Life Sciences Commons^™

Modeling Accuracy Matters: Aligning Molecular Dynamics With 2d Nmr Derived Noe Restraints, Milan Patel

Honors Scholar Theses

Among structural biology techniques, Nuclear Magnetic Resonance (NMR) provides a holistic view of structure that is close to protein structure in situ. Namely, NMR imaging allows for the solution state of the protein to be observed, derived from Nuclear Overhauser Effect restraints (NOEs). NOEs are a distance range in which hydrogen pairs are observed to stay within range of, and therefore experimental data which computational models can be compared against. To that end, we investigated the effects of adding the NOE restraints as distance restraints in Molecular Dynamics (MD) simulations on the 24 residue HP24stab derived villin headpiece subdomain to …

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 …

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 …

Posttranslational Modification And Protein Disorder Regulate Protein-Protein Interactions And Dna Binding Specificity Of P53, Robin Levy

USF Tampa Graduate Theses and Dissertations

p53 is an intrinsically disordered transcription factor that suppresses tumor development by arresting the cell cycle and promoting DNA repair. p53 deletions or mutations can lead to cancer due to the inability of cells to respond to stress. The protein levels and post-translational modification state of p53 changes in response to cellular stress like DNA damage. Previous studies have shown that p53 can undergo coupled folding and binding with the E3 ubiquitin ligase, Mdm2, and the histone deacetylase, p300. In normal cells, p53 is kept at a low level by Mdm2, which marks it with ubiquitin, targeting p53 for proteasome …

Conserved Glycine Residues Control Transient Helicity And Disorder In The Cold Regulated Protein, Cor15a, Oluwakemi Sowemimo

USF Tampa Graduate Theses and Dissertations

COR15A is a cold regulated disordered protein from Arabidopsis thaliana that contributes to freezing tolerance in plants by protecting membranes. It belongs to the (LEA) Late Embryogenesis Abundant group of proteins that accumulate during the later stage of seed development and are expressed in various parts of the plant. During freezing-induced cellular dehydration, COR15A transitions from a disordered structure to a mostly α-helical structure that binds and stabilizes chloroplast membranes when cells dehydrate due to freezing. We hypothesize that increasing the transient α-helicity of COR15A under normal conditions will increase its ability to bind and protect chloroplast membranes when cells …

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 …

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 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 …

Profiling Fatty Acid Composition Of Brown Adipose Tissue, White Adipose Tissue And Bone Marrow Adipose Tissue Of Healthy And Diet-Induced Obese Mice, Urszula Osinska Warncke

Browse all Theses and Dissertations

Bone marrow adipose tissue (MAT) contains a fat depot of unknown function. Characterization of this tissue in healthy and pathophysiological conditions may be an important step in order to understand MAT contribution to development of osteoporosis and perhaps to develop means to improve bone health by manipulation with a phenotype of MAT. Here we demonstrate that MAT, brown adipose tissue (BAT), and white adipose tissue (WAT) derived from healthy mice differ in fatty acid and phospholipid composition. The profile of MAT changed to resemble a less-metabolically active tissue upon metabolic impairment as seen in diet-induced obesity. Additionally, PPARγ activation alters …

Camp-Dependent Protein Kinase A Selects The Excited State Of The Membrane Substrate Phospholamban, Larry R. Masterson

Larry Masterson

Segmental Isotopic Labeling Of The Hsp70 Molecular Chaperone Dnak Using Expressed Protein Ligation, Eugenia M. Clerico, Anastasia Zhuravleva, Robert G. Smock, Lila Gierasch

Lila Gierasch

Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (in this case the E. coli Hsp70, DnaK) where only one of the two constituent domains was labeled, in this case with NMR active isotopes, allowing visualization of the single domain in the context of the two domain protein. Several technical obstacles were overcome, including …

A Synthetic Biological Engineering Approach To Secretion- Based Recovery Of Polyhydroxyalkanoates And Other Cellular Products, Elisabeth Linton

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The costs associated with cellular product recovery commonly account for as much as 80% of the total production expense. As a specific example, significant recovery costs limit commercial use of polyhydroxyalkanoates (PHA), which comprise a class of microbially-accumulated polyesters. PHAs are biodegradable compounds that are of interest as a sustainable alternative to petrochemically-derived plastics. Secretion-based recovery of PHAs was studied to decrease PHA production costs. Type I and II secretory pathways are commonly used for the translocation of recombinant proteins out of the cytoplasm of E. coli. Proteins were targeted for translocation using four signal peptides (HlyA, TorA, GeneIII, …

Dose-Dependent Effects Of Oxygen On Metabolism In Rat Cortico-Hippocampal Brain Tissue Slices, Jennifer Lynne Hollyfield

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Studies have shown that 95% oxygen increases neuronal excitability and ROS production. We wanted to investigate the dose-dependent effects of oxygen on brain slice metabolism. We exposed rat brain cortico-hippocampal tissue slices to 0.40, 0.95, and 4.50 ATA O2 for 60 minutes, made dual-phase tissue extracts, and used multi-nuclear NMR experiments to elucidate the slice metabolism. We found that low doses of oxygen may shift metabolism toward anaerobic glycolysis. Elevated lactate suggests this shift, along with elevated ratios of NAD+/NADH which may drive the reactions toward the production of lactate. The results also suggest that high doses of oxygen may …

Nmr Analyses Show Tcdd Elicits Differences In Hepatic Metabolism In Female C57bl/6 Mice And Sprague-Dawley Rats, Meghan Katherine Makley

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TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) elicits tissue-, sex-, and species-specific effects. This study compares the hepatic response to an oral dose of TCDD in immature ovariectomized (i.o.) C57BL/6 mice (30 µg/kg) and i.o. Sprague-Dawley rats (10 µg/kg), at 72, 120, and 168 h post-dose. Hepatic lipid extracts were analyzed by ¹³C and ³¹P NMR, and aqueous extracts by ¹H and ³¹P NMR.

Consistent with increased lipid content in mice (p≤0.05), TCDD induced increases in hepatic triacylglycerides (TAG), cholesterol, and fatty acids. Principal component analysis of ¹³C spectra show treatment groups separate in mice, but not rats. …