Structural Biology Commons^™

Structure Of Clostridium Perfringens Type Iv Pili, Alexander R. Meyer

Honors Program: Senior Projects (Public)

Type IV pili (T4P) are thin, hair-like bacterial appendages composed of protein subunits polymerized into a helical fiber. T4P perform diverse functions such as host cell adhesion, biofilm formation, natural competence, and twitching motility. While T4P are well characterized in Gram-negative bacteria, they have more recently been found in Gram-positive bacteria as well. In this work we aimed to solve the crystal structure of the type IV major pilin protein PilA2 from Clostridium perfringens, the predominant pilus subunit which makes up about 99% of the pilus fiber. We report expression, purification, and crystallization conditions which are sufficient for X-ray …

The C. Neoformans Cell Wall: A Scaffold For Virulence, Christine Chrissian

Dissertations, Theses, and Capstone Projects

Cryptococcus neoformans is a globally distributed opportunistic fungal pathogen and the causative agent of life threatening cryptococcal meningoencephalitis in immunocompromised individuals, resulting in ~180,000 deaths each year worldwide. A primary virulence-associated trait of this organism is the production of melanin. Melanins are a class of diverse pigments produced via the oxidation and polymerization of aromatic ring compounds that have a characteristically complex, heterogenous, and amorphous structure. They are synthesized by representatives of all biological kingdoms and share a multitude of remarkable properties such as the ability to absorb ultraviolet (UV) light and protect against ionizing radiation. Melanin production in fungi …

Design Of Fibril Forming Collagen Mimetic Peptides: Heterotrimers And Nucleation Domains, Sally Tan

Theses and Dissertations

This paper attempts to design collagen mimetic peptides where the triple-helical region mimics that of human Type I Collagen. With consideration for chain selection and chain register, we utilize the NC2 domain of heterotrimeric Type IX Collagen as a nucleation domain for triple-helix folding.

Synthesis And Biological Evaluation Of Grp94 Selective Inhibitors, Zach Priebe, Kyler Pugh, Brian S. J. Blagg

Summer Research

Grp94 is one of four chaperones which belong to the Heat Shock Protein 90 (Hsp90) family. Hsp90 isoforms exist as homodimers and assist in the folding and maturation of over 400 client proteins. These proteins are highly sought-after therapeutic targets as they are known to chaperone misfolded proteins involved in various signaling pathways. During the protein folding cycle, ATP binds to the N-Terminal domain of Hsp90, which allows nascent polypeptides to undergo maturation. To prevent the folding and maturation of oncogenic proteins, researchers developed N-terminal inhibitors to prevent ATP binding. Unfortunately, these inhibitors (termed pan-inhibitors) were not isoform selective, which …

Photoreceptor Phosphodiesterase (Pde6): Structure, Regulatory Mechanisms, And Implications For Treatment Of Retinal Diseases, Rick H. Cote, Richa Gupta, Michael J. Irwin, Xin Wang

Faculty Publications

The photoreceptor phosphodiesterase (PDE6) is a member of large family of Class I phosphodiesterases responsible for hydrolyzing the second messengers cAMP and cGMP. PDE6 consists of two catalytic subunits and two inhibitory subunits that form a tetrameric protein. PDE6 is a peripheral membrane protein that is localized to the signaling-transducing compartment of rod and cone photoreceptors. As the central effector enzyme of the G-protein coupled visual transduction pathway, activation of PDE6 catalysis causes in a rapid decrease in cGMP levels that results in closure of cGMP-gated ion channels in the photoreceptor plasma membrane. Because of its importance in the phototransduction …

Computational Analysis And Prediction Of Intrinsic Disorder And Intrinsic Disorder Functions In Proteins, Akila I. Katuwawala

Theses and Dissertations

COMPUTATIONAL ANALYSIS AND PREDICTION OF INTRINSIC DISORDER AND INTRINSIC DISORDER FUNCTIONS IN PROTEINS

By Akila Imesha Katuwawala

A dissertation submitted in partial fulfillment of the requirements for the degree of Engineering, Doctor of Philosophy with a concentration in Computer Science at Virginia Commonwealth University.

Virginia Commonwealth University, 2021

Director: Lukasz Kurgan, Professor, Department of Computer Science

Proteins, as a fundamental class of biomolecules, have been studied from various perspectives over the past two centuries. The traditional notion is that proteins require fixed and stable three-dimensional structures to carry out biological functions. However, there is mounting evidence regarding a “special” class …

Arabinoxylan Structural Profiling Of Cool-Season Pasture Grasses Via High-Performance Anion-Exchange Chromatography With Pulsed Amperometric Detection (Hpaec-Pad) Analysis Of Endoxylanase Digests, Glenna Erin Joyce

Theses and Dissertations--Animal and Food Sciences

Arabinoxylan (AX) is a major structural polysaccharide found in the cell walls of monocots such as cereal grains and pasture grasses. The variety of AX structural components and substitution patterns contribute to AX structural diversity between different monocot species as well as plant tissues.

The rumen is the first digestion site of masticated food material in cattle and provides 70% of energy to host through fermentation of forage. There are many species of pasture grasses that act as a forage source. Differences in AX structure found in these pasture grasses may impact rumen microbial fermentation. Understanding the AX structure of …

Interactions Of Post-Pks Enzymes Of The Mithramycin Biosynthetic Pathway, Ryan Wheeler

Theses and Dissertations--Pharmacy

Combinatorial biosynthesis is a powerful tool for generating new, more active drug analogues to combat disease. But in order for combinatorial biosynthesis to be employed to its full potential, a deep understanding of the enzymes that produce the parent molecule must be had. The goals of the work presented in this thesis are to characterize the reaction catalyzed by MtmW, the final enzyme in the mithramycin (MTM) biosynthetic pathway, and to discover the interaction between MtmW and MtmOIV.

MtmW is an aldol-ketoreductase responsible for reducing the most distal carbonyl on the MTM pentyl side chain. It forms an octamer that …

'Induction Of Apoptosis, Cytotoxicity And Radiosensitization By Novel 3,4-Dihydroquinazolinone Derivatives, Eman Ramadan, Amira Khalil

Pharmacy

Twenty new quinazolinone derivatives bearing a piperonyl moiety were designed and synthesized. The structures of the target compounds were in agreement with the microanalytical and spectral data. Compounds 4-10, 13, 14 and 17-27 were screened for their cytotoxic activity against HepG-2 and MCF-7 cancer cell lines. The target compounds showed IC₅₀ in the range of 2.46-36.85 µM and 3.87-88.93 µM for HepG-2 and MCF-7, respectively. The promising compounds 7, 19, 26 and 27 were selected to measure their EGFR inhibitory activity. The IC₅₀ values of the promising compounds were in the range of 146.9-1032.7 nM for EGFR in …

Substrate Trafficking Within The Type Vii Secretion Systems Of Pathogenic Mycobacteria, Zachary A. Williamson

Theses and Dissertations--Molecular and Cellular Biochemistry

Tuberculosis (TB), primarily caused by infection of Mycobacterium tuberculosis (Mtb) in the lungs, is the deadliest infectious bacterial disease killing 1.5 million people annually. A major determinant of virulence is active secretion through three specialized type VII secretion (ESX) systems; ESX-1, ESX-3, and ESX-5. A large group of substrates exported by the ESX systems is the PE (Proline-Glutamine) and PPE (Proline-Proline-Glutamate) families of proteins, which are highly expanded in the pathogenic species of Mycobacteria and encompass over 7% of Mtb’s genome coding capacity. PE and PPE proteins interact together to form PE-PPE heterodimers, and are secreted through …

Photoreceptor Phosphodiesterase (Pde6): Activation And Inactivation Mechanisms During Visual Transduction In Rods And Cones, Rick H. Cote

Faculty Publications

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary

intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes …

Investigation Of Multidrug Efflux Transporter Acrb In Escherichia Coli: Assembly, Degradation And Dynamics, Prasangi Irosha Rajapaksha

Theses and Dissertations--Chemistry

The Resistant Nodulation Division (RND) super family member, tripartite AcrA-AcrB-TolC efflux pump, is a major contributor in conferring multidrug-resistance in Escherichia coli. The structure of the pump complex, and drug translocation by functional rotation mechanism have been widely studied. Despite of all these data, the dynamics of the assembly process of the pump and AcrB during functional rotation in the process of drug efflux remains poorly understood. My thesis focuses on understanding the pump assembly process, dynamics of AcrB in functional rotation mechanism, and also investigate the mechanism of degradation of AcrB facilitated by a C-terminal ssrA tag.

In the …

Computational Insights On Medicinal Chemistry Targeting Cyp450s, Alexander D. Fenton

Theses and Dissertations--Chemistry

Modern-day medicinal chemistry has provided researchers with a wide variety of tools to not only gather greater insight from their data, but also to generate data in new ways. One such tool is the construction of computational protein models from crystallographic datasets, and their subsequent use to understand the structure-activity relationships of protein-ligand complexes. These models can be utilized for their predictive power to inform the synthesis of, and improvement of, lead compounds. It is the goal of this work to employ such models to the CYP450 enzyme system such that potent and selective inhibitors can be designed, evaluated biologically, …

Cryogenic Electron-Microscopy (Cryo-Em) Analysis Of Mscs, Ynai, Connexin 26 And Bioinformatic Analysis Of Cysteine And Serine In Experimental Protein Structures, Claudio Catalano

Theses and Dissertations

Functional insights into macromolecules lie in the arrangement of their molecular structures. Investigating the atomic framework of a biomolecule could unwind its biochemical mechanism, providing aid to many relevant biological questions, thus facilitating drug discovery processes. Two approaches have been used to gain knowledge at the atomic level of a protein function: experimental and computational methodologies.

From an experimental perspective, membrane proteins (MPs) were investigated. Such proteins represent ~20% of the human proteome, and ~60% of them are drug targets since they are exposed to the surface of cells and are responsible for a wide range of fundamental functions like …

Characterization Of Cytochrome P450 3a4 Structure And Ligand Binding Mechanism In Anionic Nanodiscs, David T. Sweeney

Theses and Dissertations

The Cytochromes P450 superfamily are ubiquitous throughout life and are present in some viruses. Humans encode for 57 of these enzymes that are involved in the metabolism of ~90% of all chemicals. These membrane-bound monooxygenase enzymes utilize a heme protoporphyrin at their active site to activate molecular oxygen and oxidize their substrates through an intermediate deemed nature's most powerful oxidizer. They are a powerful system at our disposal yet understanding of their structure and ligand binding mechanisms in the presence of a lipid bilayer remains poorly understood. The focus of this work is on Cytochrome P450 3A4 (CYP3A4). We set …

On The Structure And Function Of Mitochondrial Uncoupling Proteins: The Case Of Ucp2, Afshan Ardalan

Theses and Dissertations (Comprehensive)

Uncoupling proteins (UCPs) are regulated proton transporters of the mitochondrial inner membrane. UCP-mediated proton leak negatively impacts the rate of ATP synthesis. Despite the importance of their physiological role(s) in certain tissues, molecular aspects of UCPs’ structure-function relationships are not fully understood. The current study explores the tertiary and quaternary structure of UCP2, as well as its proton transport mechanism in lipid membranes. The proteins were expressed in the E. coli inner membrane, purified and reconstituted into liposomes. Proteins were characterized by semi-native SDS-PAGE. Circular dichroism spectroscopy (CD) and fluorescence quenching assays were utilized to study the conformation of proteins …

Structure Based Prediction Of A Novel Gpr120 Antagonist Based On Pharmacophore Screening And Molecular Dynamics Simulations, Ajay Pal Mr, Gemma K. Kinsella, James Curtin

Articles

The G-protein coupled receptor, GPR120, has ubiquitous expression and multifaceted roles in modulating metabolic and anti-inflammatory processes. Recent implications of its role in cancer progression have presented GPR120 as an attractive oncogenic drug target. GPR120 gene knockdown in breast cancer studies revealed a role of GPR120-induced chemoresistance in epirubicin and cisplatin-induced DNA damage in tumour cells. Higher expression and activation levels of GPR120 is also reported to promote tumour angiogenesis and cell migration in colorectal cancer. Some agonists targeting GPR120 have been reported, such as TUG891 and Compound39, but to date development of small-molecule inhibitors of GPR120 is limited. …