Biochemistry Commons^™

Defining The Role Of Phosphorylation And Dephosphorylation In The Regulation Of Gap Junction Proteins, Hanjun Li

Theses & Dissertations

Gap junctions are intercellular channels that permit the free passage of ions, small metabolites, and signaling molecules between neighboring cells. In the diseased human heart, altered ventricular gap junction organization and connexin expression (i.e., remodeling) are key contributors to rhythm disturbances and contractile dysfunction. Connexin43 (Cx43) is the dominant gap junction protein isoform in the ventricle which is under tight regulation by serine/tyrosine phosphorylation. Phosphorylation and dephosphorylation regulate many aspects of Cx43 function including trafficking, assembly and disassembly, electrical and metabolic coupling at the plaque, as well as to modulate the interaction with other proteins.

Serine phosphorylation has long been …

Functional And Structural Characterization Of The Mevalonate Diphosphate Decarboxylase And The Isopentenyl Diphosphate Isomerase From Enterococcus Faecalis, Chun-Liang Chen

Open Access Dissertations

Enterococcus faecalis causes a diverse range of nosocomial infections (in wounds, the gastrointestinal tract, the blood stream and the endocardium), and multidrug-resistant strains have become a serious issue across countries. Vancomycin, a FDA-approved drug for the disruption of the bacterial cell wall biosynthesis, has been utilized to treat infectious diseases caused by Enterococci; however, the prevalence of vancomycin-resistant enterococci (VRE) threatens communities all over the world. We aim at developing novel therapeutic strategies to control bacterial growth of Enterococci, and we focus on targeting two essential enzymes involved in poly-isoprenoid biosynthesis in Enterococcus faecalis; one is the mevalonate diphosphate decarboxylase …

Influence Of Ph And Acidic Side Chain Charges On The Behavior Of Designed Model Peptides In Lipid Bilayer Membranes, Venkatesan Rajagopalan

Graduate Theses and Dissertations

The molecular properties of transmembrane proteins and their interactions with lipids regulate biological function. Of particular interest are interfacial aromatic residues and charged residues in the core helix whose functions range from stabilizing the native structure to regulating ion channels. This dissertation addresses the pH dependence and influence of potentially negatively charged tyrosine, glutamic acid or aspartic acid side chains. We have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as favorable host peptide framework. We have substituted W5 with Tyr (Y5GWALP23) and Leu residues with Glu (L12E, L14E or L16E) or Asp (L14D or L16D), and have incorporated specific 2H-labeled alanine residues within the …

Investigating The Impact Of Small Molecule Ligands And The Proteostasis Network On Protein Folding Inside The Cell, Karan Hingorani

Doctoral Dissertations

The folded forms of most proteins are critical to their functions. Despite the complexity of the cellular milieu and the presence of high-risk deleterious interactions, there is a high level of fidelity observed in the folding process for entire proteomes. Two important reasons for this are the presence of the quality control machinery consisting of chaperones and degradation enzymes that work jointly to optimize the population of the folded state and interaction partners that re-enforce the functional state and add to the competitive advantage of an organism. While substantial effort has been directed to understand protein folding and interactions in …

Characterizing The Inhibition Of Katanin Using Tubulin Carboxy-Terminal Tail Constructs, Corey E. Reed

Masters Theses

Understanding how the cellular cytoskeleton is maintained and regulated is important to elucidate the functions of many structures such as the mitotic spindle, cilia and flagella. Katanin p60, microtubule-severing enzymes from the ATPase associated with cellular activities (AAA+) family, has previously been shown in our lab to be inhibited by free tubulin as well as α- and β-tubulin carboxy-terminal tail (CTT) constructs. Here we investigate the inhibition ability of several different tubulin CTT sequences. We quantify the effect of the addition of these constructs on the severing and binding activity of katanin. We find that some constructs inhibit katanin better …

Tuning Into Toxins And Channels: The Characterizations Of Tv1 And A Human Cardiac Sodium Channel Voltage-Sensor Domain, Mohammed H. Bhuiyan

Dissertations, Theses, and Capstone Projects

In nature, peptide toxins are an abundant resource, produced both by marine and terrestrial organisms. A major target of these peptide toxins is the group of the highly important voltage-gated ion channels. Due to their high specificity and affinity, peptide toxins have been used for over a decade in discovery and characterization of voltage-gated ion channels. Although peptide toxins have been extensively characterized structurally, the structural characterization of eukaryotic voltage-gated sodium channels has seen much less progress, due to their large size and high hydrophobicity. Voltage-gated sodium channels play crucial roles in many physiological processes, and when these processes are …

Investigation Of An Energetic Coupling Between Ligand Binding And Protein Folding, Nathan W. Gardner

Open Access Dissertations

The cellular environment presents a protein with many small molecules with which it may interact. Many novel interactions between proteins and non-substrate metabolites are being uncovered through proteome-wide screens. The homodimeric Escherichia coli cofactor-dependant phosphoglycerate mutase (dPGM) was identified as an ATP binding protein in a proteome-wide screen, but dPGM does not use ATP for catalysis. This dissertation elucidates the effect of ATP and other non-substrate metabolites on dPGM. Initial investigations revealed a partially unfolded, monomeric intermediate of dPGM that forms during equilibrium unfolding. ATP binding was found to occur at the active site of dPGM and to be energetically …

Structural And Functional Characterization Of Acetoacetate Decarboxylase-Like Enzymes, Lisa Mueller

Theses and Dissertations

The acetatoacetate decarboxylase-like superfamily (ADCSF) is a largely unexplored group of enzymes that may be a potential source of new biocatalysts. Bioinformatic analysis has grouped these approximately 2000 enzymes into seven different families based on comparison of predicted active site residues. To date, only the prototypical ADCs (Family I) that catalyze the decarboxylation of acetoacetate have been studied. Analysis of gene context suggests that Family V contains predominantly enzymes predicted to be involved in secondary metabolism. On average, these share about 20% sequence identity to the true ADCs. To learn more about the diversity of chemistries performed by members of …

Pinpointing The Molecular Basis For Metal Ion Effects On Plasminogen Activator Inhibitor-1 (Pai-1), Joel Cullen Bucci

Doctoral Dissertations

Plasminogen activator inhibitor type-1 (PAI-1) specifically inhibits the proteases tissue type plasminogen activator and urokinase plasminogen activator to control the activation of fibrinolysis. Vitronectin interacts with PAI-1 primarily through the somatomedin B (SMB) domain to stabilize and localize PAI-1 to sites of injury. Our laboratory observed that transition metals such as copper²⁺ have VN dependent, reciprocal effects on how long PAI-1 remains active. We aim to determine the molecular basis for effects of copper²⁺ on PAI-1 activity. We employed a computational algorithm (MUG) to predict metal binding clusters, and introduced mutations hypothesized to create metal binding deficiency. We …

Interaction Of Spliceosomal U2 Snrnp Protein P14 With Its Branch Site Rna Target, William Perea Vargas

Dissertations, Theses, and Capstone Projects

Newly transcribed precursor messenger RNA (pre-mRNA) molecules contain coding sequences (exons) interspersed with non-coding intervening sequences (introns). These introns must be removed in order to generate a continuous coding sequence prior to translation of the message into protein. The mechanism through which these introns are removed is known as pre-mRNA splicing, a two-step reaction catalyzed be a large macromolecular machine, the spliceosome, located in the nucleus of eukaryotic cells. The spliceosome is a protein-directed ribozyme composed of small nuclear RNAs (snRNA) and hundreds of proteins that assemble in a very dynamic process. One of these snRNAs, the U2 snRNA, is …

Mechanism Of Calcium-Dependent Chloride Channel Activation By The Secreted Regulator Clca1, Zeynep Yurtsever

Arts & Sciences Electronic Theses and Dissertations

The calcium-activated chloride channel regulator (CLCA) proteins are key signaling molecules, which are implicated in various diseases through their tissue-specific expression. Human CLCA1 protein, overexpressed in airway epithelia under pathophysiological conditions, is centrally involved in the manifestation of IL-13-driven mucus cell metaplasia (MCM), a hallmark feature of asthma and chronic obstructive pulmonary disease (COPD), for which there are currently no available therapeutics. Elucidating the poorly understood molecular basis of CLCA1 function is thus required to design specific inhibitors of CLCA1 activity to treat MCM in asthma and COPD.

Originally misannotated as ion channels, CLCA proteins are secreted soluble proteins that …

Structure And Dynamics Of A Small Multidrug Resistance Transporter, Emre, Chao Wu

Arts & Sciences Electronic Theses and Dissertations

EmrE is a small multidrug resistance transporter in E. coli. It effluxes a wide range of antibiotics, thus contributing to the evolving epidemic of drug resistance. Despite its small size, EmrE is a fully functional transporter making it an ideal model system for a comprehensive study of the multidrug transport mechanism. In the transport cycle, EmrE must alternate between outward- and inward-facing conformations upon substrate binding to translocate substrates across the membrane. High-resolution structures of EmrE in complex with substrates facing different sides of the membrane will shed light on the coupling mechanism between substrate binding and transport. However, the …

Influence Of Cholesterol On Single Arginine-Containing Transmembrane Helical Peptides, Jordana K. Thibado

Chemistry & Biochemistry Undergraduate Honors Theses

An essential component of animal cells, cholesterol exerts significant influence on the physical properties of the cell membrane and in turn, its constituents. One such category of constituents, the membrane proteins, are responsible for diverse and essential biological functions and often contain polar amino acids. Although sparse within the hydrophobic interior of lipid-bilayer membranes, polar amino acid residues are highly conserved and may play pivotal roles in determining specific structural and functional properties of key proteins. To gain greater understanding of the lipid membrane environment, and more broadly, cellular function, a model peptide framework termed “GWALP23” (acetyl-GGALWLALALAL¹²AL^{14 …}

Biophysical Studies Of The Allosteric Regulatory Mechanism Of Syk Tandem Sh2 Domains Interacting With Immunoreceptor Tyrosine-Based Activation Motifs, Chao Feng

Open Access Dissertations

The non-receptor spleen tyrosine kinase (Syk) is an important player in signal transduction from immunoreceptors to various downstream targets. It is widely expressed in both haematopoietic and epithelial cells. Syk disorder is closely related with many inflammatory and autoimmune diseases, as well as cancers.

Syk associates with immunoreceptors through its tandem SH2 domains (tSH2), which contain two SH2 domains connected by interdomain A. The association of Syk with immunoreceptors is regulated by Y130 phosphorylation in interdomain A. The unphosphorylated tSH2 can bind with the doubly phosphorylated immunoreceptor tyrosine-based activation motif (dp-ITAM) of the cytoplasmic domains of immunoreceptors with very high …

Learning The Abc's Of Ribose Transport Using Biophysical Methods, Satchal K. Erramilli

Open Access Dissertations

ATP-binding cassette transporters comprise a large superfamily of proteins that are involved in a variety of biological phenomenon, from bacterial metabolism to cellular homeostasis, antigen-presentation, and drug resistance. These proteins are implicated in a variety of clinically relevant phenomenon, including the human diseases cystic fibrosis, macular degeneration, and cancer. Understanding their structure-function can guide therapeutics and contribute to our overall understanding of these biological phenomena.

This study focuses on understanding the motor protein of the bacterial ribose ABC transporter in the context of transport. This complex is required for the uptake of the nucleotide precursor, ribose. Using biophysical methods, we …

Characterization Of Cu-Rich Aggregates In Neurogenic Niches Of The Rodent Brain By X-Ray Fluorescence Microscopy, Brendan T. Sullivan

Open Access Dissertations

Copper is an essential element in the brain playing several critical roles ranging from neurotransmitter synthesis to ATP production. As Cu is typically present in micromolar concentrations and has a spatially capricious distribution in the brain, determining concentrations has historically been challenging. X-ray fluorescence microscopy (XRF) offers excellent spatial resolution (down to 30~nm) and detection limits (sub parts per million), making it an excellent tool for analyzing metal distributions in the brain. Using XRF, it is demonstrated that Cu-rich aggregates with concentrations in the hundreds of millimolar are present in the subventricular zone of rats and mice. As the subventricular …

The Interaction Between Eukaryotic Translation Initiation Factor Eif4g And 3’ Cap Independent Translation Element Of Barley Yellow Dwarf Virus Is Affected By Multiple Initiation Factors, Pei Zhao

Dissertations, Theses, and Capstone Projects

Barley Yellow Dwarf Virus (BYDV) lacks a 5’ (7-methyl guanosine) cap as well as a 3’poly A tail. Like many plant viruses, BYDV contains a cap independent translation element (CITE) in the 3’ untranslated region of the viral mRNA. BTE (Barley Yellow Dwarf Virus like cap-independent translation element) is one of the well characterized CITEs. BTE mediated translation primarily depends on eukaryotic initiation factor eIF4G. BTE binds to eIF4G; however, the details of BTE initiated translation are still unclear. Three eIF4G deletion mutants with different domain organization were used to investigate BTE interaction with eIF4G: eIF4G601-1196 is the eIF4G fragment …

Calcineurin: From Activation To Inhibition, Erik C. Cook

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin is a Ser/Thr phosphatase whose function is implicated in critical physiological processes such as immune system activation, fetal heart development, and long-term depression in neurons. Calcineurin has been implicated in the progression of Alzheimer’s disease and cardiac hypertrophy. It is not well understood how calcineurin is activated on a molecular level by Ca²⁺ and its activating protein calmodulin. Previous data from our lab show that calmodulin interaction induces the folding of the intrinsically disordered regulatory domain of calcineurin in two discrete and distant regions into α-helical conformations and that this folding is critical for complete activation of calcineurin. …

Functional Effects Of Calcium Regulation Of Thin Filaments At Single Particle Resolution, Christopher Solis-Ocampo

Electronic Theses and Dissertations

Heart disease is the leading cause of death in the United States. Understanding heart function at the molecular level is critical for developing of more effective treatments. In the cardiac muscle, the thin filament is composed by troponin (Tn), tropomyosin (Tm), and F-actin. It provides Ca2+-dependent regulation of contraction by modulating myosin attachment and force generation in a cooperative scheme. However, this mechanism remains unclear. To understand thin filament activation, we studied the binding and functional properties of Tn and Tm to F-actin at single particle resolution by employing fluorescence image colocalization, in vitro motility assays, and Förster resonance energy …

Probing Allosteric, Partial Inhibition Of Thrombin Using Novel Anticoagulants, Stephen S. Verespy Iii

Theses and Dissertations

Thrombin is the key protease that regulates hemostasis; the delicate balance between procoagulation and anticoagulation of blood. In clotting disorders, like deep vein thrombosis or pulmonary embolism, procoagulation is up-regulated, but propagation of clotting can be inhibited with drugs targeting the proteases involved, like thrombin. Such drugs however, have serious side effects (e.g., excessive bleeding) and some require monitoring during the course of treatment. The reason for these side effects is the mechanism by which the drugs’ act. The two major mechanisms are direct orthosteric and indirect allosteric inhibition, which will completely abolish the protease’s activity. Herein we sought an …

Lipid-Protein Interactions Via Fluorescence Quenching In Small Unilamellar Vesicles, Talia Kordahi

Williams Honors College, Honors Research Projects

Studying peptide interactions with model membranes have aided in understanding protein-lipid interactions more clearly. In this report, fluorescent small unilamellar vesicles (SUVs) of a known composition were made. Two peptides, RND1 and MARCKS, were also fluorescently labeled and added to a solution of SUVs. The interactions between these were observed via fluorescence quenching, read out with a spectrofluorimeter. In addition, two environments of buffer were tested: 1X PBS and 0.1X PBS. After obtaining data, it was concluded that MARCKS quenched the SUVs more than RND1. This could be due to the presence of more lysine amino acids in MARCKS, which …

A Biophysical Study Of The Ion Transport Mechanism In Uncoupling Protein 2 By Investigating The Role Of Lysine Residues In Its Matrix Network, Stephanie O. Uwumarenogie

Theses and Dissertations (Comprehensive)

ABSTRACT

Uncoupling protein 2 (UCP2) is one of five UCP homologues found in the inner mitochondrial membrane that transports protons from the intermembrane space to the mitochondrial matrix. In turn, the proton motive force is dissipated and less ATP is produced in the mitochondria. UCP2 is proposed to influence insulin secretion in type II diabetes, and decrease the amount of reactive oxygen species produced in the mitochondria, however the detailed mechanism of ion (proton and anions) transport in UCP2 and other UCP homologues are not fully understood. Sequence alignment analysis performed on proteins in the mitochondrial carrier family (MCF) including …