As a highly versatile signal, Ca2+ operates over a wide temporal range to regulate many different cellular processes, impacting nearly every aspect of cellular life including excitability, exocytosis, motility, apoptosis, and transcription. While it has been well recognized that Ca2+ acts as both a second messenger to regulate cell-cell communication upon external stimuli and as a first messenger to integrate extracellular with intracellular signaling in various cell types. Molecular bases for such regulation and related human diseases are largely hampered by the challenges related to key membrane proteins. In the present study, we first investigated the regulatory role of intracellular Ca2+ ([Ca2+]i) on Connexin45 (Cx45) gap junction through a ubiquitous Ca2+ sensor protein-Calmodulin (CaM). Using bioluminescence resonance energy transfer assay, this study provides the first evidence of direct association of Cx45 and CaM in a Ca2+-dependent manner in cells. Complementary approaches including bioinformatics analysis and various biophysical methods identified a putative CaM-binding site in the intracellular loop of Cx45 with high Ca2+/CaM-binding affinity and Ca2+-dependent binding mode that is different from alpha family of connexins. To understand the role of extracellular calcium in regulation of gap junction hemichannels, we would like to prove a possible Ca2+-binding site predicted by our computational algorithm MUGSR in Connexin 26 (Cx26) through mutagenesis study, metal binding affinity measurement, conformational changes examination of purified Cx26 protein from Sf9; however, we failed to achieve this goal due to either the limitation of available methods or lethal effect of mutating the predicted Ca2+-binding ligand. Additionally, in this study, we identified a putative Ca2+-binding site in metabotropic glutamate receptor 5 (mGluR5) and demonstrated the importance of this Ca2+-binding site in activation of mGluR5 and modulating the actions of other orthosteric ligands on mGluR5. In addition, we successfully solved the first crystal structure of the extracellular domain of Ca2+-sensing receptor (CaSR) bound with Mg2+ and an unexpected Trp derivative. The extensive study of mechanism of CaSR function specifically through Mg2+-binding site and the unexpected ligand-binding site was done using several cell-based assays in wild type CaSR and mutants. Studies in this dissertation provides more information on how Ca2+ regulates gap junction channels, modulates mGluR5 activities and structural basis for regulation of CaSR by Mg2+ and an unexpected Trp derivative co-agonist.

Calcium (Ca2+), a crucial effector for many biological systems, has been associated with diseases such as cardiovascular disease, Alzheimer’s, Parkinson’s, cancer, and osteoporosis. It is important to develop calcium sensors to measure intracellular Ca2+ dynamics at various biological and pathological states. Our lab has engineered such probes by designing a Ca2+ binding site into fluorescent proteins such as Enhanced Green Fluorescent Protein (EGFP) and mCherry. In this thesis, we aim to improve optical properties and metal binding properties of green EGFP-based sensor CatchER and mCherry based red sensors by site-directed mutagenesis and protein engineering, various spectroscopic methods and cell imaging. The green EGFP-based sensor CatchER, with a Ca2+ binding pocket charge of -5, displays the greatest affinity for Ca2+ and has the greatest fluorescence intensity change with Ca2+ when compared to its variants with a less negative binding pocket charge. In addition, we have also designed several SR/ER targeting CatchER variants using Ryanodine receptor and Calnexin transmembrane domains. These constructs were shown to display a strong presence in the SR/ER lumen and further designed for a new luminal orientation. Further, we have shown that the optical properties of two red calcium sensors can be significantly improved by modifying the local environment of the chromophore.

This thesis begins with a brief review about the role and importance of the small molecules containing fluorine atoms in medicine and imaging. Then, the first part of the thesis will discuss the synthesis, purification and characterization of pentamethine cyanine dyes. The structure identification of the final dyes is done by using 1H NMR, 13C NMR, 19F NMR, and mass spectrometry. The studies performed after full characterization were the determination of optical and physicochemical properties. After these properties were performed, the fluorophores were evaluated to be good candidates for in vivo testing.

This thesis outlines two projects that examine interactions between DNA and cyanine dyes: 1) Investigation of monomethine cyanine dye through the synthesis and optical characteristics with a goal to synthetically manipulate these systems to develop a fluorescent biological probe. Several asymmetric and one symmetrical monomethine cyanine dyes were synthetically prepared and their optical properties versus structural changes were explored. Representative dyes were selected for spectroscopically investigating DNA binding to enhance existing probe designs. 2) Early stage development of a non-metalated photosensitizer for photodynamic therapy (PDT) by screening experimentally derived cleavage activities of a number of polymethine cyanine dyes with various adjoining heterocycles. Structural characteristics of these dyes which promoted cleavage in the dark were identified and eliminated to allow for more selective photo-induced cleavage to enhance the candidacy of these compounds as photosensitizing agents. All compounds were purified through recrystallization or column chromatography and were characterized via 1H and 13C nuclear magnetic resonance (NMR). Compounds from the first chapter were further characterized using mass spectrometry.

Influenza infection remains constant threat to human health and results in huge financial loss every year. Rapid and accurate detection of influenza can help governments and health organizations monitor influenza activity and take measurements when necessary. In addition, influenza detection in a timingly manner can help doctors make diagnosis and provide effective treatment. On the other hand, novel inhibitors of influenza virus are in high demand because circulating strains have started to develop resistance to currently available anti-viral drugs. Influenza virus has two surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA), which play important roles in the influenza infection. The binding of HA to sialic acid-containing carbohydrates on cell surface initiates virus internalization, while cleavage of terminal sialic acid by NA facilitates viral particle release. In this dissertation, we focus on the development of glycan microarray that is comprised of a panel of NA resistant sialosides, and demonstrate the application of microarray to capture influenza virus at ambient temperature without the addition of NA inhibitors. We also describe a novel electrochemical biosensor for the detection of influenza virus. In addition, we have developed a new class of bivalent NA inhibitors that show promising inhibitory activities against influenza viruses.

D-Arginine dehydrogenase (DADH) catalyzes the oxidation of D-arginine to imino arginine using FAD as the cofactor. The enzyme is part of a recently discovered two-enzyme complex from Pseudomonas aeruginosa involved in arginine utilization. Function of the enzyme within the organism is unknown. Work on this enzyme has been undertaken to understand the structure as well as its reaction mechanism so as to eventually assign a function to the enzyme within the physiological context. In the reductive half-reaction 2 e- and 1 H+ are transferred from the amino acid substrate to FAD cofactor. In the oxidative half-reaction the reducing equivalents from the FAD cofactor are passed to an electron acceptor that is yet to be discovered. The enzyme has been established to have no reactivity with O2. Choline oxidase (CHO) from Arthrobacter globiformis is a well characterized member of Glucose-Methanol-Choline Superfamily that reacts with molecular O2. It catalyzes the oxidation of choline to glycine betaine mediated by betaine aldehyde intermediate using FAD as the cofactor and O2 as the oxidant to regenerate oxidized FAD for further reaction. Glycine betaine, the product of the reaction is an important osmolyte that regulates nutrients for plants under stressful conditions. Therefore it is of commercial interest to genetically engineer crops that do not typically possess competent pathways for glycine betaine synthesis. In this dissertation molecular details concerning the reductive half-eaction of DADH and oxidative half-reaction of CHO have been studied using a combination of steady state kinetics, rapid kinetics, pH, multiple substrates, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects or computational approaches. In DADH, the oxidation of amino acid substrate by FAD has been shown to most likely proceed via hydride transfer mechanism in the reductive half-reaction with Glu87, Tyr53, Tyr249 and His48 emerging as key players in substrate binding, catalysis or for up keeping the integrity of the FAD cofactor. In CHO, the oxidative half-reaction proceeds without stabilization of any reaction intermediates with H atom from reduced FAD and H+ from solvent or solvent exchangeable site occurring in the same kinetic step.

Nucleic acids, as one of the most important macromolecules in living systems, play critical roles in storing, transferring, regulating genetic information, directing proteins synthesis, and catalysis. Understanding the structure of nucleic acid can bring us valuable information for mechanistic study and for drug discovery as well. Among all experimental methods, X-ray crystallography is the most powerful tool in structural biology study to reveal the 3D structure of macromolecules, which has provided over 80% of the highly detailed structural information to date. However, this great technology comes with two disturbing features, crystallization and phasing. The covalent selenium modification of nucleic acids has been proven to be a powerful tool to address both issues in nucleic acid crystallography. First part of this dissertation focuses on the development of novel selenium-modified nucleic acids (SeNA) for crystallization and phasing of B-form DNA containing structures. The novel 2’-SeMeANA modification is the first and currently the only selenium modification, which is fully compatible with X-ray crystallographic study of B-form DNA. Since selenium derivatization at 2’-arabino position dose not affect the B-type 2’-endo sugar conformation, this strategy is suitable for incorporating selenium into DNA for structural studies of B-DNA, DNA-protein complexes, and DNA-drug complexes. Specific base pairing is essential to many biological processes, including replication, transcription, and translation. It is crucial to NA (nucleic acid) sequence-based diagnostic and therapeutic applications as well. By utilizing the unique steric and electronic property of selenium, we designed, synthesized the novel 2-Se-U RNA modification, and demonstrated its highly specific base-pairing property by both biophysical and crystallographic methods. Our studies of 2-Se-U-containing RNAs suggest that this single-atom replacement can largely improve base pairing fidelity against U/G wobble pair, without significant impact on U/A pair.

Metal ions and complexes utilized as cleavage agents have influenced many synthetic approaches of scientists to assist in the cleavage and transformation of biomolecules. These metal-based synthetic cleavage agents have potential applications in biotechnology or as molecular therapeutic agents. Herein, we have examined Ce(IV) metal ion and complexes as acidic hydrolytic agents in lipid hydrolysis reactions (Chapter 2 and 3), and a copper(II) complex that photo-oxidizes DNA upon exposure to ultraviolet light (Chapter 4). In Chapter 2 we examined the hydrolysis of sphingomyelin vesicles by Ce(NH4)2(NO3)6 (Ce(IV)) and compared the results to twelve d- and f-block metal salts, hydrolysis of mixed lipid vesicles and mixed micelles of sphingomyelin by Ce(IV), and hydrolysis of phosphatidylcholine vesicles by Ce(IV), using either MALDI-TOF mass spectrometry or colorimetric assays. In Chapter 3, we described the study of a Ce(IV) complex based on 1,3-bis[tris(hydroxymethyl)methylamino]propane as a potential acidic hydrolytic agent of phospholipids using colorimetric assays and NMR spectroscopy. The hydrolytic agent provided markedly enhance hydrolysis at lysosomal pH (~ 4.8), but suppress hydrolysis when pH was raised to near-neutral pH (~ 7.2). This was due to the pKa values of the donor atoms of the ligand, in which the metal’s electrophilicity was reduced to a greater extent at ~ pH 7.2 compared to ~ pH 4.8. Chapter 4 describes the synthesis and study of a Cu(II) complex based on a hexaazatriphenylene derivative for photo-assisted cleavage of double-helical DNA. Scavenger and chemical assays suggested the formation of DNA damaging reactive oxygen species, hydroxyl and superoxide radicals, and hydrogen peroxide, in the photocleavage reactions. Thermal denaturation and UV-vis absorption studies suggested that the Cu(II) complex binds in a non-intercalative fashion to duplex DNA.

α-Amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) and α-aminomuconate-ε-semialdehyde dehydrogenase (AMSDH) are two neighboring enzymes in the L-tryptophan and 2-nitrobenzoic acid degradation pathways. The substrates of the two enzymes, α-amino-β-carboxymuconate-ε-semialdehyde (ACMS) and α-aminomuconate-ε-semialdehyde (2-AMS), are unstable and spontaneously decay to quinolinic acid and picolinic acid, respectively. ACMSD utilizes a divalent zinc metal as cofactor and is a member of the amidohydrolase superfamily. In this dissertation work, we have identified an important histidine residue in the active site that plays dual roles in tuning metal selectivity and activating a metal bound water ligand using mutagenesis, resonance Raman, EPR, crystallography, and ICP metal analysis techniques. The crystal structures of ACMSD from Pseudomonas fluorescens (PfACMSD) have been solved as homodimers in our laboratory while human ACMSD (hACMSD) was annotated as a monomer by another group. To resolve this structural difference, we used two conserved active site arginine residues as probes to study the oligomeriztion state of ACMSD and demonstrated that these two arginine residues are involved in substrate binding and that both Pf- and h- ACMSD are catalytically active only in the dimeric state. Subsequently, we solved the crystal structure of hACMSD and found it to be a homodimer in both catalytically active and inhibitor-bound forms. AMSDH is an NAD+ dependent enzyme and belongs to the aldehyde dehydrogenase superfamily. Due to the high instability of its substrate, AMSDH has not been studied at the molecular level prior to our work. We have cloned and expressed PfAMSDH in E. coli. The purified protein has high activity towards both 2-AMS and 2-hydroxymuconate semialdehyde (2-HMS), a stable substrate analog. We have successfully crystallized AMSDH with/without NAD+ and solved the crystal structure at up to 1.95 Å resolution. Substrate bound ternary complex structures were obtained by soaking the NAD+ containing crystals with 2-AMS or 2-HMS. Notably, two covalently bound catalytic intermediates were captured and characterized using a combination of crystallography, stopped-flow, single crystal spectroscopy, and mass spectrometry. The first catalytic working model of AMSDH has been proposed based on our success in structural and spectroscopic characterization of the enzyme in five catalytically relevant states in this dissertation work.

A novel series of near-infrared fluorescent contrast agents was developed and characterized. Their physicochemical and optical properties were measured. By altering functional groups of cyanine fluorophores, the selective targeting of endocrine glands, exocrine glands, cartilage and bone using NIR fluorescence to visualize the targeted tissue has been reported. These agents have high specificity for tissue targeting inherent to the chemical structure of the fluorophore. After a single low-dose intravenous injection these agents have high specificity for tissue targeting inherent to the chemical structure of the fluorophore. The results lay the foundation for future improvements in optical imaging in endocrine surgery, tissue engineering, joint surgery, and cartilage-specific drug development.