic photosynthesis. In this work, a stable framework consisting of a three-dimensional DNA tetrahedron has been used for the design of a bio-mimic of the Oxygen-Evolving Complex (OEC) found in natural Photosystem II (PSII). PSII is a large protein complex that evolves all the oxygen in the atmosphere, but it cannot be used directly in artificial systems, as the light reactions lead to damage of one of Photosystem II's core proteins, D1, which has to be replaced every half hour in the presence of sunlight. The final goal of the project aims to build the catalytic center of the OEC, including the Mn4CaCl metal cluster and its protein environment in the stable DNA framework of a tetrahedron, which can subsequently be connected to a photo-stable artificial reaction center that performs light-induced charge separation. Regions of the peptide sequences containing Mn4CaCl ligation sites are implemented in the design of the aOEC (artificial oxygen-evolving complex) and are attached to sites within the tetrahedron to facilitate assembly. Crystals of the tetrahedron have been obtained, and X-ray crystallography has been used for characterization. As a proof of concept, metal-binding peptides have been coupled to the DNA tetrahedron which allowed metal-containing porphyrins, specifically Fe(III) meso-Tetra(4-sulfonatophenyl) porphyrin chloride, to be encapsulated inside the DNA-tetrahedron. The porphyrins were successfully assembled inside the tetrahedron through coordination of two terminal histidines from the orthogonally oriented peptides covalently attached to the DNA. The assembly has been characterized using Electron Paramagnetic Resonance (EPR), optical spectroscopy, Dynamic Light Scattering (DLS), and x-ray crystallography. The results reveal that the spin state of the metal, iron (III), switches during assembly from the high-spin state to low-spin state. / Dissertation/Thesis / Ph.D. Biochemistry 2012

abstract: X-ray diffraction is the technique of choice to determine the three-dimensional structures of proteins. In this study it has been applied to solve the structure of the survival motor neuron (SMN) proteins, the Fenna-Mathews-Olson (FMO) from Pelodictyon phaeum (Pld. phaeum) protein, and the synthetic ATP binding protein DX. Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease resulting in muscle atrophy and paralysis via degeneration of motor neurons in the spinal cord. In this work, we used X-ray diffraction technique to solve the structures of the three variant of the of SMN protein, namely SMN 1-4, SMN-WT, and SMN-Δ7. The SMN 1-4, SMN-WT, and SMN-Δ7 crystals were diffracted to 2.7 Å, 5.5 Å and 3.0 Å, respectively. The three-dimensional structures of the three SMN proteins have been solved. The FMO protein from Pld. phaeum is a water soluble protein that is embedded in the cytoplasmic membrane and serves as an energy transfer funnel between the chlorosome and the reaction center. The FMO crystal diffracted to 1.99Å resolution and the three-dimensional structure has been solved. In previous studies, double mutant, DX, protein was purified and crystallized in the presence of ATP (Simmons et al., 2010; Smith et al. 2007). DX is a synthetic ATP binding protein which resulting from a random selection of DNA library. In this study, DX protein was purified and crystallized without the presence of ATP to investigate the conformational change in DX structure. The crystals of DX were diffracted to 2.5 Å and the three-dimensional structure of DX has been solved. / Dissertation/Thesis / Ph.D. Biochemistry 2013

<p> <b>Introduction.</b> The overarching goal of this study is to determine whether a long-lived, transgenic helminth parasite might express a potentially therapeutic antibody, using HIV-1 as the model target of a broadly neutralizing antibody expressed and secreted by schistosomes. </p><p> <b>Methods.</b> Cultured schistosomes were transfected by square wave electroporation with a plasmid encoding a human immunoglobulin G1 that is broadly neutralizing for HIV-1. </p><p> <b>Results.</b> Following introduction of an expression plasmid into cultured schistosomes by square wave electroporation, and extraction of total RNA and soluble lysates of the parasites, transcripts encoding both the light and heavy chains of the anti-HIV-1 broadly neutralizing antibody b12 and fragments of the human IgG1 antibody b12 were detected by reverse transcription PCR and western blot analysis, respectively. </p><p> <b>Conclusions.</b> These findings revealed that a human antibody, which is known to be broadly neutralizing for HIV-1, was expressed in schistosomes. Whereas this investigation is a work in progress, the findings thus far provide impetus to explore whether schistosomes transgenic with the gene encoding b12 might be harnessed to inhibit HIV-1 infection in vivo.</p><p>

This study investigates the recognition of structured RNAs by two essential enzymes, proteinaceous RNase P (PRORP) and lysine-specific demethylase-1 (LSD1). PRORP binds and cleaves human mitochondrial precursor tRNAs in a fundamental step for the generation of mature mitochondrial transcripts. The enzyme recognizes specific structured domains of the pre-tRNAs in a manner similar to but distinct from the unrelated nuclear RNase P complex. In order to move towards a crystal structure of the PRORP-RNA complex, minimal constructs were generated and biochemically validated with binding studies and in vitro activity assays and used in crystallization studies. LSD1 modulates gene expression through enzymatic histone demethylation and also serves as a protein scaffold in various large protein complexes. The long noncoding RNA (lncRNA) telomeric repeat-containing RNA (TERRA) has previously been demonstrated to recruit LSD1 to deprotected telomeres where it promotes the recruitment of the nuclease MRE11. Here, it is shown that LSD1 specifically recognizes the G-quadruplex structure formed by TERRA and other RNAs and a G-quadruplex RNA binding region is identified in the regulatory SWIRM domain of LSD1. Together, these studies advance our understanding of the role of structured RNAs in RNA/protein interactions.

The replication stress response (RSR) maintains genome stability and promotes the accurate duplication of the genome. ssDNA binding proteins are integral components of the RSR and have been extensively studied for years. However, the mechanisms by which they specifically direct enzymes to the right substrates and how they regulate replication fork remodeling is less well understood. My thesis projects have focused on the regulation of fork remodeling pathways by ssDNA binding proteins. In Chapter III, I discovered how RPA enforces SMARCAL1 substrate specificity to promote appropriate fork reversal. In chapter IV, I identified a new RPA-like ssDNA binding protein, RADX, at replication forks. I characterized the function of RADX as a negative regulator of RAD51 mediated fork reversal and described RADX loss as a chemo-resistance mechanism in BRCA2-deficient U2OS cells. In chapter V, I further explored the mechanisms by which RADX regulates RAD51. Utilizing RADX as a tool, I interrogated RAD51 functions in different pathways of fork protection and discovered the differential requirements of RAD51 in fork reversal and fork protection. Overall, my thesis has made significant contributions to our understanding of the processes of fork reversal and fork protection and has identified a potential chemo-resistance mechanism for BRCA2-mutant cancers.

DNA replication is constantly challenged by several sources of replication stress that can affect the accuracy and timely completion of this essential biological process. The DNA damage response (DDR) exists to address these sources of stress and promote rapid and accurate duplication of nearly 7 billion base pairs of DNA with each round of replication in humans. The SNF2 family of DNA translocases contains multiple proteins within the DDR that are recruited to stalled replication forks to promote repair and replication restart. SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent, regulator of chromatin, subfamily A-like 1), ZRANB3 (zinc-finger RANBP2-type containing 3), and HLTF (helicase-like transcription factor) share similar replication fork remodeling capabilities, such as fork reversal. Despite their biochemical similarities, genetic studies indicate they must have unique cellular activities. In my dissertation work, I sought to differentiate the functions of these highly similar enzymes in cells. I used a combination of biochemical, molecular biological, and genomics approaches to investigate the cellular requirement for each of these proteins with a specific focus on SMARCAL1. Here, I present data showing that SMARCAL1 has an important function at telomeres, which present an endogenous source of replication stress. SMARCAL1-deficient cells accumulate telomere-associated DNA damage and have greatly elevated levels of extrachromosomal telomere DNA (C-circles). Although these telomere phenotypes are often found in tumor cells employing the alternative lengthening of telomeres (ALT) pathway for telomere elongation, SMARCAL1 deficiency does not yield other ALT phenotypes such as telomere recombination. The activity of SMARCAL1 at telomeres can be separated from its genome maintenance activity in bulk chromosomal replication since it does not require its interaction with replication protein A (RPA). Importantly, this telomere maintenance function is not shared by ZRANB3 or HLTF. These results provide the first identification of an endogenous replication stress source that SMARCAL1 is needed to resolve and defines differences between members of this class of replication fork repair enzymes.

<p> It is well known that enzymes differ from small-molecule catalysts by use of non-covalent interactions to position active site residues, but our understanding of the relative importance of residues in this positioning is limited. Active site residues participate directly in covalent and non-covalent interactions with substrates, but second shell residues may also contribute indirectly to catalysis through positioning and structuring. In triosephosphate isomerase (TIM), a key glycolytic enzyme, a highly conserved glutamate residue at position 97 has been suggested to be important for catalysis and may be important for positioning a key active site lysine residue (K13). In <i> Trypanosoma brucei brucei</i> (TBB), a kinetoplastid which causes African sleeping sickness, Glu97 has been shown to be catalytically important. Mutations of Glu97 to Gln, Asp and Ala have been shown to lead to approximate 24-, 18-, and 6280-fold <i>k</i>_cat decreases, respectively. Whereas this glutamate residue is involved in the catalysis of the enzyme, the nature of its involvement in the structure of the enzyme is unclear. To evaluate the role of this residue in the structure of the enzyme, we performed structural and denaturation evaluations using intrinsic and ANS fluorescence. Our results suggested that the Glu97Asp and Glu97Gln mutations did not significantly perturb the structure of the enzyme compared with the wild-type, but may have slight structural effects, based on spectral center of mass and &lambda;_max values. Denaturation evaluations suggested that the Glu97Asp and Glu97Gln did not significantly destabilize the enzyme based on [GdnHCl]1/2. The effect of the Glu97Ala mutation, however, was less clear. Overall, our results suggested that although Glu97 is important to catalysis, Glu97Asp and Glu97Gln mutants do not appear to significantly perturb structure, but may have slight effects. Future directions include continued investigation of Glu97Ala, and evaluation of the structural effects of double mutants at the Glu97 and Lys13 positions. </p><p>

<p>Locusta migratoria apolipophorin III is an 18 kDa exchangeable apolipoprotein present in the hemolymph of locusts. In addition to its known role in diacylglycerol transport, the protein has antimicrobial properties targeting bacterial membranes. Apolipophorin III contains eight lysine residues, which interact with negatively charged membrane phospholipids. To better understand the importance of these lysines, a site-directed mutagenesis approach was employed. A series of lysine to glutamine variants were generated targeting lysines clusters in helix 2 and 5. The variants showed decreased helical structure and stability in particular when multiple residues were substituted. Ionic interactions with negatively charged phosphatidylglycerol (PG) were weakened in particular when seven of the eight lysines were substituted. In contrast, binding to phosphatidylcholine and lipoprotein was significantly improved when all lysines were substituted. This suggested that lysine residues are critical for protein structure and PG binding, but not for phosphatidylcholine and lipoprotein binding interaction.

<p> Electrical stimulation of tissues has been found to have many uses in pain management, antibacterial treatment, and wound healing. <i> In vivo,</i> it is known to stimulate epidermal migration and increase fibroblast cell proliferation. Here the effects of electrical field (EF) stimulation on collagen, elastin, and collagenase expression in human dermal fibroblasts are studied. The cells are stimulated in bioreactor using square wave voltage pulses controlled by potentiostat for up to 24 h period. The pulse voltage (0&ndash;10V), pulse bias (0, +,&minus;), pulse time (10&ndash;1000 ms), rest time (0.1&ndash;10 s) was varied. The effects of EF stimulation is evaluated in terms of protein expression level and changes in cell morphology. The results show that the expressions of these proteins are correlated and are doubled when EF stimulation larger than 3V and positive bias is applied. The shorter pulse time stimulates the cells more effectively, while the rest time between pulses has smaller effect.</p>

<p> Apolipoprotein E is a 299-amino acid protein with two structural domains: an NT (1-191) and C-terminal (201-299) domain that bear high affinity binding sites to the LDL receptor and lipid, respectively. In the plasma and brain, apoE associates with phospholipids and cholesterol to form discoidal nascent HDL which are remodeled into spherical HDL by the action of lecithin cholesterol acyltransferase (LCAT). We examined the conformation of apoE4 in spherical-HDL generated by the action LCAT on discoidal reconstituted HDL (rHDL) bearing single Cys variants employing fluorescence spectroscopy and site-specific crosslinking. The site-specific crosslinking studies showed the absence of intermolecular dimers which would be formed if two spatially proximal molecules are crosslinked. We also observed a loss of excimer between pyrene molecules attached covalently to single Cys variants of apoE4. The absence of dimers and loss of excimer indicate that apoE4 molecules in spherical rHDL are oriented in an anti-parallel fashion. This study provides more insight into the conformation of apoE4 in spherical HDL.</p><p>