The mixed lineage leukemia (MLL) proteins regulate an array of developmental and differentiation processes. Similar to other members of the SET1 family, association of MLL1-4 with Ash2L, RbBP5 and WDR5, collectively termed the MLL core complex, is required for MLL mediated histone H3 Lys-4 di/tri-methylation. Each member of the core complex has a unique role in modulating the activity of MLL1. WDR5 is key in nucleating the formation of the core complex by acting as a structural scaffold, whereas Ash2L and RbBP5 are responsible for stimulating MLL methyltransferase activity. Currently, the structural and biochemical mechanisms utilized by the core complex to regulate MLL1 activity are unknown. Through structural and biochemical dissection of the core complex we have assigned specific functions to core complex subunits and have identified the minimal structural requirements for methyltransferase activity. Furthermore, through structure based drug design, we have identified a peptidomimetic inhibitor of MLL1 methyltransferase activity.

The post-translational modifications (PTMs) of histones regulate many cellular processes including transcription, replication, DNA repair, recombination, and chromosome segregation. A large number of combinations of PTMs are possible, with methylation being one of the most complex, since it is found in three states and is recognized in a sequence specific context. Methylation of histones at key lysine residues has been shown to work in concert with other modifications to provide a Histone Code that may determine heritable transcriptional conditions in normal and disease states. On the most basic level it is pivotal to understand how and by which proteins the numerous PTMs are recognized, as well as mechanisms for downstream signal propagation. To address this need we developed a high-throughput method that allows analysis of up to 600 PTMs in a single experiment. This approach was utilized to characterize macromolecules interacting with the specific modifications on histone tails and to screen for the marks that bound to Malignant Brain Tumor (MBT) proteins, important chromatin regulators implicated in cancer. All MBTs recognized either mono- or dimethyllysine histone marks, and using structure-based mutants we identified a triad of residues that were responsible for this discrimination. These results provide the foundation for the rational design of highly selective MBT inhibitors. Additionally, this thesis describes combinatorial recognition of histone modifications, as proposed in the original Histone Code hypothesis. We demonstrate that Tudor domains of UHRF1, a protein involved in epigenetic maintenance of DNA methylation, is able to read a dual modification state of histone H3 in which it is trimethylated at lysine 9 and unmodified at lysine 4. This study provides an elegant example of the combinatorial readout of histone modification states by a single domain. Together, our findings offer mechanistic insights into the recognition of methylated histone tails by MBT domains and Royal Family in general.

Tridentate "pincer" ligands provide a unique balance of stability and reactivity in organometallic chemistry. The development of diarylamido-based PNP pincer ligands has led to many applications in catalysis, including the potential to facilitate unique chemical transformations at transition metal centers. The main objective of this thesis was to explore transition metal chemistry supported by the PNP pincer framework for both early and late transition metals. In Chapter I, the history behind the design and synthesis of pincer complexes is described. The advantages and disadvantages of various pincer ligands are reviewed to show the reasoning behind the synthesis of the PNP pincer framework. Chapter II discusses the synthesis of novel Hf and Ta complexes involving the PNP ligand. Reactions of (PNP)HfCl3 with large alkyl Grignards led to double alkylation and triple alkylation was achieved with methyl Grignard. (PNP)HfMe3 and (PNP)Hf(CH2SiMe3)2Cl displayed remarkably irregular coordination environments about hafnium, in contrast to the approximately octahedral structure of (PNP)HfCl3. (PNP)HfMe3 was found to be thermally stable at 75 degrees C, whereas thermolysis of (PNP)Hf(CH2SiMe3)2Cl under similar conditions led to a mixture of products. The major decomposition product is believed to be a Hf alkylidene complex on the basis of in situ NMR spectroscopic observations (e.g., delta 248.2 ppm in the 13C{1H} NMR spectrum). The reaction of (PNP)TaF4 with an excess of ethyl Grignard led primarily to the double alkylation product, (PNP)Ta(CH2CH3)2F2. Repeating this reaction in the presence of excess ethyl Grignard and dioxane resulted in the formation of an ethylene complex, (PNP)Ta(=CHCH3)(C2H4). In Chapter III, a C-C reductive elimination study is described comparing two pincer ligand scaffolds: Me(PNP) ligand and TH(PNP) ligand. The tied ligand has previously been found to be more sterically demanding than the untied ligand, which has allowed for faster N-C cleavage, faster oxidative addition and a more selective alkyne dimerization catalyst. This study reveals that the tied ligand complex, TH(PNP)Rh(C6H4CF3)(Ph), undergoes slower reductive elimination of p-Ph-C6H4CF3 (< 4% after 7 h at 38 degrees C; t1/2 = 7.7 h at 64 degrees C; t1/2 = 2.13 h at 75 degrees C) than Me(PNP)Rh(C6H4CF3)(Ph) (t1/2 = 15.6 min at 38 degrees C).

The post-translational modifications (PTMs) of histones regulate many cellular processes including transcription, replication, DNA repair, recombination, and chromosome segregation. A large number of combinations of PTMs are possible, with methylation being one of the most complex, since it is found in three states and is recognized in a sequence specific context. Methylation of histones at key lysine residues has been shown to work in concert with other modifications to provide a Histone Code that may determine heritable transcriptional conditions in normal and disease states. On the most basic level it is pivotal to understand how and by which proteins the numerous PTMs are recognized, as well as mechanisms for downstream signal propagation. To address this need we developed a high-throughput method that allows analysis of up to 600 PTMs in a single experiment. This approach was utilized to characterize macromolecules interacting with the specific modifications on histone tails and to screen for the marks that bound to Malignant Brain Tumor (MBT) proteins, important chromatin regulators implicated in cancer. All MBTs recognized either mono- or dimethyllysine histone marks, and using structure-based mutants we identified a triad of residues that were responsible for this discrimination. These results provide the foundation for the rational design of highly selective MBT inhibitors. Additionally, this thesis describes combinatorial recognition of histone modifications, as proposed in the original Histone Code hypothesis. We demonstrate that Tudor domains of UHRF1, a protein involved in epigenetic maintenance of DNA methylation, is able to read a dual modification state of histone H3 in which it is trimethylated at lysine 9 and unmodified at lysine 4. This study provides an elegant example of the combinatorial readout of histone modification states by a single domain. Together, our findings offer mechanistic insights into the recognition of methylated histone tails by MBT domains and Royal Family in general.

Although some physiological effects of toxic metal poisoning have been known for centuries, the specific chemical interactions between biological molecules and mercury(I), mercury(II) or lead(II) are not well understood. To date, only thirteen crystal structures of inorganic mercury-amino acid complexes and six crystal structures of lead-amino acid complexes have been reported with varying degrees of characterization. In order to improve our understanding of the coordination chemistry of mercury and lead in biological environments, a systematic method for the isolation of inorganic metal-amino acid complexes from acidic aqueous solutions has been developed. With this method we have prepared five new lead-amino acid complexes (with L-valine, L-isoleucine, L-phenylalanine, and L-arginine) and four new mercury-amino acid complexes (with L-alanine, D-alanine, L-proline, and N-methyl-L-alanine). These metal-amino acid complexes have been comprehensively characterized in the solid state, solution state and gas phase. The development of this isolation technique in conjunction with the exploration of a number of characterization techniques for studying metal-amino acid interactions greatly enhances the known methods by which metal-biological molecule systems are studied.

The mixed lineage leukemia (MLL) proteins regulate an array of developmental and differentiation processes. Similar to other members of the SET1 family, association of MLL1-4 with Ash2L, RbBP5 and WDR5, collectively termed the MLL core complex, is required for MLL mediated histone H3 Lys-4 di/tri-methylation. Each member of the core complex has a unique role in modulating the activity of MLL1. WDR5 is key in nucleating the formation of the core complex by acting as a structural scaffold, whereas Ash2L and RbBP5 are responsible for stimulating MLL methyltransferase activity. Currently, the structural and biochemical mechanisms utilized by the core complex to regulate MLL1 activity are unknown. Through structural and biochemical dissection of the core complex we have assigned specific functions to core complex subunits and have identified the minimal structural requirements for methyltransferase activity. Furthermore, through structure based drug design, we have identified a peptidomimetic inhibitor of MLL1 methyltransferase activity.

Fluorescent protein based genetically encoded fluorescent reporters play an improtant role in understanding the cellular physiology by directly monitoring real-time cellular signaling pathways with fluorescent microscope. Quantitative analysis of Ca2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca2+-dependent signaling under physiological and pathological conditions. Here, we developed a novel class of genetically encoded indicators by designing a Ca2+ binding site in the enhanced green fluorescent protein (EGFP). One of them, CatchER (Calcium sensor for detecting high concentration in the ER), exhibits unprecedented Ca2+ release kinetics with an off-rate estimated at around 700 s-1 and appropriate Ca2+ binding affinity, likely due to local, Ca2+-induced conformational changes around the designed Ca2+ binding site and reduced chemical exchange between two chromophore states. CatchER reported considerable differences in ER Ca2+ dynamics and concentration among epithelial HeLa, kidney HEK 293, and muscle C2C12 cells, enabling us to monitor SR luminal Ca2+ in flexor digitorum brevis (FDB) muscle fibers to determine the mechanism of diminished SR Ca2+ release in aging mice. Moreover, the structure of CatchER has been investigated by nuclear magnetic resonance spectroscope (NMR) and high-resolution X-ray crystal structures to understand the novel mechanism of Ca2+ induced fluorescent enhancement of GFP. It is crucial to investigate the metal selectivity of Ca2+/Mg2+ of these metalloproteins to understand cellular physiology. The major Mg2+ binding sites of proteins have been reviewed and classified based on structural differences, and identified several key factors to determine Mg2+/Ca2+ selectivity with binding constants difference up to 104 in several types of metalloproteins. Thrombin is involved in numerous cellular signaling pathways and plays a crucial role in blood coagulation. I designed a novel class of single EGFP-based thrombin sensors by inserting a thirty-amino acid short peptide with a thrombin cleavage site into the fluorescent sensitive location of EGFP. These designed protease sensors exhibited optimized kcat/Km up to 104 magnitudes higher than that of small peptide based absorption indicator EGR-pNA. The measured Km value is in below 10 mM, in the same magnitude as that of natural thrombin substrate Fibrinogen A.

Adenosine triphosphate (ATP) is the energy currency of all living cells and its production is a key reaction in the energy metabolism of living organisms. Cells produce most of the ATP they require through ATP synthase, a unique molecular rotary motor driven by the movement of protons across the lipid membrane. In E.coli, ATP synthase is composed of a soluble domain called F1, which houses the catalytic sites, and a transmembrane domain called F0 that shuttles protons across the membrane to drive ATP production in the F1 sector. The F0 domain is built of three subunit types: subunit a and a dimer of subunit b form the stator of the motor, while a decameric c ring forms the rotor. The dynamic interface between a and c10 forms the proton channel. The ultimate goal of this work is to determine the structure of the proton transport machinery and understand the molecular mechanism of proton translocation in ATP synthase. We have characterized some of the key events in the stepwise assembly of the F0--complex. We have designed and validated a model protein, consisting of genetically fused subunits a and c, for structural studies. We have made progress towards determining the structure of the proton channel, including the development of a novel procedure for purification of subunit a and the a/c fusion protein, and crystallization of subunit a. Medical applications of this work include the potential development of novel antibiotic compounds, as well as the characterization and potential treatment of three human diseases caused by disruptions in proton transport through F0.

In this thesis, fundamental questions about the nature of the solvent/counter-ion region of x-ray crystal structures are raised. The ambiguity in the identity and occupancy of the molecular and atomic species in this region is explored experimentally. Anomalous scattering is proposed as a possible method for resolving this ambiguity. To this effect, the properties of rubidium I and thallium I are compared and contrasted to each other and to other group I metals. Finally, the structures of two modified B-DNA dodecamers are determined to explore the effect of monovalent cations on B-DNA structure. The modifications in these structures harbor tethered cations that are covalently linked to the DNA in the major groove. In one structure, the tethered cation causes axial bending of the DNA molecule, while in the second structure the molecule remains linear. We posit that the discrepancy between the two structures is due to lattice packing forces. In addition, we show evidence for the displacement of thallium I cations from the major groove of the bent structure.

Nitrochemicals are widely used as explosives, biocides and drugs. In addition, 3-nitro-tyrosine and other nitrated protein residues are important markers for many cardiovascular, neurodegenerative, and malignant conditions. Because of the wide presence of the nitrocompounds as toxins, potential nitrogen/carbon sources, and metabolic intermediates, different organisms have evolved to produce enzymes that can biodegrade nitrocompounds. The structural studies of the enzymes, which catalyze the removal of nitro group from nitrochemicals, are of considerable interest for both applied and fundamental reasons. The insights into the reaction mechanism of these enzymes can be used for designing efficient biocatalysts for bioremediation and for developing antibiotics for disease resistant microbes. Nitroalkane oxidase (NAO) produced by