Protein arginine methyltransferase 1 (PRMT1) is a key posttranslational modification enzyme that catalyzes the methylation of specific arginine residues in histone and nonhistone protein substrates, regulating diverse cellular processes such as transcriptional initiation, RNA splicing, DNA repair, and signal transduction. Recently the essential roles of PRMT1 in cancer and cardiovascular complications have intrigued much attention. Developing effective PRMT inhibitors therefore is of significant therapeutic value. The research on PRMT inhibitor development however is greatly hindered by poor understanding of the biochemical basis of protein arginine methylation and lack of effective assays for PRMT1 inhibitor screening. Herein, we report our effort in the kinetic mechanism study as well as the fluorescent probe and inhibitor development for PRMT1. New fluorescent reporters were designed and applied to perform single-step analysis of substrate binding and methylation of PRMT1. Using these reporters, we performed transient-state fluorescence measurements to dissect the rate constants along the PRMT1 catalytic coordinate. The data give evidence that the chemistry of methyl transfer is the major rate-limiting step, and that binding of the cofactor SAM or SAH affects the association and dissociation of H4 with PRMT1. Importantly, we identified a critical kinetic step suggesting a precatalytic conformational transition induced by substrate binding. On the other hand, we discovered a type of naphthyl-sulfo (NS) compounds that block PRMT1- mediated arginine methylation at micromolar potency through a unique mechanism: they directly target the substrates but not PRMT enzymes for the observed inhibition. We also found that suramin, an anti-parasite and anti-cancer drug bearing similar functional groups, effectively inhibited PRMT1 mediated methylation. These findings about novel PRMT inhibitors and their unique inhibition mechanism provide a new way for chemical regulation of protein arginine methylation. Addionally, to dissect the interplaying relationship between different histone modification marks, we investigated how individual lysine acetylations and their different combinations at the H4 tail affect Arg-3 methylation in cis. Our data reveal that the effect of lysine acetylation on arginine methylation depends on the site of acetylation and the type of methylation. While certain acetylations present a repressive impact on PRMT-1 mediated methylation (type I methylation), lysine acetylation generally is correlated with enhanced methylation by PRMT5 (type II dimethylation). In particular, Lys-5 acetylation decreases activity of PRMT1 but increases that of PRMT5. Furthermore, hyperacetylation increases the content of ordered secondary structures of H4 tail. These findings provide new insights into the regulatory mechanism of Arg-3 methylation by H4 acetylation, and unravel that complex intercommunications exist between different posttranslational marks in cis.

In addition to the well known quantum confinement effects resulted from size and shape, interfacial bond structure is another factor, affecting the properties of the nanomaterial that is rarely studied. Inspired by the “Au-S-Au” staple motif discovered from the crystal structure of monothiol protected Au102 nanocluster (Science, 2007, 318, 430), dithiol molecules (e. g. 1, 2-dithiol, 1, 4-dithiol, etc.) with molecular structural constraint have been employed to create dithiolate protected clusters or mixed monothiolate and dithiolate protected clusters. The structure and properties of the Au clusters are expected to change due to two effects: The entropy gain of dithiol over monothiol protection and the constraint to the formation of the thiol bridging motif. DMPS (1, 2-dithiol molecule) stabilized clusters with characteristic absorption bands have been obtained, and characterized by multiple techniques. Monolayer reaction on gold core surface between the monothiol tiopronin and dithiol DMPS has been performed, and the mechanism has been probed. Mixed phenylethanethiolate and durene-dithiolate (1, 4-dithiol molecule) protected Au130 clusters with rich electrochemical features have been created, and the optical and electrochemical energetics have been successfully correlated based on core and core-ligand energy states. Furthermore, the impact of 1, 4-dithiolate-Au bonding on the near infrared luminescence has been studied.

In Streptococcus pyogenes, the protein SiaA (HtsA) is part of a heme uptake pathway system and involved in heme transfer from Shp to the ABC transporter. SiaA mutants, in which alanine replaces the axial histidine (H229) and methionine (M79) ligands, as well as a lysine (K61) and cysteine (C58) located near the heme propionates, are reported. Studies on a mutant of a cysteine expected to be at a distance from the propionates (C47A) are also reported. The coordination state and spin state of the selected mutants were determined via Resonance Raman studies. The pKa values of mutants ranged from 9.0 to 9.4, which were close to the pKa of the WT SiaA (9.7). The midpoint reduction potential of lysine (K61A) mutant was determined by spectroelectrochemical titration to be 61 ± 3 mV vs. SHE, similar to the WT protein (68 ± 3 mV). The addition of guanidinium hydrochloride resulted in protein denaturation that could show more than one process and occurred over days. The ease of protein unfolding was directly related to the extent of interaction of the residues with the heme: changes in the axial ligands resulted in far greater changes in heme protein stability than changes in the residues near the heme propionates. The causative agent of diphtheriae, Corynebacterium diphtheriae, imports heme via an ABC uptake transporter. In this research, two of the five proteins in the heme uptake pathway of C. diphtheriae were studied. These proteins were HmuT, lipoprotein component of the ABC transporter, and HtaA, the heme receptor. UV-visible spectroscopy and fluorescence spectroscopy showed that HmuT protein as isolated bound a porphyrin, rather than heme. Electrospray ionization mass spectrometry (ESI-MS) studies suggested that two tetrapyrroles were bound. To assess stability of this protein towards heme release, thermal denaturation studies were performed. For HtaA, UV-visible and fluorescence spectroscopy also showed the protein as isolated was also bound a porphyrin, rather than heme. Homology studies showed that HtaA protein is quiet distant from homologous heme uptake proteins and could be a member of novel heme binding domain family.

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca2+ ([Ca2+]o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca2+-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca2+-binding site (Site 1) in the ECD-mGluR1α using a recently developed computational algorithm. This predicted site (D318, E325, D322 and the bound L-Glu) is situated in the hinge region in the ECD-mGluR1α adjacent to the reported Glu-binding site. Mutagenesis studies indicated that binding of L-Glu and Ca2+ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca2+ ([Ca2+]i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca2+-sensing capability largely intact. Mutating the predicted Ca2+-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca2+]o and [Gd3+]o but also, in some cases, to Glu. In addition, the Ca2+ effects on drugs targeting mGluR1α were investigated. Ca2+ enhances L-Quis response of the receptor by increasing L-Quis binding to ECD-mGluR1α and promotes the potency of Ro 67-4853, a positive allosteric modulator of mGluR1α. Increasing Ca2+ concentration, the inhibitory effects of a competitive antagonist ((s)-MCPG) and a non-competitive negative allosteric modulator (CPCCOEt), were eliminated. Furthermore, we also identified another potential Ca2+ binding pocket (Site 2) consists of S165, D208, Y236 and D318, which completely overlapped with L-Glu. Thapsigargin (TG) induced ER Ca2+ depletion reduced surface expression of mGluR1α, and D208I and Y236I also decreased the receptor trafficking to plasma membrane suggesting the role of Ca2+ binding in protein folding and trafficking in the ER. Further, to measure ER Ca2+, a series of genetically encoded biosensors were designed by placing a Ca2+ binding pocket at the chromophore sensitive region of red florescent protein mCherry. The designed sensors are able to bind Ca2+ and monitor Ca2+ concentration change both in vitro and in cells. The findings in this dissertation open up new avenues for developing allosteric modulators of mGluR function that target related human diseases.

The synthesis of four homologous anthraquinones (AQ I-IV) bearing increasing lengths of polyethylene glycol (PEG) side chains and their binding to AT- and GC-rich DNA hairpins are reported. The molecules were designed such that the cationic charge is at a constant position and the ethylene glycol units chosen to allow significant increases in size with minimal changes in hydrophobicity. The mode and affinity of binding were assessed using circular dichroism (CD), nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). The binding affinity decreased as the AQ chain length increased along the series with both AT- and GC-rich DNA. ITC measurements showed that the thermodynamic parameters of AQ I-IV binding to DNA exhibited significant enthalpy-entropy compensation. The enthalpy became more favorable while the entropy became less favorable. The correlation between enthalpy and entropy may involve not only the side chains, but also changes in the binding of water and associated counterions and hydrogen bonding. The interactions of AQ I-IV with GC-rich DNA have been studied via molecular dynamics (MD) simulations. The geometry, conformation, interactions, and hydration of the complexes were examined. As the side chain lengthened, binding to DNA reduced the conformational space, resulting in an increase in unfavorable entropy. Increased localization of the PEG side chain in the DNA groove, indicating some interaction of the side chain with DNA, also contributed unfavorably to the entropy. The changes in free energy of binding due to entropic considerations (-3.9 to -6.3 kcal/mol) of AQ I-IV were significant. The kinetics of a homologous series of anthraquinone threading intercalators, AQT I-IV with calf thymus DNA was studied using the stopped-flow. The threading mechanisms of the anthraquinones binding to DNA showed sensitivity to their side chain length. Fitting of the kinetic data led to our proposal of a two step mechanism for binding of AQT I, bearing the shortest side chain, and a three step mechanism for binding of the three longer homologs. Binding involves formation of an externally bound anthraquinone-DNA complex, followed by intercalation of the anthraquinone for AQT I-IV, then isomerization to another complex with similar thermodynamic stability for AQT II-IV.

DNA photosensitizers are compounds that are capable of binding in to DNA strands through groove binding, intercalation, or electrostatic interactions. Excitation of these agents by light generates reactive oxygen species which causes extensive photo-oxidative damage to genomic DNA. Physiological concentrations of NaCl and KCl are ~ 150 mM and 260 mM within the cell nucleus where DNA is contained. Unfortunately, the ability of most photosensitizers to bind to double-helical DNA is reduced and photocleavage yields are diminished as concentrations of salt increase. The aim of this project is to observe the photocleavage of pUC19 plasmid DNA induced by N1,N1-bis(9-anthrymethyl)triethylenetetraamine tetrahydrochloride (AL-VIII 23) 1 or N,N-dimethyl-N’-(9-methylanthracenyl)ethylenediamine (NMEA) 2 in presence of salt. Spectroscopic titrations and DNA melting assays were used to study binding modes and affinities of both dyes to the helix upon the addition of salt.

The synthetic methodology of tri- and pentamethine carbocyanines and their interactions with biomolecules will be discussed in two chapters. The first chapter describes the preparation of halogenated carbocyanine dyes that display multiple charges; furthermore, these particular compounds were examined for their ability to bind G-quadruplex DNA with selectivity over duplex DNA and have potential for developing novel chemotherapeutic agents. The second section discusses the synthetic methods utilized to prepare trimethine cyanine fluorophores. This chapter will show how varying the N-indolenyl substituients’ hydrophobicity from ethyl to phenylpropyl influences the binding to Human Serum Albumin (HSA); additionally, alternating the terminal heterocyclic moieties of the cyanine dye has a direct quantitative effect on the biomolecular interaction. These identical compounds were recognized to be structurally analogous to agents that commonly interact with Protein Arginine Methyl Transferase (PRMT) and these compounds display low IC50 values toward inhibition of PRMT1 with unique NIR imaging properties.

Cocatalyzed reactions using Brønsted acids and chiral N-heterocyclic carbenes to yield highly enantioselective products have been reported recently in many journals. The development of new chiral N-heterocyclic carbenes is a competitive field among synthetic chemist. In a recent study we found that conjugated trienes could be cyclized using Brønsted acids and chiral N-heterocyclic carbenes. The synthesis of novel chiral N-heterocyclic carbene precursors, and the precursors to novel conjugated trienes are reported herein.

The ability to generate chiral building blocks is of paramount importance to organic chemists. This problem presents itself most notably at the interface of chemistry and biology, where molecules of only a single enantiomer can induce function to many biological systems. In this context, recent developments in the field of organocatalysis, most notably the employment of chiral N-heterocyclic carbenes (NHCs) have shown much promise. Our group has recently shown that one possible chiral NHC catalyzed Stetter cyclization product of a conjugated triene, a highly functionalized cyclopentenone, contains both a chiral center and an adjacent conjugated diene. This structure can be easily elaborated to a bicyclic structural motif present in some biologically active natural products from the ginkgolide family, and is difficult to access by other means. The synthesis of novel vinyl stannanes and other coupling substrates involved in the development of the aforementioned reaction discovery are described in this report.

The high surface to volume ratio which is a property of nanoscale devices means the interfacial effects from these devices on the mass transport of analyte can be significant. Quartz nanopipette effect on the mass transport behavior of inorganic monovalent salts such as potassium chloride is shown to differ from those of conical nanopore. Quartz nanopipettes demonstrate a more significant interfacial impact on the mass transport behavior of inorganic salts. This is evidenced by significant impacts on ionic transport even at high electrolyte concentration where nanopore interfacial effects do not significantly impact the ion transport. Nanopipettes have been use to precipitate salts such as lithium chloride in bulk concentrations three orders of magnitude below the saturation concentration. These novel interfacial interactions have opened new avenues for crystallization of more complex organic biomolecules using inorganic systems as model systems on which to base the approach for these more complex systems.