Biochemistry, Biophysics, and Structural Biology Commons^™

Synthesis And Evaluations Of “1,4-Triazolyl Combretacoumarins” And Desmethoxy Analogs, Tashrique A. Khandaker, Jessica D. Hess, Renato Aguilera, Graciela Andrei, Robert Snoeck, Dominique Schols, Padmanava Pradhan, Mahesh K. Lakshman

Publications and Research

1,4-Triazolyl combretacoumarins have been prepared by linking the trimethoxyarene unit of combretastatin A4 with coumarins, via a 1,2,3-triazole. For this, 4-azidocoumarins were accessed by a sequential two-step, one-pot reaction of 4-hydroxycoumarins with (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), followed by reaction with NaN₃. In the reaction with BOP, a coumarin-derived phosphonium ion intermediate seems to form, leading to an O⁴-(benzotriazolyl)coumarin derivative. For the CuAAC reaction of azidocoumarins with 5-ethynyl-1,2,3-trimethoxybenzene, catalytic [(MeCN)₄Cu]PF₆ in CH₂Cl₂/MeOH with 2,6-lutidine, at 50 ^oC, was suitable. The 4-azidocoumarins were less reactive as compared to PhN₃ and …

Reduced Alphabet Of Prebiotic Amino Acids Optimally Encodes The Conformational Space Of Diverse Extant Protein Folds, Armando D. Solis

Publications and Research

Background

There is wide agreement that only a subset of the twenty standard amino acids existed prebiotically in sufficient concentrations to form functional polypeptides. We ask how this subset, postulated as {A,D,E,G,I,L,P,S,T,V}, could have formed structures stable enough to found metabolic pathways. Inspired by alphabet reduction experiments, we undertook a computational analysis to measure the structural coding behavior of sequences simplified by reduced alphabets. We sought to discern characteristics of the prebiotic set that would endow it with unique properties relevant to structure, stability, and folding.

Results

Drawing on a large dataset of single-domain proteins, we employed an information-theoretic measure …

Probing The Formation Of Reactive Oxygen Species By A Porous Self- Assembled Benzophenone Bis-Urea Host, Baillie A. Dehaven, Hannah K. Liberatore, Alexander Greer, Susan D. Richardson, Linda S. Shimizu

Publications and Research

Herein, we examine the photochemical formation of reactive oxygen species (ROS) by a porous benzophenone-containing bis-urea host (1) to investigate the mechanism of photooxidations that occur within the confines of its nanochannels. UV irradiation of the self-assembled host in the presence of molecular oxygen generates both singlet oxygen and superoxide when suspended in solution. The efficiency of ROS generation by the host is lower than that of benzophenone (BP), which could be beneficial for reactions carried out catalytically, as ROS species react quickly and often unselectively. Superoxide formation was detected through reaction with 5,5- dimethyl-1-pyrroline N-oxide in the presence of …

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 …

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 …

Global Biochemical And Structural Analysis Of The Type Iv Pilus From The Gram-Positive Bacterium Streptococcus Sanguinis, Jamie-Lee Berry, Ishwori Gurung, Jan Haug Anonsen, Ingrid Spielman, Elliot Harper, Alexander M. J. Hall, Vivianne J. Goosens, Claire Raynaud, Michael Koomey, Nicolas Biais, Steve Matthews, Vladimir Pelicic

Publications and Research

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis. In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis. We found that the two major pilins, PilE1 and PilE2, (i) follow …

Arabinose Substitution Effect On Xylan Rigidity And Self-Aggregation, Utsab Shrestha, Sydney Smith, Sai Venkatesh Pingali, Hui Yang, Mai Zahran, Llyod Breunig, Liza Wilson, Daniel Cosgrove, Hugh O'Neill, Loukas Petridis

Publications and Research

Substituted xylans play an important role in the structure and mechanics of the primary cell wall of plants. Arabinoxylans (AX) consist of a xylose backbone substituted with arabinose, while glucuronoarabinoxylans (GAX) also contain glucuronic acid substitutions and ferulic acid esters on some of the arabinoses. We provide a molecular-level description on the dependence of xylan conformational, selfaggregation properties and binding to cellulose on the degree of arabinose substitution. Molecular dynamics simulations reveal fully solubilized xylans with a low degree of arabinose substitution (lsAX) to be stiffer than their highly substituted (hsAX) counterparts. Small-angle neutron scattering experiments indicate that both wild-type …

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 …

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 …

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 …

Putative Cellular And Molecular Roles Of Zika Virus In Fetal And Pediatric Neuropathologies, Rajendra Ghardbaran, Rajendra Ghardbaran, Latchman Somenarain

Publications and Research

Although the World Health Organization declared an end to the recent Zika virus (ZIKV) outbreak and its association with adverse fetal and pediatric outcome, on November 18, 2016, the virus still remains a severe public health threat. Laboratory experiments thus far supported the suspicions that ZIKV is a teratogenic agent. Evidence indicated that ZIKV infection cripples the host cells' innate immune responses, allowing productive replication and potential dissemination of the virus. In addition, studies suggest potential transplacental passage of the virus and subsequent selective targeting of neural progenitor cells (NPCs). Depletion of NPCs by ZIKV is associated with restricted brain …

Deletion Of Mgr2p Affects The Gating Behavior Of The Tim23 Complex, Oygul Mirzalieva, Shinhye Jeon, Kevin Damri, Ruth Hartke, Layla Drwesh, Keren Demishtein-Zohary, Abdussalam Azem, Cory D. Dunn, Pablo M. Peixoto

Publications and Research

The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast appears to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal amino acid signal. However, the mechanism of lateral release is unknown. Here, we used patch clamp electrophysiology …

Characterizing Chromosomal Aberrations In Cells Deficient For Both Atm And Msh2, Yeliz Inalman

Dissertations and Theses

Ataxia telangiectasia mutated (ATM) and mutS homologue 2 (MSH2) are important DNA repair proteins that participate in DNA repair pathways to maintain genomic integrity. Mice deficient for ATM and MSH2 mice are viable. However, ATM^-/- mice show growth retardation, neurological defects, and spontaneous lymphomagenesis. MSH2^-/- mice suffer from aggressive lymphoid tumors between two to five months of age and have increased microsatellite instability, which predisposes MSH2^-/- mice to carcinomas. However, mice deficient in both ATM and MSH2 are unable to survive beyond postnatal day 21 (P21). The observed lethality in ATM^-/-MSH2^-/- mice may result …

Cryptic Diversity In The Mexican Highlands: Thousands Of Uce Loci Help Illuminate Phylogenetic Relationships, Species Limits And Divergence Times Of Montane Rattlesnakes (Viperidae: Crotalus ), Christopher Blair, Robert W. Bryson Jr, Charles W. Linkem, David Lazcano, John Klicka, John E. Mccormack

Publications and Research

With the continued adoption of genome‐scale data in evolutionary biology comes the challenge of adequately harnessing the information to make accurate phylogenetic inferences. Coalescent‐based methods of species tree inference have become common, and concatenation has been shown in simulation to perform well, particularly when levels of incomplete lineage sorting are low. However, simulation conditions are often overly simplistic, leaving empiricists with uncertainty regarding analytical tools. We use a large ultraconserved element data set (>3,000 loci) from rattlesnakes of the Crotalus triseriatus group to delimit lineages and estimate species trees using concatenation and several coalescent‐based methods. Unpartitioned and partitioned maximum …

Luciferin Production And Luciferase Transcription In The Bioluminescent Copepod Metridia Lucens, Michael Tessler, Jean P. Gaffney, Jason M. Crawford, Eric Trautman, Nehaben A. Gujarati, Philip Alatalo, Vincent A. Pierbone, David F. Gruber

Publications and Research

Bioluminescent copepods are often the most abundant marine zooplankton and play critical roles in oceanic food webs. Metridia copepods exhibit particularly bright bioluminescence, and the molecular basis of their light production has just recently begun to be explored. Here we add to this body of work by transcriptomically profiling Metridia lucens, a common species found in temperate, northern, and southern latitudes. In this previously molecularly-uncharacterized species, we find the typical luciferase paralog gene set found in Metridia. More surprisingly, we recover noteworthy putative luciferase sequences that had not been described from Metridia species, indicating that bioluminescence produced by these copepods …

Optimizing Spacer Length For Positioning Functional Groups In Bio-Waste, Revathi Iyengar, Maria Faure-Betancourt, Saleh Talukdar, Jinting Ye, Abel E. Navarro

Publications and Research

The goal of this study was to determine the optimal chain length needed for tethering functional groups on bio-wastes. The purpose of modifying the surface of bio-waste is to improve their affinity for phenols. To this end, four different aminated green tea leaves, with the amine group located at the end of 6, 8, 10, and 12 carbons were synthesized. Green approaches to functionalization lead to fewer reactive sites. Optimizing spacer length is one way to ameliorate this. The aminated tea leaves were prepared by a tosylation reaction followed by displacement with a diamine used in excess. The tea leaves …

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 …

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 …

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. …

Directly Light-Regulated Binding Of Rgs-Lov Photoreceptors To Anionic Membrane Phospholipids, Spencer T. Glantz, Erin E. Berlew, Zaynab Jaber, Benjamin S. Schuster, Kevin H. Gardner, Brian Y. Chow

Publications and Research

We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helixinthelinker regionbetween the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction …

Water Pharmacophore: Designing Ligands Using Molecular Dynamics Simulations With Water, Sang Won Jung, Minsup Kim, Steven Ramsey, Tom Kurtzman, Art E. Cho

Publications and Research

In this study, we demonstrate a method to construct a water-based pharmacophore model which can be utilized in the absence of known ligands. This method utilizes waters found in the binding pocket, sampled through molecular dynamics. Screening of compound databases against this water-based pharmacophore model reveals that this approach can successfully identify known binders to a target protein. The method was tested by enrichment studies of 7 therapeutically important targets and compared favourably to screening-by-docking with Glide. Our results suggest that even without experimentally known binders, pharmacophore models can be generated using molecular dynamics with waters and used for virtual …

Retraction Of Dna-Bound Type Iv Competence Pili Initiates Dna Uptake During Natural Transformation In Vibrio Cholerae, Courtney K. Ellison, Triana N. Dalia, Alfredo Vidal Ceballos, Joseph Che-Yen Wang, Nicolas Biais, Yves V. Brun, Ankur B. Dalia

Publications and Research

Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation, and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation1 , however, their mechanism of action has remained unclear due to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus labeling method, we define the mechanism for type IV competence pilus-mediated DNA uptake …

Dextran Hydrogels By Crosslinking With Amino Acid Diamines And Their Viscoelastic Properties, Naphtali O'Connor, Andrei Jitianu, G Nunez, Quentin Picard, Madeline Wong, D Akpatsu, Adam Negrin, Rajendra Ghardbaran, D Lugo, S Shaker, Mihaela Jitianu, Stephen Redenti

Publications and Research

Amine functionalized polysaccharide hydrogels such as those based on chitosan are widely examined as biomaterials. Here we set out to develop a facile procedure for developing such hydrogels by crosslinking dextran with amino acid diamines. The dextran-amino acid gels were formed by the addition of the amino acid diamines to a dextran and epichlorohydrin solution once it became homogeneous. This was demonstrated with three amino acid diamines, lysine, lysine methyl ester, and cystine dimethyl ester. Hydrogel networks with albumin entrapped were also demonstrated. These hydrogels were characterized by FTIR, SEM, rotational rheometry, swelling studies and cell biocompatibility analysis. These hydrogels …

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 …