Biochemistry Commons^™

Ubiquitin, Cavitand, And Nmr: 15n-1h Hsqc Analysis Of Cavitand Binding Domains On Ubiquitin, Grant Stucky

CMC Senior Theses

Ubiquitin and its derivatives through side chain modification are present in many of the degradation pathways for proteins found in neurodegenerative diseases. A non-toxic method for determining the amount of these proteins could have biosensing applications, but the binding domains of such proteins are not well characterized. ¹⁵N-¹H HSQC analysis of ubiquitin’s interactions with different concentrations of a small negatively charged host molecule called cavitand provides insight into the binding interactions with side chains on the protein such as lysine and arginine. This is done comparing the HSQC spectra of different concentrations of cavitand and ubiquitin to …

Technological Development Of In-Cell Nmr: Microfluidic, Metabolomic, And Fluxomic Approaches, Nicholas Sciolino

Electronic Theses & Dissertations (2024 - present)

In the fields of structural biology, metabolomics, and fluxomics, there is a driving, technique-based concern when it comes to high-resolution spectrometry and its marriage to biological and physiological relevance. Sample preparation often involves cellular perturbation, lysate separation schemes, combinations with non-biological reagents, and timeline slack. Conceptually, the farther we get from the cell, the greater the potential disparity between what exists in vivo and our in vitro manipulations. This is particularly true when seeking to collect data on transfected targets of interest, and on sensitive metabolic equilibria that occur on the order of minutes. The question remains: if we are …

Illuminating Function And Enabling Inhibition At The Protein-Membrane Interface Of Glutathione Peroxidase 4., Courtney L. Labrecque

Theses and Dissertations

Membrane proteins act as gateways to cells and are responsible not only for signaling, lipid transport, and cellular homeostasis, but they are also integral in disease pathologies. One class of membrane proteins, peripheral membrane proteins (PMPs), reside in aqueous compartments of the cell until they are recruited to the membrane to perform their function. Glutathione peroxidase 4 (GPx4) is a PMP enzyme responsible for engaging the membrane and reducing lipid hydroperoxides, thereby preventing cellular death via ferroptosis. Due to its central function surrounding ferroptosis, GPx4 is an interesting drug target that has recently gained interest for inflammatory diseases as well …

Investigations Of A Nox2 Activator, P47phox, Px Domain Membrane Anchoring Interactions And Inhibition, Angela Develin

Theses and Dissertations

The regulation of cellular signaling across plasma membranes is a well-organized process through cross-talk of biomolecules such as proteins and membranes. Peripheral membrane proteins (PMPs) are a class of water soluble, membrane associated, proteins that play a critical role in transducing such signals. Due to the dynamic nature of their membrane interactions, relatively smooth surfaces, and general lack of deep ligand binding pockets, PMPs are commonly associated with an approximate 85% of the human proteome that is classified as ‘undruggable’ by current drug discovery methodologies. One such PMP, p47^phox, contains a PX domain which is essential in activation …

Characterization Of Cl-Par-4: Wt Vs. Mutant, Samjhana Pandey, Krishna K. Raut, Andrea M. Clark, Antoine Baudin, Lamya Djemri, David S. Libich, Steven M. Pascal

The Graduate School Posters

Intrinsically disordered proteins (IDPs) play important roles in regulation of cell signaling pathways as well as cellular processes. Dysregulation of these proteins is associated with several human diseases. Prostate apoptosis response-4 (Par-4), a proapoptotic tumor suppressor protein, is categorized as an intrinsically disordered protein and downregulation of this protein has been reported in myriad of cancers including glioma, breast cancers, and prostate cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) plays an active role in tumor suppression by inhibiting several cell survival pathways.

Here, we employed site-directed mutagenesis to introduce a point mutation in the cl-Par-4 wildtype (WT) to generate the …

Par-4: An Attractive Target For Cancer Therapy, Krishna K. Raut, Antoine Baudin, David S. Libich, Lijun Liu, Scott Lovell, Steven M. Pascal

College of Sciences Posters

Lack of early diagnosis, cancer recurrence, metastasis, and adverse side effects are some of the major problems in the treatment of cancers. Par-4, a tumor suppressor protein, is an attractive target for cancer therapy as it selectively kills cancer cells. Cl-Par-4 is the active fragment of Par-4 that enters the nucleus and selectively induces apoptosis in cancer cells. It has also been reported that Par-4 increases the susceptibility of cancer cells to chemotherapy and reverses cancer recurrence. Further, Par-4 has been shown to play a dual role: inhibition of EMT (Epithelial-mesenchymal transition) as well as assistance in the reverse process, …

Applications Of Nuclear Magnetic Resonance Spectroscopy: From Drug Discovery To Protein Structure And Dynamics., Mark Vincent C. Dela Cerna

Electronic Theses and Dissertations

The versatility of nuclear magnetic resonance (NMR) spectroscopy is apparent when presented with diverse applications to which it can contribute. Here, NMR is used i) as a screening/ validation tool for a drug discovery program targeting the Phosphatase of Regenerating Liver 3 (PRL3), ii) to characterize the conformational heterogeneity of p53 regulator, Murine Double Minute X (MDMX), and iii) to characterize the solution dynamics of guanosine monophosphate kinase (GMPK). Mounting evidence suggesting roles for PRL3 in oncogenesis and metastasis has catapulted it into prominence as a cancer drug target. Yet, despite significant efforts, there are no PRL3 small molecule inhibitors …

Bodipy-Based Light-Responsive Materials, Zhaoxiong Wan

Dissertations

The energy required for life on our planet is provided by light. In science, light is an important and powerful tool for measuring and modifying substances. For example, under the effect of illumination, light-responsive materials can change their physical and chemical properties. Photochemistry is a branch of research to study the effects of light irradiation on chemical structures and reactions. Some photochemical changes could lead to chemical bond dissociation (such as in photosynthesis) and structure isomerization (such as in the retina). Photochemistry has a strong relationship with human lives and it is essential in many ways. Among photophysics, fluorescence emission …

Paddling Along The Voltage Gated Sodium Channel Galaxy With Sea Anemone Toxins: Structural Studies Of The Interaction Between The Paddle Motif From Nav1.5div And Sea Anemone Toxin, Adel K. Hussein

Dissertations, Theses, and Capstone Projects

Voltage gated sodium channels (VGSC) are membrane proteins that serve an important function in the central nervous system (CNS), peripheral nervous system (PNS), and cardiac muscles amongst others. The main function of VGSC is in the propagation of electrical signals by depolarizing excitable cells. Nine mammalian VGSC subtypes have been characterized, Na_V1.1 – Na_V1.9, that are expressed in a tissue specific manner, each with unique gating properties. Numerous diseases have been linked to defects in VGSC including epilepsy, mental retardation, long QT syndrome, and Brugada disease. Furthermore, these channels are one of the primary targets of …

Investigations Of The Structure And Protein-Protein Interactions Of Chlamydia Trachomatis Scc4, Thilini Oshadhi Senarath Ukwaththage

LSU Doctoral Dissertations

Chlamydia trachomatis (CT) is the most common, sexually transmitted bacterial disease (STD) in the world. In the developmental cycle of CT, specific chlamydia chaperone 4 (Scc4) is a unique protein with essential and multiple roles. Hence, Scc4 is significant as a virulence target for therapeutic approaches to treat chlamydial infections. A novel approach was discovered to purify tag free Scc4 by utilizing a 6X-histidine-tag on Scc1 in the co-expressed Scc4:Scc1 complex by capturing the complex on nickel-charged immobilized metal affinity chromatography resin, followed by dissociation of Scc4 with sarkosyl. Using triple resonance NMR experiments, backbone and sidechain resonances …

Synthetic Pathways For Potential Platinum 1,10-Phenanthroline Compounds, Jacob Young

Mahurin Honors College Capstone Experience/Thesis Projects

There are extremely important uses for platinum when it comes to the treatment of cancers and dementias. One potential therapeutic combines platinum with a structure known as phenanthroline. This platinum phenanthroline structure is normally extremely water-insoluble, rendering it difficult to study and use in biological systems. The Williams’ Biochemistry Laboratory endeavored to find a simpler synthetic pathway for a water-soluble phenanthroline product. While unsuccessful in identifying a product as of yet, many synthetic pathways have been ruled out, and more hypothetical pathways are being formulated for testing.

Among the strategies analyzed are varying solvent, reagents, apparatus, methodology, and temperature in …

Investigating The Disorder And Compaction Of Designed Minielastin Using Nuclear Magnetic Resonance., Ma Faye Charmagne Aquino Carvajal

Electronic Theses and Dissertations

Minielastins are elastin-based proteins with alternating hydrophobic and cross-link modules similar to tropoelastin. Tropoelastin is the ~70 kDa soluble monomeric precursor of elastin. The extracellular matrix protein, elastin provides elasticity to tissues and organs such as lungs, arteries and ligaments. The elastic properties of natural elastin are believed to be entropic in origin. In vivo, the elastin matrix is approximately 50% water by weight. Without water, elastin is brittle and hard. Minielastins, like tropoelastin, undergo a liquid-liquid phase transition upon an increase in temperature. Factors such as hydrophobicity, chain length and concentration affect the coacervation temperature, T_c. The …

Structure And Bioactivity Of A Rationally Designed Nicotinic Acetylcholine Receptor Antagonist Targeting The Alpha-3 Beta-2 Subtype, Leanna Marquart

Boise State University Theses and Dissertations

The work presented in this thesis contributes to ongoing development of an efficient workflow for identification of lead compounds, based on the molecular scaffold of α-conotoxin MII, for drug therapies targeting nicotinic acetylcholine receptors. Nicotinic acetylcholine receptors (nAChRs) are pentameric, ligand-gated ion channels with distribution throughout the central nervous system and are implicated in a variety of neurological diseases including schizophrenia, nicotine addiction, Alzheimer’s disease, and Parkinson’s disease. However, ligand effect on nAChR function is not well understood. Consequently, drug therapies for neurological diseases either do not exist, or have short-lasting efficacy and/or severe side effects. Barriers to the comprehension …

Xenobiotic Exposure Requires Mitochondrial Metabolism For Redox Homeostasis And Survival In Astrocytes, Jordan Rose

Department of Biochemistry: Dissertations, Theses, and Student Research

Astrocytes are integral components of glutamatergic neurotransmission, providing essential metabolic processes for neuronal homeostasis and acting as the first line of defense against xenobiotics crossing the blood brain barrier. Arsenic is a xenobiotic with widespread natural and industrial prevalence, and has been linked to impaired neurodevelopment and neuronal death. Given the integrated metabolic nature of astrocytes and neurons, we sought to explore how arsenic impacts astrocyte metabolism in order to better understand the mechanisms of xenobiotic toxicity in the mammalian brain.

We demonstrate that astrocyte viability depends upon de novoglutathione (GSH) synthesis during arsenic exposure, and sub-lethal arsenic exposure …

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 …

Automatic 13C Chemical Shift Reference Correction Of Protein Nmr Spectral Data Using Data Mining And Bayesian Statistical Modeling, Xi Chen

Theses and Dissertations--Molecular and Cellular Biochemistry

Nuclear magnetic resonance (NMR) is a highly versatile analytical technique for studying molecular configuration, conformation, and dynamics, especially of biomacromolecules such as proteins. However, due to the intrinsic properties of NMR experiments, results from the NMR instruments require a refencing step before the down-the-line analysis. Poor chemical shift referencing, especially for ¹³C in protein Nuclear Magnetic Resonance (NMR) experiments, fundamentally limits and even prevents effective study of biomacromolecules via NMR. There is no available method that can rereference carbon chemical shifts from protein NMR without secondary experimental information such as structure or resonance assignment.

To solve this problem, we …

Structure Of The Picornavirus Replication Platform: A Potential Drug Target For Inhibiting Virus Replication, Meghan Suzanne Warden

Chemistry & Biochemistry Theses & Dissertations

Picornaviruses are small, positive-stranded RNA viruses, divided into twelve different genera. Members of the Picornaviridae family cause a wide range of human and animal diseases including the common cold, poliomyelitis, foot and mouth disease, and dilated cardiomyopathy. The picornavirus genome is replicated via a highly conserved mechanism involving a presumed cloverleaf structure located at the 5’ noncoding region of the virus genome. The 5’ cloverleaf consists of three stem loops (B, C, and D) and one stem (A), which interact with a variety of virus and host cell proteins during replication. In this dissertation, human rhinovirus serotype 14 (HRV-14) SLB …

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 …

The Effects Of Carrier Ligands On Cisplatin Binding To Cysteine And Methionine, Adam C.R Smith

Masters Theses & Specialist Projects

We have reacted several derivatives of the anticancer drug cisplatin with N-acetyl-Lcysteine (N-AcCys) and N-acetyl-L-methionine (N-AcMet), which are two of the primary amino acid targets of platinum. NMR spectroscopy was used to monitor the reactions and determine the effect the different ligands would have on the platinum reactivity. Several of the platinum compounds were tested at pH of 4 and 7, and with platinum:amino acid ratios of 1:1, 2:1 and 1:2. Competition reactions between cysteine and methionine were done to confirm which would react with the platinum compound first. [Pt(dien)(NO3)]+ reacts faster with methionine than with cysteine at both pH …

Reactivity Of Ketyl And Acetyl Radicals From Direct Solar Actinic Photolysis Of Aqueous Pyruvic Acid, Alexis J. Eugene, Marcelo I. Guzman

Chemistry Faculty Publications

The variable composition of secondary organic aerosols (SOA) contributes to the large uncertainty for predicting radiative forcing. A better understanding of the reaction mechanisms leading to aerosol formation such as for the photochemical reaction of aqueous pyruvic acid (PA) at λ ≥ 305 nm can contribute to constrain these uncertainties. Herein, the photochemistry of aqueous PA (5-300 mM) continuously sparged with air is re-examined in the laboratory under comparable irradiance at 38° N at noon on a summer day. Several analytical methods are employed to monitor the time series of the reaction, including (1) the derivatization of carbonyl (C═O) functional …

Structural Characterization Of The Interactions Of Nicotinamide And Analogs With Human Sirt6 By Saturation Transfer Difference (Std) Nmr And Site Directed Mutagenesis, Beatriz Elena Bolivar-Vega

Legacy Theses & Dissertations (2009 - 2024)

The allosteric regulation of SIRT6 by nicotinamide (NAM), along with the growing evidence of this enzyme's key role in the immune response, prompted the mechanistic study of SIRT6 inhibition by pyrazinamide (PZA) and analogs. In our current study, PZA, an analog of NAM, was revealed to have a modest modulatory effect on SIRT6, an enzyme that regulates the NF-κB signaling pathway at the transcriptional level (a relevant pathway to inflammation). Similarly, the analogs of PZA, 5-Cl PZA, 5-MeO PZA, and POA exhibited a modulatory effect against SIRT6 in our in vitro studies, enabling identification of a potential new target for …

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 …

The Nmr Solution Structure And Function Of Rpa3313: A Hypothetical Protein From R. Palustris, Austin J. Lowe, Jonathan Catazaro, Cheryl Arrowsmith, Robert Powers

UCARE: Research Products

Protein function elucidation often relies heavily on amino acid sequence analysis and other bioinformatics approaches. The reliance is further extended to structure homology modeling for ligand docking and protein-protein interaction mapping. However, sequence analysis of RPA3313 exposes a large, unannotated class of hypothetical proteins mostly from the Rhizobiales order. In the absence of sequence and structure information, further functional elucidation of this class of proteins has been significantly hindered. A high quality NMR structure of RPA3313 reveals that the protein forms a novel split βαβ fold with a conserved ligand binding pocket between the first β-strand and the N-terminus of …

Structural Elucidation Of Aggr-Activated Regulator, Aar, In Enteroaggregative Escherichia Coli, Andrew Heindel

Senior Honors Projects, 2010-2019

Travelers’ Diarrhea is the number one cause of childhood death in the world. Enteroaggregative Escherichia coli (EAEC) is one of the main causes of this disease. EAEC adhere to the surface of the intestine and stack in a brick-like pattern. Via an unstudied quorum-sensing mechanism, these bacteria express a variety of virulence factors that lead to diarrhea. The long-term goal of this research is to elucidate the mechanism by which EAEC changes from benign to virulent. A previously-unstudied open-reading frame in EAEC, AggR activated repressor (Aar), has recently been hypothesized to act as one of the major transcription factors influencing …

Conformational Heterogeneity And Exchange Within The Junction Of The Human U2-U6 Snrna Complex Measured By 19f Nmr Spectroscopy, Caijie Zhao

Dissertations, Theses, and Capstone Projects

Pre-mRNA splicing, the process by which non-coding sequences are removed and coding sequences are ligated, plays an important role in the maturation of precursor messenger RNA molecules prior to their translation into proteins. In eukaryotes, splicing is catalyzed by the spliceosome, a large and dynamic nuclear macromolecular "machine" that comprises five small nuclear (sn)RNA molecules and more than one hundred proteins. Among the RNA components, the complex formed between the U2 and U6 snRNA molecules is implicated in catalytic activity. In this dissertation, I have used ¹⁹F NMR techniques to characterize the conformation ("fold") and dynamics of a protein-free …

Sharp Rna Recognition Motif Optimizations, Extensions, And Mutations For Use In 2d And 3d Nmr Experiments, Shaun M. Christie

Williams Honors College, Honors Research Projects

SMRT/HDAC Associated Repressor Protein interacts with the long noncoding RNA, produced by SRA, by binding at the RRMs. Three projects were done to prepare the truncated proteins for use in 2D and 3D NMR experiments. The first focuses on RRM 3 and its optimization during the purification process. The second focuses on RRM 2-4, which was found to be missing two alpha helices that may be important for protein stability. These helices can also interact with RRM 3 as well due to the tight association of RRMs 3 and 4. The two step PCR extension of RRM 2-4 was assumed …

Some Of The Most Interesting Casp11 Targets Through The Eyes Of Their Authors, Andriy Kryshtafovych, John Moult, Arnaud Basle, Alex Burgin, Timonthy K. Craig, Robert A. Edwards, Deborah Fass, Marcus D. Hartmann, Mateusz Korycinski, Richard J. Lewis, Donald Lorimer, Andrei N. Lupas, Janet Newman, Thomas S. Peat, Kurt H. Piepenbrink, Janani Prahlad, Mark J. Van Raaij, Forest Rohwer, Anca M. Segall, Victor Seguritan, Eric J. Sundberg, Abhimanyu K. Singh, Mark A. Wilson, Torsten Schwede

Department of Biochemistry: Faculty Publications

The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features, and assess whether these features were correctly reproduced in the predictions submitted to CASP11.

Effects Of Statin Drugs And Tocotrienol Rich Fraction Supplementation In Chronic Hemodialysis Patients And Metabolomic Profile, Eno Latifi

Wayne State University Theses

Chronic kidney disease (CKD) is known as a heterogeneous disorder which currently is on the rise and lately has been classified as a public health issues in the United State and worldwide. CKD is an irreversible and progressive disease which can lead to kidney failure, and this is depicted by the advanced stage of the disorder when it reaches the point, that is classified as end stage of renal disease (ESRD) (Stage 5 of CKD) (eGRF <15 mL/min/ 1.73 m2 working capacity), where both organs are in a total or permanent kidney failure. End-Stage renal disease patients, on hemodialysis have been associated to experience an accelerated form of atherosclerosis, which is induced by inflammation, impairment of antioxidant system and elevated oxidative stress. Since the problem effecting ESRD patients is multifactorial, the objective of this investigation is to explore and look at the effects of supplementing with vitamin E-tocotrienol rich fraction (TRF), a micronutrient which has anti-inflammatory, antioxidant, and lipid lower capabilities into tackling these comorbid conditions experienced by this population. Therefore the aims of this investigation will be to explore changes in lipid profiles, inflammatory markers, and oxidative status, as well as look at any changes in metabolomic profiles. It was hypothesized that by supplementing with TRF a vitamin E, for 16 weeks in ESRD patients undergoing hemodialysis, it may help reverse and/or improve, oxidative status, inflammatory markers, increase antioxidants status and improve lipid profiles.

The study was double-blinded, randomized, parallel, placebo-controlled design trial, of 81 adult patients undergoing chronic hemodialysis at Great Lake Dialysis Clinic, Detroit MI, where …

The Development And Applications Of Nmr Metabolomics Analysis Of Bacterial Metabolomes, Steven M. Halouska

Department of Chemistry: Dissertations, Theses, and Student Research

Metabolomics is a relatively new field that involves the study of metabolic responses that are occurring within a biological system. Metabolite profiles of an organism, tissue extract, and biofluids are important indicators to determine the physiological state of a biological profile. Comparison of such profiles from different phenotypes can be used to identify specific metabolic changes leading to the understanding of metabolic pathways, disease progression, drug toxicity and efficacy, and cellular responses to different intracellular and extracellular conditions. Metabolomics investigations often use sophisticated analytical techniques such as NMR spectroscopy to provide an unbiased and comprehensive approach to evaluate metabolic perturbation …

Utilizing Nmr Spectroscopy And Molecular Docking As Tools For The Structural Determination And Functional Annotation Of Proteins, Jaime Stark

Department of Chemistry: Dissertations, Theses, and Student Research

With the completion of the Human Genome Project in 2001 and the subsequent explosion of organisms with sequenced genomes, we are now aware of nearly 28 million proteins. Determining the role of each of these proteins is essential to our understanding of biology and the development of medical advances. Unfortunately, the experimental approaches to determine protein function are too slow to investigate every protein. Bioinformatics approaches, such as sequence and structure homology, have helped to annotate the functions of many similar proteins. However, despite these computational approaches, approximately 40% of proteins still have no known function. Alleviating this deficit will …