Biochemistry Commons^™

Investigation Of The Role Of Heparin-Binding Pocket In Amyloid Fibrils Formation Of Fgf-1, I Gusti Ayu Agung Septiari

Graduate Theses and Dissertations

Human acidic fibroblast growth factor (aFGF/hFGF-1) is one of the promising molecules to be investigated to generate an in-depth understanding of the pathological mechanism of Alzheimer's disease (AD) neurodegenerative disorder characterized by the presence of amyloid fibrils. Some in vivo and human brain tissue studies proved the correlation of high-level expression of FGF-1-induced neuroinflammation and the occurrence of AD. The presence of amyloid fibrils as a hallmark of AD can be related to the generic property of the proteins to form amyloid fibrils; High level of FGF-1, in this case, may contribute to the formation of amyloid fibrils. As a …

Effect Of Charged Lipids On The Ionization Behavior Of Glutamic Acid Containing Transmembrane Helices, Brooke Nunn

Chemistry & Biochemistry Undergraduate Honors Theses

Transmembrane proteins make up critical components of living cells. Protein function can be greatly impacted by the charged state of its respective components, the side chains of amino acid residues. Thus far, in the lipid membrane, little is known about the properties of residues such as glutamic acid. To explore these properties, I have included glutamic acid in a suitable model peptide-lipid system for fundamental biophysical experiments. Within the system, I have placed a glutamic acid residue instead of leucine in the L14 position of the helical hydrophobic peptide GWALP23 (acetyl-GGALWLALALALAL¹⁴ALALWLAGA-amide). Substitutions of glutamine and aspartic acid serve …

Effect Of Ph And Lipid Composition On Membrane-Spanning Helices With Glutamic Acid Examined By Solid-State Nmr, Kelsey Marr

Chemistry & Biochemistry Undergraduate Honors Theses

Transmembrane proteins constitute about 30% of the proteins in a mammalian cell and are involved in major biological processes. The dynamic properties of membrane proteins and the ionization states of particular side chains are important for biological function. The biophysical properties of membrane proteins nevertheless can be difficult to decode, particularly for glutamic acid in the lipid environment of cell membranes. To study the ionization of glutamic acid in transmembrane peptides, guest glutamic acid residues were substituted into the well-defined model helix of GWALP23 (acetyl-GGAL4WLALALALALAL16ALWLAGA-amide). These guest residues were placed at position L16 or L4 and specific 2H-labeled alanine residues …

Understanding Cytochrome C Maturation In Anaerobic Archaea, Blake Wojciechowski

Biological Sciences Undergraduate Honors Theses

Methanoperedens nitroreducens (MPEBLZ), an archaeal methanotroph and close relative of Methanosarcina acetivorans, contain numerous cytochrome c proteins. However, difficulty in using these organisms as a model for cytochrome cresearch has created a pressure to express cytochrome c proteins in an organism that is much easier to work with. A punitive cytochrome c protein (MPEBLZ_04274) from M. nitroreducens was attempted to be cloned into a model methanogen M. acetivorans as well as Escherichia coli. Cytochrome c proteins are important for many metabolic processes within anaerobic archaea. In order for a mature cytochrome c to be formed heme must …

Cloning, Expression, Purification And Characterization Of Heparin-Binding Pocket Of Recombinant Fgf1, Quratulayn Ashraf

Graduate Theses and Dissertations

Fibroblast growth factors are polypeptide members of the FGF family, which to date comprises of at least 22 members. They belong to a group of growth factors and are involved in a variety of cellular processes including wound healing, angiogenesis, differentiation and development (organogenesis). Amongst FGF members, human acidic FGF-1 and basic FGF-2 are the most characterized. FGF-1 and FGF-2 are known to share more than 80% sequence similarity and have an identical structural fold. However, their biological roles are quite different. FGFs bind to heparin and heparan sulfate ligands through their heparin-binding pockets. The interactions are primarily electrostatic in …

Characterization Of Avidin And Case9 Single Protein Molecules By A Solid-State Nanopore Device, Haopeng Li

Graduate Theses and Dissertations

The shape and charge of a protein play significant roles in protein dynamics in the biological system of humans and animals. Characterizing and quantifying the shape and charge of a protein at the single-molecule level remains a challenge. Solid-state nanopores made of silicon nitride (SiNx) have emerged as novel platforms for biosensing such as diagnostics for single-molecule detection and DNA sequencing. SSN detection is based on measuring the variations in ionic conductance as charged biomolecules translocate through nanometer-sized channels driven by an external voltage applied across the membrane. In this paper, we observe the translocation of asymmetric cylindrical structure CRISPR-Cas9 …

Production And Purification Of Basic Fibroblast Growth Factor Fused To Two Collagen Binding Domains Expressed In E. Coli Bl21 Using Flask And Fed-Batch, Hazim Aljewari

Graduate Theses and Dissertations

Delivering effective and non-toxic doses of bioactive materials that can aid in activating tissue regeneration to wounded tissue has proven to be an enormous challenge. This study was designed to produce a potential therapeutic recombinant protein by fusing two collagen binding domains to basic fibroblast growth factors (bFGF) through a collagenase cleavage site linker, so it can release the bFGF in a wound site by the action of this enzyme. The novel fusion protein was expressed in Escherichia coli BL-21 (E. coli) using traditional flask shaker and fed-batch cultivation. Cell lysate was purified by FPLC using Immobilized metal affinity chromatography …

Asymmetric Synthesis Of The C29-C34 Moiety Of Fragment A Of The Antascomicin B & Thermal Azole Based Claisen Rearrangements, Dharma Theja Nannapaneni

Graduate Theses and Dissertations

The dissertation describes asymmetric synthesis towards C29-C34 moiety of fragment A of the Antascomicin B and Thermal azole based Claisen rearrangements. In chapter 1, we describes asymmetric synthesis towards C29-C34 moiety of fragment A of the Antascomicin B. The non-immunosuppressant Rapamycin, Ascomycin, and Tacrolimus (FK506), strongly binds with FKBP12, the ligand FKBP12 complexes responsible for immunosuppressive activity. Antascomicin B structurally related to Rapamycin, Ascomycin, and Tacrolimus (FK506), binds strongly to FKBP12, yet does not shown immunosuppressive activity. The ligand FKBP12 binding complexes shown to have potent neuroprotective and neurogenerative properties in mouse models of Parkinson’s disease. The linear synthesis of …

Probing Of Carbohydrate-Protein Interactions Using Galactonoamidine Inhibitors, Jessica B. Pickens

Graduate Theses and Dissertations

Glycoside hydrolases are ubiquitous and one of the most catalytically proficient enzymes known, and thus understanding their mechanisms are crucial. Most research has focused on the interaction of the glycon of substrates and their inhibitors within the active site of glycoside hydrolases. The inhibitors employed to probe these interactions generally had small aglycons (i.e. a hydrogen atom, amidines, small aliphatic groups, or benzyl groups). Here, the interactions of the aglycon with glycoside hydrolases are examined by probing the active sites with a library of 25 galactonoamidines. The studies described in this dissertation aim to increase the understanding of stabilization of …

Toward Understanding The Mechanism Of Protein Targeting In The Chloroplast Signal Recognition Particle Pathway, Mercede Furr

Graduate Theses and Dissertations

Protein targeting is a vital cellular function. The signal recognition particle (SRP) pathway is a universally conserved targeting system present in the cytosol and used to co-translationally target many proteins to the inner membrane of prokaryotes and the endoplasmic reticulum of eukaryotes. The chloroplast has a homologous SRP system which post-translationally targets light harvesting chlorophyll binding proteins (LHCPs) to the thylakoid membrane for integration. The chloroplast SRP (cpSRP) is a heterodimer with a 54 kDa subunit equivalent to SRP54 in the canonical pathway. In addition, cpSRP contains a novel 43 kDa subunit which is a unique and irreplaceable component. cpSRP43 …

Rotational Tuning Of Transmembrane Helix Properties Based On The Precise Placements Of Aromatic And Charged Residues, Matthew J. Mckay

Graduate Theses and Dissertations

Designed model transmembrane peptides and oriented 2H and 15N solid-state nuclear magnetic resonance (NMR) spectroscopy were used to analyze how simple sequence modifications can influence peptide structure, behavior and dynamics as well as for determining the pKa of glutamic acid at the membrane interface. The GW5,19ALP23 (acetyl-GGALW(LA)6LWLAGA-amide) peptide framework adopts a well-defined tilted orientation in lipid bilayers (DLPC, DMPC and DOPC) and undergoes low amounts of dynamic motion. The sequence was initially modified by moving the Trp residues outwards to positions 4 and 20. This new sequence GW4,20ALP23 (acetyl-GGAW(AL)7AWAGA-amide) displays high amounts of signal averaging of NMR observables caused by …

Single Molecule Fluorescence Studies Of Protein Structure And Dynamics Underlying The Chloroplast Signal Recognition Particle Targeting Pathway, Dustin R. Baucom

Graduate Theses and Dissertations

The work presented in this dissertation explores the structural dynamics in the chloroplast signal recognition particle pathway. Findings include cpSRP shows scanning functionality similar to that in the cytosolic SRP with the ribosome. The intrinsically disordered C-terminal tail of the Albino3 protein has some transient secondary structure. Upon binding to cpSRP43 in solution, separate secondary structure formation was identified in the C-terminal tail of Albino3. Finally, to increase efficiency of analyzing fluorescence time traces for this work, a modular software was produced.

Influence Of Single And Multiple Histidine Residues And Their Ionization Properties On Transmembrane Helix Dynamics, Orientations And Fraying, Fahmida Afrose

Graduate Theses and Dissertations

Since aromatic and charged residues are often present in various locations of transmembrane helices of integral membrane proteins, their impacts on the molecular properties of transmembrane proteins and their interactions with lipids are of particular interest in many studies. In this work, I used solid-state deuterium NMR spectroscopy in designed model peptide GWALP23 [GGALW(LA)6LWLAGA] with selective deuterium labels to addresses the pH dependence and influence of single and multiple “guest” histidine residues in the orientation and dynamic behaviors of transmembrane proteins. The mutations include Gly to His (G2/22 to H2/22), Trp to His (W5/19 to H5/19) and Leu to His …

The Effects Of Eicosapentaenoic Acid (Epa) And Docosahexaenoic Acid (Dha) On Brown Adipogenesis In Stem Cell Culture, Darynne Dahlem

Animal Science Undergraduate Honors Theses

Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are major maternal dietary supplements due to their positive benefits on neurological tissue growth during the first 12 weeks of gestation. Previous studies show that EPA and DHA inhibit muscle formation but promote adipogenesis. However, no research has addressed the question whether high intake of EPA and DHA affects brown fat development during gestation. The objective of this study was to measure the effect of EPA and DHA supplement on brown adipogenesis and potential pathways related to mitochondrial biosynthesis using fibroblasts as in vitro model. Using Oil-Red-O staining …

Autofluorescence To Study The Effects Of Acid Concentration On Cellular Metabolism In Vitro, Robin L. Raley

Chemistry & Biochemistry Undergraduate Honors Theses

Ultraviolet (UV) radiation-induced sunburns and their accompanying afflictions are a growing public health concern in the United States. There is a need for techniques that can accurately and non-invasively characterize the physiology of sunburned skin tissue directly after UV-damage and applying a topical skin treatment to relieve pain and promote healing. Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetime imaging (FLIM) can be used to investigate metabolic processes in live cells through endogenous fluorescence of the cofactors, NADH and FAD. These methods employ the optical redox ratio of FAD/(NADH+FAD), mean NADH lifetime, and the separation of the free and bound …

The Synthesis, Purification, And Characterization Of The P3 Peptoid, Myles Joyce

Chemical Engineering Undergraduate Honors Theses

The relatively young field of nano-systems has vast applications over various different fields. Peptoids (peptide analogues) and their uses are consistently investigated components of these nano-studies. A particularly interesting group of peptoids are able to self-assemble into secondary structures under the right conditions. The P3 peptoid is able to form microspheres if properly synthesized and purified. Microspheres are frequently studied due to their low surface area to volume ratios. This unique aspect of microspheres makes them an excellent candidate for drug delivery systems; however, these systems must also be robust so that the desired compound can be encapsulated within the …

Targeting Sec61Α By Ipomoeassin F Leads To Highly Cytotoxic Effect, Zhijian Hu

Graduate Theses and Dissertations

Ipomoeassin F is a flagship congener of a resin glycoside family that inhibits growth of many tumor cell lines with only single-digital nanomolar IC50 values. However, biological and pharmacological mechanisms of ipomoeassin F have been undefined. To facilitate exploration of the biological and pharmacological properties, we performed sophisticate SAR (Structure–activity relationship) studies of ipomoeassin F to understand its pharmacophore and structure properties so that we can design favorable probes for further biological investigation. By applying appropriate deviates that possess fluorescent groups and similar bio-activity, the target protein was found to be localized in endoplasmic reticulum (ER). Through biotin affinity pull …

Proteomics Of Carbon Fixation Energy Sources In Halothiobacillus Neapolitanus, Jonathan Hunter, Maria Marasco, Ilerioluwa Sowande, Newton P. Hilliard Jr.

Journal of the Arkansas Academy of Science

Through the use of proteomics, it was uncovered that the autotrophic, aerobic purple sulfur bacterium Halothiobacillus neapolitanus displays changes in cellular levels of portions of its carbon dioxide uptake and fixation mechanisms upon switch from bicarbonate to CO₂(g) as carbon source. This includes an increase in level of a heterodimeric bicarbonate transporter along with a potential switch between form I and form II of RubisCO. Additional changes are seen in several sulfur oxidation pathways, which may indicate a link between sulfur oxidation pathways as an energy source and carbon uptake/fixation mechanisms.

The First In Vivo Human Methionine Sulfide Proteome And The Impact Of Smoking, Abdullah Qassab

Graduate Theses and Dissertations

Reactive oxygen species are naturally generated within the human body and they are known to modulate signaling pathway and mediate other physiological activities. However, excessive generation of ROS and the inability of body defense system in detoxifying them results in the so called “oxidative stress”. Methionine has powerful antioxidant properties due to the presence of electronegative sulfur in its structure. Therefore, Met is readily oxidized, and methionine sulfoxide has been linked to several pathological conditions.

The urinary proteome is an attractive candidate for the discovery of biomarkers to diagnose and classify health conditions because of the non-invasive collection procedure. However, …

Ca2+ - Induced Structural Change Of Multi-Domain Collagen Binding Segments Of Collagenases Colg And Colh From Hathewaya Histolytica, Christopher Eric Ruth

Graduate Theses and Dissertations

Hathewaya histolytica, previously renamed as Clostridium histolyticum, secretes at least two collagenases, ColH and ColG, that allow for degradation of extracellular matrices of animal tissue. Hathewaya histolytica virulence factors are proposed to undergo domain rearrangement upon secretion from the bacteria to the host. In order to accomplish this, collagenases seek the least ordered regions in collagen to efficiently disassemble the fibril. Two types of domains, Polycystic Kidney Disease-like (PKD) and Collagen Binding Domain (CBD), direct the collagenases ability to disassemble the fibril. Calcium dependent structural change have been reported to increase in thermal stability and in tighter collagen binding for …

Characterization Of A Variant Of Tuberous Sclerosis Complex 2 And Its Interaction With Rheb, Sowmya Sivakumar

Graduate Theses and Dissertations

Protein-protein interactions are vital in maintaining proper function and homeostasis in cells. Some signaling pathways are regulated by G-proteins that work like switches to activate and deactivate pathways. Mutations in these proteins, their effectors or the interaction between proteins may cause dysregulation of signals that can lead to many diseases.

Rheb, Ras homology enriched in brain, is a Ras family GTPase that is vital in regulation of the mTOR (mammalian target of rapamycin) pathway that signals cell proliferation and growth. Due to the low intrinsic GTPase activity of Rheb, a GTPase activating protein (GAP), Tuberous Sclerosis Complex 2 (TSC2) down …

Engineering The Structure Of The Human Acidic Fibroblast Growth Factor To Enhance Its Stability And Cell Proliferation Activity, Julie Davis Eberle

Graduate Theses and Dissertations

Human acidic fibroblast growth factor (hFGF1) is a protein well known for its role in cell growth and differentiation. To elicit these cell-signaling processes, hFGF1 non-selectively binds to any one of the seven cell surface hFGF receptor isoforms. Due to its significant involvement in tissue repair activity, hFGF1 is a prime candidate for novel wound healing therapeutics. However, one drawback toward its use as a novel wound healing therapeutic is the poor inherent thermal stability of hFGF1, as it has been found to unfold near physiological temperature. The cause of this instability is strong electrostatic repulsion created by a dense …

Optimizing Genetic Manipulation Of Methanogens Through Faster Cloning Techniques, Merrisa Jennings

Biological and Agricultural Engineering Undergraduate Honors Theses

Methanogenesis is the biological production of methane. Only anaerobic archaea known as methanogens are capable of such a metabolic feat. They have strict living conditions and substrate sources which determine their rate of metabolism. This is of particular importance from a greenhouse gas reduction perspective or biogas capturing perspective. One of the best ways to optimize methanogen methane production is via genetic manipulation. The current procedures are timely though, therefore a faster cloning processes should be developed. The objective of this study was to optimize a premade genetic transformation kit known as the Gibson Kit. The Gibson Kit was supposed …

Determination Of The Effects Of Different Maillard Reaction Products On The Taxonomic Composition Of The Gut Microbiota, Nesreen Hamdan Aljahdali

Graduate Theses and Dissertations

The Maillard Reaction (MR) is a non-enzymatic chemical reaction which results in linkage between the amino group of amino acids and the carbonyl group of reduced sugars. This reaction generates Maillard reaction products (MRPs) which are not present naturally in foods, and are responsible for a range of colors, odors, flavors, and other sensory properties. Conflicting reports of MRPs impacts on human health are probably due to the fact that bioconversion of these digestible molecules by the gut microbiota has been marginally taken into account. This study aimed to determine the effects of different MRPs on rodent’s gut microbiota through16S …

Membrane Surface Engineering For Biochemical Applications, Anh T. Vu

Graduate Theses and Dissertations

Synthetic membranes have been frequently used for many fields, such as, the food and beverage, biopharmaceutical, and biofuel industries. In the beer industry, microfiltration frequently suffers from fouling due to the interaction between different species. It is shown that polyphenols can form cross-links with protein molecules, forming insoluble aggregates. However, by adding an optimal amount of polysaccharides these aggregates can be disrupted thus reducing fouling by the aggregates. Confocal laser scanning microscopy (CLSM) is a powerful technique to locate the foulants inside the wet membrane in order to understand more about the behavior of fouling in microfiltration.

Membrane surface modification …

Membranes For Food And Bioproduct Processing, Alexandru Marius Avram

Graduate Theses and Dissertations

Modified membranes for process intensification in biomass hydrolysis

Production of biofuels and chemicals from lignocellulosic biomass is one of the leading candidates for replacement of petroleum based fuels and chemicals. However, conversion of lignocellulosic biomass into fuels and chemicals is not cost effective compared to the production of fuels and chemicals from crude oil reserves. Some novel and economically feasible approaches involve the use of ionic liquids as solvents or co-solvents, since these show improved solvation capability of cellulose over simple aqueous systems. Membranes offer unique opportunities for process intensification which involves fractionation of the resulting biomass hydrolysate leading to …

Removal Of Endocrine Disrupting Compounds Using Membrane Bioreactor, Mohanad Ali Abdulsahib Kamaz

Graduate Theses and Dissertations

The presence of endocrine disrupting compounds (EDCs) and pharmaceutically active compounds (PhAC) such as pesticides, personal care products, antibiotics and pharmaceutical compounds, in sewage, industrial, and domestic waters has extensively become the major concern for health and environmental organizations. These compounds have the ability to interact with mammalian endocrine system and disrupting their functions. The traditional activated sludge processes are designed to degrade solids, organic carbon and nitrogen loading. Although several treatment steps in a wastewater treatment plant can contribute to partial removal of EDCs, effective removal has been a challenge due to their resistant chemical and biological degradation and …

Molecular And Biochemical Studies Of Several Novel Estrogen Receptor Alpha-Interacting Proteins In Breast Cancer Cells, Ahmed Edan Dhamad

Graduate Theses and Dissertations

Breast cancer is the second leading cause of cancer-related death in women, and approximately 70% of incidences are estrogen receptor (ER)-positive breast cancer. ERα and its interacting proteins play a key role in the development and progression of breast cancer. However, how ERα regulates its target gene expression and hence cell proliferation is not fully understood. To enhance our understanding of the molecular mechanism by which ERα regulates gene expression, we used a quantitative proteomic method to identify cellular proteins that interact with ERα. The first group of proteins that were identified to associate with ERα are heat shock proteins …

Structural Characterization Of Bacterial Collagenases, Ryan Bauer

Graduate Theses and Dissertations

Clostridium histolyticum secretes collagenases ColG and ColH to cause extensive tissue destruction during myonecrosis. The collagenases are multi-domain enzymes consisting of a N-terminal collagenase module, s1, polycystic kidney disease (PKD)-like domains (s2 in ColG; s2a and s2b in ColH) and collagen-binding domains (CBD) (s3a and s3b in ColG; s3 in ColH). The individual CBD and PKD-like domains chelate calcium to modulate stability and domain rearrangement. Though used by bacteria to break down collagen in the extracellular matrix during infection, therapeutic use of the enzyme to break down excess connective tissue has been approved by the FDA. Meanwhile, the use of …

Characterization Of A Tuberous Sclerosis Complex 2 Variant And Its Interaction Involving The Ras-Related Protein Rheb, Nosaiba Shokr

Graduate Theses and Dissertations

Structure-function relationships of any complex underlie the molecular details of the biological interactions. Rheb, Ras Homology Enriched in Brain, is a Ras-related protein, and it is genetically identified as a molecular switch. Rheb is regulated by cycling between the biologically active GTP and inactive GDP-bound forms. This regulation is partially controlled by an interaction with Tuberous Sclerosis Complex2 (TSC2), a GTPase activating protein (GAP). Upon interaction, TSC2 stimulates Rheb GTP hydrolysis and diminishes the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway is involved in proteins synthesis, cell cycle, and signaling. Mutation in TSC2 causes abnormal activation of Rheb …