251 |
Enzymatic Mechanisms and Chemical Probes of the Myst Family of Histone AcetyltransferasesYang, Chao 01 August 2013 (has links)
As an important posttranslational modification, protein acetylation plays critical roles in many biological processes such as gene transcription, DNA damage repair, apoptosis and metabolism. The acetylation occurs on the ε-amino group of specific lysine residues, and is catalyzed by histone acetyltransferases (HATs). In cellular contexts, HATs are found to target hundreds and thousands of substrates including histone and nonhistone proteins. Lysine acetylation changes the microenvironment of protein and may potentially alter protein activity and protein-protein interaction. The goal of this dissertation project is to investigate the impact of lysine acetylation on the catalysis of MYST HATs, and to establish the strategy for labeling substrates of the MYST HATs at cellular level. To understand the regulatory mechanism of MYST HATs, a detailed study was carried out to investigate the active site lysine acetylation of two MYST HATs (MOF and Tip60). Autoradiography and immunoblotting data shows that mutation of active site lysine differentially affects the enzyme autoacetylation activity and the cognate substrate acetylation activity. In addition, deacetylated MOF and Tip60 were prepared by using the nonspecific lysine deacetylase Sirt1. Kinetic study demonstrated that the acetylation of the active site lysine on MYST HATs marginally modulates the HAT catalysis. This work provides new insights into the regulatory mechanism of MYST catalysis. In the second part of my work, we designed and synthesized a series of Ac-CoA analogs conjugated with alkynyl or azido functional groups. Meanwhile, the active site of the MOF was engineered to expand the cofactor binding capability. Fluorescence screening was carried out to characterize the enzyme activity to Ac-CoA analogs. MOF-I317A with all analogs and MOF-I317A/H273A–5HYCoA were identified and further applied in the labeling of the cognate histone H4 protein and HAT substrates in 293T cell lysate. Visualizing of the labeled substrate was achieved using the alkynyl or azido-tagged fluorescent reporters through the copper-catalyzed azide−alkyne cycloaddition. As expected, the histone H4 protein was successfully labeled by the active enzyme-cofactor pairs. More intriguingly, multiple protein bands in cell lysate were labeled and observed. This work provides a new versatile strategy in exploring the substrates of MYST HATs at the proteomic level.
|
252 |
A Structural and Mechanistic Study of Two Members of Cupin Family ProteinLiu, Fange 18 June 2013 (has links)
is a functionally diverse large group of proteins sharing a jelly roll β-barrel fold. An enzymatic member 3-hydroxyanthranilate-3,4-dioxygenase (HAO) and a non-enzymatic member pirin, which is a human nuclear metalloprotein of unknown function present in all human tissues, were selected for structural and functional studies in this dissertation work. HAO is an important enzyme for tryptophan catabolism and for 2-nitrobenzoic acid biodegradation. In this work, seven catalytic intermediate were captured in HAO single crystals, enabling for the first time a nearly complete structural snapshot viewing of the entire molecular oxygen activation and insertion mechanism in an iron- and O2-depedent enzyme. The rapid catalytic turnover rate was found achieved in large part by protein dynamics that facilitates O2 binding to the catalytic iron, which is bound to the enzyme by a facile 2-His-1-carboxylate ligand motif. An iron storage and chaperon mechanism was also discovered in the bacterial source of this enzyme, which led to a proposed novel biological function of a mononuclear iron-sulfur center. Although human pirin protein shares the same structural fold with HAO, its iron ion is coordinated by a 3-His-1-carboxylate ligand motif. Pirin belongs to a subset of proteins whose members are playing regulatory functions in the superfamily. In this work, pirin is shown to act as a redox sensor for the NF-κB transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to pro-inflammatory signals which controls the expression of a vast array of genes involved in immune and stress responses.
|
253 |
Macromolecular Interactions in West Nile Virus RNA-TIAR Protein Complexes and of Membrane Associated Kv Channel PeptidesZhang, Jin 01 July 2013 (has links)
Macromolecular interactions play very important roles in regulation of all levels of biological processes. Aberrant macromolecular interactions often result in diseases. By applying a combination of spectroscopy, calorimetry, computation and other techniques, the protein-protein interactions in the system of the Shaw2 Kv channel and the protein-RNA interactions in West Nile virus RNA-cellular protein TIAR complex were explored. In the former system, the results shed light on the local structures of the key channel components and their potential interaction mediated by butanol, a general anesthetic. In the later studies, the binding modes of TIAR RRM2 to oligoU RNAs and West Nile virus RNAs were investigated. These findings provided insights into the basis of the specific cellular protein–viral RNA interaction and preliminary data for the development of strategies on how to interfere with virus replication
|
254 |
Development of a Novel DNA Microchip for Pathogen DetectionMaw, Khin Lay 13 April 2010 (has links)
Although DNA microarray can detect multiple DNA samples simultaneously, current detection techniques involve PCR and other traditional procedures. In this study, a sensitive, specific and rapid detection method, which eliminates PCR and other lengthy processes, for pathogenic DNA is presented. This technology is based on the hybridization of target DNA to the immobilized probe, extension of probe DNAs using the target-DNA as a template and signal generation by streptavidin-horseradish peroxidase and substrate. This method is highly specific and sensitive, allowing single-nucleotide-base mismatches discrimination and the detection at femtomole level. The experiments are designed to achieve short hybridization time. Therefore, satisfactory signal can be detected within minutes, allowing the rapid detection of multiple pathogenic DNA. Most importantly, the E. coli genomic DNA can be detected using this technology. In conclusion, this detection method is useful for applications including on-site pathogenic disease detection, crime scene investigation, and pathogen inspection in the environment.
|
255 |
Modulation and Recognition of Nucleic Acid StructuresSpring, Alexander M 21 June 2012 (has links)
The fidelity of an organism’s genome is central to biology. DNA, however, is constantly being damaged and modified by a variety of sources. As a result of these changes, repair enzymes, polymerases, and other interrogating biomolecules must be able to recognize, repair, and adapt to a multitude of different structures and dynamics presented. Manipulation of natural systems via the development and introduction of novel bases and DNA structures only adds to this complexity. In addition, specific RNA sequences are becoming more prevalent therapeutic and diagnostic targets. These include retroviruses and other viruses that maintain their genome with RNA. Unlike DNA, RNA poses a unique challenge as targets due to their highly diverse secondary and tertiary structures. In this manuscript, three different nucleic acid systems were chosen to investigate how intramolecular and intermolecular interactions impact their own structure as well as giving further insight into how nucleic acids are recognized and distorted by interrogating damage specific enzymes as well as structure specific proteins.
|
256 |
Two-site DNA Minor Groove Binding CompoundsSheldon Deuser, Shelby Diane 12 July 2012 (has links)
DNA minor groove binding compounds have had limited therapeutic uses, in part due to problems with sequence specificity. A two-site model has been developed to enhance specificity, in which compounds bind to two short AT sites separated by one or two GC base pairs. Using thermal melting, heterocyclic dications with this capability were tested with various oligonucleotides for binding affinity and specificity. Compounds of interest were further probed using circular dichroism, mass spectrometry, biosensor-SPR, and molecular modeling. Several compounds were found to “jump” a GC base pair, binding to AT sites in the minor groove of DNA with a two-site recognition mode. One compound was also found to recognize a single intervening GC base pair. Compounds with terminal, non-polar amidine extensions were found to have increased DNA binding compared to analogs with terminal amidines. This unique, two-site DNA recognition mode offers novel design principles to recognize entirely new DNA motifs.
|
257 |
Design and Synthesis of Thiamine Analogs as Anti-Cancer TherapeuticsDinh, Hieu T. 11 August 2012 (has links)
Cancer is one of the leading causes of death. There have been many investigations into therapeutic ways to prevent and reverse cancerous growth. We report a new approach in this thesis, which is to investigate the functions of Vitamin B1 (thiamine) in cancerous cells and their regulation. A number of thiamine analogs were synthesized to carry out the structure-activity relationship (SAR) studies with two transporters THTR1 and THTR2. Initial results show that the modifications of thiazole reduced the uptake of thiamine.
|
258 |
Studying the DNA Binding and Conformation of Metal-Binding Site Mutations in PirinRehmani, Imran J 07 August 2012 (has links)
The transcription factor NF-κB interacts with many other co-regulator proteins that modulate its binding and transcriptional activity. One of these co-regulators, Pirin, is an iron-dependent metalloprotein that has been shown to enhance the DNA binding of NF-κB homodimers. Here, we characterize the interactions between Pirin and its known NF-κB binding partners and examined the role of Bcl-3, a protein that is required for Pirin’s interaction with p50. In addition, we use site-directed mutagenesis to alter conserved residues within Pirin’s metal binding environment and observed how it affected the DNA binding and conformation of the Pirin-NF-κB complex. These studies show that, while a similar enhancing effect on DNA binding is observed, the interactions of Pirin with different NF-κB members are distinct from each other and could possibly have different physiological purposes.
|
259 |
Rational Design and Application of Genetically Encoded Fluorescent Reporters in Cellular PhysiologyTang, Shen 01 May 2012 (has links)
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.
|
260 |
Dissecting Key Determinants for Calcium and Calmodulin Regulation of GAP Junction and Viral ProteinChen, Yanyi 07 May 2012 (has links)
Calcium and calmodulin are implicated in mediating the Ca2+-dependent regulation of gap junctions that are essential for the intercellular transmission of molecules such as nutrients, metabolites, metal ions and signal messengers (< 1000 Da) through its specialized cell membrane channels and communication to extracellular environment. To understand the key determinants for calcium and calmodulin regulation of gap junction, in this study, we identified a calmodulin binding domain in the second half of the intracellular loop of Cxonnexin50 (the major gap junction protein found in an eye lens) using peptide fragments that encompass predicted CaM binding sites and various biophysical methods. Our study provides the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx50 in a Ca2+-dependent manner. Furthermore, two novel CaM binding regions in cytosolic loop and C-termini of Connexin43 (the most ubiquitous connexin) have been shown to interact with CaM with different binding modes in the presence of Ca2+ using high resolution NMR. Our results also elucidate the molecular determinants of regulation of gap junction by multiple CaM targeting regions and provide insight into the molecular basis of gap junction gating mechanism and the binding of CaM to the cytoslic region Cx43-3p as the major regulation site. Upon response to the cytosolic calcium increase, CaM binds to the cytosolic loop to result in the conformational change of gap junction and close the channel. It is possible for CaM to use an adjacent region as an anchor close to the regulation site to allow for fast response. Since a large number of residues in the Cxs mutated in human diseases reside at the highly identified CaM binding sites in Cxs, our studies provide insights into define the critical cellular changes and molecular mechanisms contributing to human disease pathogenesis as part of an integrated molecular model for the calcium regulation of GJs. In addition, we have applied the grafting approach to probe the metal binding capability of predicted EF-hand motifs within the streptococcal hemoprotein receptor (Shr) of Streptococcus pyrogenes as well as the nonstructural protein 1 (nsP1) of Sindbis virus and Poxvirus. This fast and robust method allows us to analyze putative EF-hand proteins at genome-wide scale and to further visualize the evolutionary scenario of the EF-hand protein family. Further, mass spectrometry has also been applied to probe modification of proteins such as CaM labeling by florescence dye and 7E15 by PEG.
|
Page generated in 0.106 seconds