Biochemistry, Biophysics, and Structural Biology Commons^™

Minimalistic Peptide-Based Supramolecular Systems Relevant To The Chemical Origin Of Life, Daniela Kroiss

Dissertations, Theses, and Capstone Projects

All forms of life are based on biopolymers, which are made up of a selection of simple building blocks, such as amino acids, nucleotides, fatty acids and sugars. Their individual properties govern their interactions, giving rise to complex supramolecular structures with highly specialized functionality, including ligand recognition, catalysis and compartmentalization. In this thesis, we aim to answer the question whether short peptides could have acted as precursors of modern proteins during prebiotic evolution. Using a combination of experimental and computational techniques, we screened a large molecular search space for peptide sequences that are capable of forming supramolecular complexes with adenosine …

Molecular Dynamics Simulations Of Supramolecular Assemblies In Biology And Bionanotechnology, Anjela Manandhar

Dissertations, Theses, and Capstone Projects

Molecular self-assembly is an energy driven process where randomly organized building blocks interact noncovalently to form highly organized supramolecular nanostructures. In biology, the cytoskeleton is a classic example of a dynamic self-assembly, forming long filamentous structures from monomeric protein subunits. Similarly, the self-assembly process is widely exploited in nanotechnology to build bio-functional nanostructures. In this work, we studied biological (microtubule) and synthetic (peptide drug amphiphile nanotube) self-assembled systems. We utilized long time-scale molecular dynamics simulation to investigate the structural and dynamical properties of these systems.

At the molecular level, the dynamic instability (random growth and shrinkage) of the microtubule (MT) …

Hyaluronan At The Brain-Environment Interface, Donald M. Thevalingam

Dissertations, Theses, and Capstone Projects

Hyaluronan (HA; Hyaluronic Acid), a primary scaffolding component of the brain extracellular matrix, serves as an integral structural component to the brain extracellular space (ECS). The fossorial African naked mole-rat (Heterocephalus glaber; NM-R), a mammal which lives in a low-oxygen environment and is capable of tolerating hypoxia and hypercapnia, has been shown to synthesize and sustain a unique high-molecular-mass variant of hyaluronan macromolecule (HMM-HA). This body of work highlights HA’s role in mediating the interplay between brain ECM composition, ECS structure, and cell viability.

Here we employ the NM-R as a unique animal model to observe the role of the …

Exploration Of The Inhibitory Properties Of The Nucleoside Antibiotic Salicyl-Ams And Analogues Targeting Siderophore Biosynthesis In Mycobacterium Tuberculosis, Glennon Bythrow

Dissertations, Theses, and Capstone Projects

Mycobacterium tuberculosis (Mtb) is a resilient, obligate bacterial pathogen responsible for pulmonary tuberculosis disease (TB), that has upheld a significant impact on global public health throughout history. The World Health Organization (WHO) approximates nearly 10 million new TB cases arose in 2017 alone, accounting for 1.6 million deaths. There has been a notable rise in TB cases produced by multidrug‑resistant (MDR) and extensively drug-resistant (XDR) strains of Mtb. This, along with the intrinsic resistance of Mtb to many standard drugs and poor patient compliance, is deeply impacting global control of TB. Among the several strategies currently in …

Characterization Of Extracellular Vesicles From Human Diabetic Retinopathy Retinal Tissue In Vitro And From Urine Of Human Patients With Diabetic Retinopathy, Jason Mighty

Dissertations, Theses, and Capstone Projects

Diabetic Retinopathy (DR) is a neurovascular complication associated with diabetes mellitus that affects approximately 120 million people worldwide and its prevalence is expected to reach 190 million by 2030. DR diagnosis is accomplished with fundus ophthalmoscopy often when retinal damage and vision loss have already occurred. A group of biomarker being explored for early detection of diseases are extracellular vesicles (EVs), which are nanometer diameter lipid enclosed vesicles, released from all cell types and containing genetic cargo reflective of releasing cell state. EV biomarkers are currently being explored to help monitor disease predisposition, pathogenesis and response to treatment. While an …

The Role And Regulation Of Alternative Polyadenylation In The Dna Damage Response, Michael R. Murphy

Dissertations, Theses, and Capstone Projects

Cellular homeostasis is achieved by the dynamic flux in gene expression. Post-transcriptional regulation of coding and non-coding RNA offers a fast method of adapting to a changing cellular environment, including deadenylation, microRNA (miRNA) pathway, and alternative polyadenylation (APA). In this dissertation, I explored some of the mechanisms involved in the post-transcriptional regulation of gene expression. The main hypothesis in these studies is that a single APA event after DNA damage is governed by specific conditions and factors outside of current known regulators of APA, and that the resultant transcript has a role in the DNA damage response (DDR). My aims …

Structural Studies On Calcium/Calmodulin-Dependent Activation Of Eukaryotic Elongation Factor 2 Kinase, Kwangwoon Lee

Dissertations, Theses, and Capstone Projects

Eukaryotic elongation factor 2 kinase (eEF-2K) is a key modulator of the rate of protein synthesis. Activated by calcium-loaded calmodulin (Ca²⁺-CaM), eEF-2K phosphorylates its only known physiological substrate, eEF-2, on a specific threonine residue (Thr-56). Phosphorylated eEF-2 has reduced affinity for the ribosome, and results in a significant decrease in the rate of translation elongation. Modulation of the rate of translation elongation plays a crucial role in proteostasis – adequate regulation of protein synthesis, protein folding, and protein degradation that greatly influences cellular growth and survival. Binding of Ca²⁺-CaM triggers activation of eEF-2K and remains intact …

Characterization Of Immunomodulatory Microbial Factors In Medicinal Plants, Kriti Kalpana

Dissertations, Theses, and Capstone Projects

Medicinal plants are one of the biggest sources of natural products with therapeutic importance. There are currently over 28,000 plants with putative medicinal values. Plant-derived compounds have been explored extensively for various biological activities ranging from anti-cancer, immune-boosting to anti-inflammatory and anti-oxidant. Some of the most important therapeutic agents are of plant-origin, such as paclitaxel from Pacific yew (Taxus brevifolia) and artemisinin from qinghao su (a Chinese medicinal herb; a.k.a. Artemisia annua) to name a few.

The study presented in this thesis started out as classical pharmacognosy research, which focused on the identification of immunostimulatory factors in …

Solvation Thermodynamic Mapping In Computer Aided Drug Design, Steven Ramsey

Dissertations, Theses, and Capstone Projects

The displacement of water from surfaces upon biomolecular recognition and association makes a significant contribution to the free energy changes of these processes. We therefore posit that accurate characterization of local structural and thermodynamic molecular water properties can improve computational model accuracy and predictivity of recognition and association processes. In this thesis, we discuss Solvation Thermodynamic Mapping (STM) methods that we have developed using inhomogeneous fluid solvation theory (IST) to better characterize active site water structural and thermodynamic properties on protein surfaces and the open source tools that we have developed, GIST-CPPTRAJ and SSTMap, which implement these methods which we …

The Determining Factors Of The Self-Assembly Of Collagen Mimetic Peptide, Fangfang Chen

Dissertations, Theses, and Capstone Projects

We recently reported that a designed triple helix Col108 self-associates to form collagen-like mini-fibrils having a d-periodicity of 35 nm. Recombinant protein Col108 consists of 378-residue triple helix domain organized into three repeating sequence units, and a C-terminal foldon domain. The 35 nm d-period of Col108 mini-fibrils is consistent with the one unit staggered arrangement of the associating helices. To further investigate if the sequence periodicity is crucial for collagen mimetic peptide fibril formation, we studied the self-assembly of four other designed triple helical peptides. Peptide 2U108 has two repeating sequence units; peptide Col877 consists of three repeating …

Pretargeted Radioimmunotherapy Utilizing The Inverse Electron Demand Diels-Alder Reaction, Rosemery Membreno

Dissertations, Theses, and Capstone Projects

Radioimmunotherapy capitalizes on the specificity and affinity of antibodies for their antigens to localize radioactivity at the tumor site. One limitation in using antibodies is their long circulation time, which can take days to weeks to fully clear from blood circulation. When a radioisotope is directly conjugated to this immunoglobulin vector, the red marrow and other healthy organs are receiving a constant radiation dose while it is in blood circulation. This not only harms healthy organs, but limits the dose of radioactivity that can be administered thus prolonging treatment. A promising alternative to limit the radiation dose to non-tumor tissue …

Bisthioether Stapled Peptides Targeting Polycomb Repressive Complex 2 Gene Repression, Gan Zhang

Dissertations, Theses, and Capstone Projects

Interactions between proteins play a key role in nearly all cellular process, and therefore, disruption of such interactions may lead to many different types of cellular dysfunctions. Hence, pathologic protein-protein interactions (PPIs) constitute highly attractive drug targets and hold great potential for developing novel therapeutic agents for the treatment of incurable human diseases. Unfortunately, the identification of PPI inhibitors is an extremely challenging task, since traditionally used small molecule ligands are mostly unable to cover and anchor on the extensive flat surfaces that define those binary protein complexes. In contrast, large biomolecules such as proteins or peptides are ideal fits …

Ck2 Negatively Regulates 5-Ht4 Receptor Signaling In The Prefrontal Cortex And Mediates Depression-Like Behaviors, Julia Castello Saval

Dissertations, Theses, and Capstone Projects

The serotonergic system has been the major candidate in the pathophysiology of mood related disorders such as anxiety and major depressive disorder (MDD). Unfortunately, current antidepressant drugs are ineffective in 50% of the population and require chronic administration for a period of 3-6 weeks before the onset of therapeutic response. 5-HT4 receptor (5-HT₄R) agonists have emerged as potential candidates for fast antidepressant action, since an antidepressant response can be achieved after 3 days of pharmacological administration in rodents.

This dissertation aims to investigate the role of casein kinase 2 (CK2) as a regulator of 5-HT₄R expression …

Phospholipase D-Dependent Mtorc1 Activation By Glutamine, Elyssa Bernfeld

Dissertations, Theses, and Capstone Projects

Glutamine, the conditionally essential amino acid and most abundant amino acid in human sera, is a key nutrient required for sustaining cell proliferation. Glutamine is essential for nucleotide, protein, and lipid synthesis, all of which are essential for cell proliferation. The mammalian target of rapamycin complex 1 (mTORC1) is a highly conserved protein complex that acts as a sensor of nutrients, relaying signals for the shift from catabolic to anabolic metabolism. While glutamine plays an important role in activating mTORC1, the mechanism is not completely clear. Here we describe a Rag-independent mechanism of mTORC1 activation by glutamine that is dependent …

Mechanisms Adopted By Dengue-2 Viruses To Induce Autophagy In Mammalian Cells, Sounak Ghosh Roy

Dissertations, Theses, and Capstone Projects

Dengue, the most rapidly spreading flavivirus, threatens to affect almost half of the human global population. We previously showed that dengue-2 protects canine kidney cells (MDCK) from cytotoxic chemicals. We showed, independently, that cell protection, as well as viral replication and maturation, are positively regulated by PI3K-dependent autophagy. However, we had not identified the specific pathway that induces autophagy in infected cells. The current study explores the role of a specific branch of the endoplasmic reticulum (ER) stress-mediated Unfolded Protein Response (UPR), the PERK/eIF2α/ATF4 pathway in the induction of autophagy by …

Renal Risk Variants Of Apolipoprotein L-1 Form Channels At The Plasma Membrane That Lead To A Cytotoxic Influx Of Calcium, Joseph A. Giovinazzo

Dissertations, Theses, and Capstone Projects

Apolipoprotein L-1 (APOL1) is a secreted protein that provides protection against several protozoan parasites due to its channel forming properties. Recently evolved variants, G1 and G2, increase kidney disease risk when present in two copies. In mammalian cells, overexpression of G1 and G2, but not wild-type G0, leads to swelling and eventual lysis. However, the mechanism of cell death remains elusive with multiple pathways being invoked, such as autophagic cell death mediated by a BH3 domain in APOL1, which we evaluated in this study. We hypothesized that the common trigger for these pathways is the APOL1 cation channel, which is …

Towards An Atomic Level Model Of The Structure And Calmodulin Mediated Activation Of Eef-2k, Nathan E. Will

Dissertations, Theses, and Capstone Projects

Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM) dependent member of the a-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific residue (Thr-56), decreasing its affinity for the ribosome and reducing the rate of peptide chain elongation during protein translation. In contrast to the “release-of-inhibition’ mechanism operative in most CaM-dependent proteins kinases, the activation of eEF-2K is proposed to occur through a two-step process subsequent to the engagement of CaM and involves (1) auto-phosphorylation on T348 and (2) engagement of an allosteric site by phospho-T348 leading to a state with the highest activity towards the substrate eEF-2. …

Supercharged Models Of Intrinsically Disordered Proteins And Their Utility In Sensing, Peter J. Schnatz

Dissertations, Theses, and Capstone Projects

In this thesis I show that greatly increasing the magnitude of a protein’s net charge using surface supercharging transforms that protein into a ligand-gated or counterion-gated conformational molecular switch. To demonstrate this I first modified the designed helical bundle hemoprotein H4 using simple molecular modeling, creating a highly charged protein which both unfolds reversibly at low ionic strength and undergoes the ligand-induced folding transition commonly observed in signal transduction by intrinsically disordered proteins in biology. Due to the high surface charge density, ligand binding to this protein is allosterically activated by low concentrations of divalent cations and the polyamine spermine. …

Binding Of Maize Necrotic Streak Virus (Mnesv) 3’ I-Shaped Structure (3’ Iss) To Eukaryotic Translation Factors (Eifs) And Implication In Eif4f Mediated Translation Initiation, Qiao Liu

Dissertations, Theses, and Capstone Projects

5' m⁷GpppN cap and the 3' poly adenosine (A) tail of eukaryotic mRNAs are key elements for recruiting translation initiation machinery in canonical translation initiation. Unlike host mRNAs, many viruses lack these elements and yet they are translated efficiently. Plant viruses, in particular, have complex structures within their untranslated regions (UTR) that allow them to bypass some cellular translation control steps. In Maize necrotic streak virus (MNeSV) 3' UTR, an I-Shaped RNA Structure (ISS) has been reported to mediate the virus translation initiation progress. 3’ ISS binding with eIF4F has been shown to facilitate translation. 5’ -3’ kissing …

Evidence For Organelle-Like Extracellular Vesicles From A Parasite Of Drosophila And Their Function In Suppressing Host Immunity, Mary Heavner

Dissertations, Theses, and Capstone Projects

Parasitic wasps act as keystone species in natural ecosystems. Adept at suppressing immunity of their insect hosts, these natural enemies of insect pests are used for biocontrol in many parts of the world. Female parasitic wasps of the closely-related species Leptopilina heterotoma (Lh), a generalist of many Drosophilia flies, and Leptopilina boulardi (Lb), a specialist on flies of the melanogaster subgroup, produce venom and virus-like particles (VLPs) in their long gland-reservoir complexes, a secretory organ connected to ovipositors. Venom and VLPs are deposited, along with wasp eggs, into the body of the wasp’s larval fly host …

Regulation Of The Tubulin Homolog Ftsz In Escherichia Coli, Monika S. Buczek

Dissertations, Theses, and Capstone Projects

Escherichia coli is a well-known pathogen, and importantly, a widely used model organism in all fields of biological sciences for cloning, protein purification, and as a model for Gram-negative bacterial species. And yet, researchers do not fully understand how this bacterium replicates and divides. Every year additional division proteins are discovered, which adds complexity to how we understand E. coli undergoes cell division. Due to their specific roles in cytokinesis, some of these proteins may be potential targets for development of antibacterials or bacteriostatics, which are much needed for fighting the current global antibacterial deficit. My thesis work focuses on …

Mechanism And Development Of Peptide-Based Inhibitors To Human Islet Amyloid Polypeptide (Hiapp) Self-Assembly, Jayson Vedad

Dissertations, Theses, and Capstone Projects

Amyloid fibrils formed by of hIAPP_1-37 (also known as amylin) has been linked to type-II diabetes mortalities and its formation was found to be related to the three aromatic residues in hIAPP_1-37. In this dissertation, the role of aromatic amino acids, particularly that of Phe-23, and its various interactions to the self-assembly of human islet amyloid polypeptide (hIAPP)_22-29 were investigated. Using a variety of spectroscopic techniques with emphasis to vibrational spectroscopy (FT-IR and Raman spectroscopies) in conjunction with computational methods, different factors leading to aggregation as well as its inhibition were identified. Among the driving forces …

Functional Interactions Of Tau, Pin1, P53 And Mrna 3′ Processing Factors In Non-Neuronal Cells, Jorge E. Baquero

Dissertations, Theses, and Capstone Projects

ABSTRACT

mRNA 3’ end processing, an essential step in eukaryotic RNA metabolism, regulates the steady-state levels of different mRNAs and contributes to the cells rapid response to stress. Studies have described potential contributions of mRNA 3’ end processing to numerous human diseases, including cancer and Alzheimer’s disease (AD). Therefore, the main purpose of this dissertation is to further elucidate some of the roles of disease-related factors Pin1, p53 and tau in the regulation mRNA 3’ end processing in non-neuronal cells under different cellular conditions, including during the DNA damage response (DDR).

The results from Chapter II show that the prolyl …

Mechanisms For Survival And Drug Resistance In Cancer Cells, Matthew B. Utter

Dissertations, Theses, and Capstone Projects

PART I

Prostate cells are hormonally driven to grow and divide. Typical treatments for prostate cancer involve blocking the hormone androgen from activating the androgen receptor (AR) and thus inhibit growth and proliferation of the cancer. Androgen deprivation therapy (ADT) can lead to the selection of cancer cells that grow and divide independently of androgen receptor activation. Prostate cancer cells that are insensitive to androgens commonly display metastatic phenotypes and reduced long-term survival of patients. In this study, we provide evidence that androgen-insensitive prostate cancer cells have elevated phospholipase D (PLD) activity relative to the androgen-sensitive prostate cancer cells. PLD …

Pharmacological Antagonism And The Olfactory Code, Mihwa Na

Dissertations, Theses, and Capstone Projects

Mammals can detect and discriminate uncountable odors through their odorant receptors. To accommodate the countless and diverse odors, exceptionally large numbers of odorant receptor (OR) genes are expressed in mammals. In addition, the mammals utilize a combinatorial code, where an odorant molecule can activate multiple ORs; an OR also responds to a set of multiple odorants. In nature, an odor is often a complex mixture of multiple odorant molecules. The combination of the ORs activated by each constituent generates the unique olfactory code for the particular odor.

Some odorants can antagonize select ORs, as discussed in Chapter 1. An antagonist …

Structural And Biochemical Studies Of The Dna Replication Initiation Mechanism Of The Second Chromosome Of Vibrio Cholerae, Natalia Orlova

Dissertations, Theses, and Capstone Projects

Transmission of genetic information through DNA replication is one of the key processes for any living organism. Despite the extensive effort put into studies of the mechanism of DNA replication, the understanding of the process on the molecular level is still incomplete. Specifically the molecular details of the very first events of DNA replication initiation are not sufficiently understood.

The majority of bacteria possess a single circular chromosome, and in order to initiate DNA replication these organisms utilize a conserved system, consisting of a specific DNA sequence - replication origin, called oriC, and replication initiator protein DnaA. However, bacteria …

Regulation Of The Amyloid Precursor Protein By Prostaglandin J2, A Mediator Of Inflammation: Relevance To Alzheimer’S Disease, Teneka L. Jean-Louis

Dissertations, Theses, and Capstone Projects

Inflammation plays a major role in Alzheimer’s disease (AD). Investigating how specific mediators of inflammation contribute to neurodegeneration in AD is crucial. Our studies focused on cyclooxygenases, which are key enzymes in inflammation and highly relevant to AD. Cyclooxygenases (COX -1, constitutive; COX-2, inducible) have emerged as important determinants of AD pathogenesis and progression. COX-2 is highly induced in AD, correlating with AD severity, and COX-1 is also involved in AD. Cyclooxygenases are the rate-limiting enzymes that convert arachidonic acid into prostaglandins (PGs), the principal mediators of CNS neuroinflammation.

The overall GOAL of these studies was to address the mechanisms …

Computational Investigation Of The Pore Formation Mechanism Of Beta-Hairpin Antimicrobial Peptides, Richard Lipkin

Dissertations, Theses, and Capstone Projects

β-hairpin antimicrobial peptides (AMPs) are small, usually cationic peptides that provide innate biological defenses against multiple agents. They have been proposed as the basis for novel antibiotics, but their pore formation has not been directly observed on a molecular level. We review previous computational studies of peptide-induced membrane pore formation and report several new molecular dynamics simulations of β-hairpin AMPs to elucidate their pore formation mechanism. We simulated β-barrels of various AMPs in anionic implicit membranes, finding that most of the AMPs’ β-barrels were not as stable as those of protegrin. We also performed an optimization study of protegrin β-barrels …

Insight Into The Interaction Between The Peroxisome Proliferator-Activated Receptor Gamma (Pparγ) And Adipocyte Fatty Acid-Binding Protein (A-Fabp), Qian Wang

Dissertations, Theses, and Capstone Projects

The Adipocyte Fatty Acid-Binding Protein (AFABP) is mainly expressed in fat cells. It can bind fatty acids and other lipophilic substances such as eicosanoids and retinoids. The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor protein that requires ligand binding to regulate the specific gene transcription. PPARγ is expressed at extremely high levels in adipose tissue, macrophages, and the large intestine, where it controls lipid adipogenesis and energy conversion. Moreover, it has been found that AFABP and PPARγ can form a complex in vivo. It is proposed that AFABP carries the ligand and enters into the nucleus where it …

A Combined Computational Strategy Of Sequence And Structural Analysis Predicts The Existence Of A Functional Eicosanoid Pathway In Drosophila Melanogaster, Michael Scarpati

Dissertations, Theses, and Capstone Projects

With increased understanding of their roles in signal transduction and metabolism, eicosanoids have emerged as important players in human health and disease. Mammalian prostanoids and related lipid mediators perform varied functions in different tissues and organs. Synthesized through the oxygenation of C20 polyunsaturated fatty acids, mammalian eicosanoids are both pro- and anti-inflammatory. The physiological contexts in which eicosanoid family members act at the cellular level are not well understood. In this study, we examined whether the genome of Drosophila melanogaster, a powerful model for innate immunity and inflammation, codes for the enzymes required for eicosanoid biosynthesis. We report the …