Life Sciences Commons^™

Influence Of Histidine Residues, Ph And Charge Interactions On Membrane-Spanning Peptides, Ashley N. Henderson

Graduate Theses and Dissertations

Designed transmembrane peptides were employed for investigations of histidine residues within the hydrophobic environment of the lipid bilayer by means of oriented solid-state deuterium NMR spectroscopy. Using the model peptide GWALP23 sequence (GGALW(LA)6LWLAGA) as a host framework, the effects of single and double histidine mutations were explored. Replacement of leucine residue 12 to polar neutral histidine had little influence on the peptide average orientation, however under strongly acidic pH conditions in DOPC bilayers, the histidine becomes positively charged (pKa 2.5) and the GWALP23-H12 peptide exits the membrane and adopts a surface-bound orientation. Conversely, mutation of leucine 14 to neutral histidine …

Influence Of Ph And Acidic Side Chain Charges On The Behavior Of Designed Model Peptides In Lipid Bilayer Membranes, Venkatesan Rajagopalan

Graduate Theses and Dissertations

The molecular properties of transmembrane proteins and their interactions with lipids regulate biological function. Of particular interest are interfacial aromatic residues and charged residues in the core helix whose functions range from stabilizing the native structure to regulating ion channels. This dissertation addresses the pH dependence and influence of potentially negatively charged tyrosine, glutamic acid or aspartic acid side chains. We have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as favorable host peptide framework. We have substituted W5 with Tyr (Y5GWALP23) and Leu residues with Glu (L12E, L14E or L16E) or Asp (L14D or L16D), and have incorporated specific 2H-labeled alanine residues within the …

Applications Of The Gst- Affinity Tag In The Purification And Characterization Of Proteins, Wibke Beatrice Kachel

Graduate Theses and Dissertations

With the latest innovations in biological sciences, large quantities of biologically active polypeptides as well as high throughput screening methods to quickly evaluate if these biomolecules potentially have therapeutic, diagnostic, or industrial purposes are required. The synthesis and purification of peptides and small proteins continue to be demanding as the production of high yields through chemical synthesis can involve large costs. On the other hand, there are only few examples of acquiring those biomolecules through cloning and expression in bacterial systems in form of recombinant fusion proteins. Glutathione S-Transferase (GST) is not only a very commonly used affinity tag to …

Investigation Of The Inherent Chemical, Structural, And Mechanical Attributes Of Bio-Engineered Composites Found In Nature: Alligator Gar’S Exoskeleton Fish Scales, Wayne Derald Hodo

Graduate Theses and Dissertations

The U.S. Army has determined a huge cost savings of up to 51% can be accomplished by reducing the gross vehicle weight, for their personnel carrier, by 33%. To cut cost, composite materials are needed. Man-made composites can have superior material properties (high-strength, high-fracture toughness, and lightweight), but they are prone to delamination at the glued-layered interface. In contrast, fish scale is a natural composite that has the same material properties and, additionally, tend not to delaminate.

The focus of this study was to learn how nature integrates hard and soft materials at each length scale to form a layered …

Optimizing A Luciferase-Based Tool For Studying The Effects Of Fatty Acid Desaturase 7 On Singlet Oxygen Accumulation In Arabidopsis Thaliana, Abeer Muhammedali Alnasrawi

Graduate Theses and Dissertations

In plants, reactive oxygen species (ROS) are generated as a byproduct of normal metabolism, as well as in response to adverse conditions such as light stress, extreme temperatures, and exposure to pests and pathogens. Singlet oxygen (1O2) is a ROS that is formed during photosynthesis in photosystem II (PSII) of the chloroplasts. Levels of 1O2 and other ROS are tightly controlled in healthy plants, but some studies suggest that levels of fatty acid desaturase (FAD) activity in Arabidopsis thaliana can influence constitutive and/or stress-responsive ROS accumulation. In this study, a luciferase-based reporter gene that is selectively stimulated by 1O2 (AAA-ATPase: …

Production And Characterization Of Islet Amyloid Polypeptide Using Recombinant Technology, Emilio Duverna

Graduate Theses and Dissertations

Islet amyloid polypeptide (IAPP) also called amylin is an amyloid-forming protein; IAPP is a proteinaceous hormone that comprises 37 amino acid residues. It is secreted along with insulin from the pancreatic β-cells to help it regulate the uptake and removal of glucose in the bloodstream. IAPP has been observed in the amyloid deposits found in pancreatic β-cells of most patients suffering from type II diabetes mellitus. This research project aims at producing recombinant amylin peptide. To achieve this goal, we used the pBAD plasmid vector which we introduced into Escherichia coli to express the peptide. Although the vector was successfully …

Designing Fret Assays To Study Electrostatic Interactions Pertaining To The Binding Of Intrinsically Disordered Proteins, Ashley Ann Howard

Graduate Theses and Dissertations

Fibroblast growth factor receptor plays a major role in several biological processes. Without FGFR, a human cannot live. FGFR is involved in cell differentiation and wound healing. Of course, if FGFR signaling becomes unregulated, it causes severe distress in the body. Several cancers are contributed to high signaling levels, as well as developmental conditions like rickets and Kallmann’s syndrome. FGFR is thought to undergo an auto-inhibition (or self-regulatory) process in order to try to facilitate regulation. The exact method of this inhibition is currently unknown, but is proposed to involve the unstructured acid box region of FGFR. We developed a …

Local Modulation And Measurement Of Macrophage-Derived Bioactive Proteins From Implanted Biomaterials In Rat, Geetika Bajpai

Graduate Theses and Dissertations

Fibrosis around the implanted medical devices is a severe problem that can plague long-term device reliability. Activation of macrophage phenotype (macrophage polarization) has emerged as a new and possible means for reducing fibrosis in the fields of biomaterials and regenerative medicine. Macrophages are phagocytic cells that respond to microenvironmental cues that direct their phenotype. Macrophage activation has been widely studied in mouse and human in the context of tumor biology, yet little information is available regarding how macrophage activation could be used in a biomaterials context. Further, rats rather than mice are the common subjects in biomaterials experiments. A significant …

Inducing And Characterizing M2c Macrophages At A Non-Degradable Implant In Vivo, Geoffrey David Keeler

Graduate Theses and Dissertations

The implantation of biomaterials in the body leads to a reaction known as the foreign body response (FBR) which leads to the eventual encapsulation of the biomaterial. This encapsulation proves to be detrimental to certain biomaterials, such as implanted glucose sensors, which rely on interacting with the extracellular space for proper function. Altering the FBR has become of interest in an effort to increase the longevity and integration of biomaterials. One strategy for altering the FBR is by targeting the macrophage, shown to play an important role in the FBR. In this work, the microdialysis sampling technique was used to …

Dlipin-A Link Between Lipid Metabolism, Glucose Homeostasis And Growth In Drosophila Melanogaster, Sandra Schmitt

Graduate Theses and Dissertations

Lipins are a family of highly conserved proteins found from yeasts to humans. Lipins have dual functions, serving as phosphatidate phosphatase enzymes (PAP) in the synthesis of neutral fats (triacylglycerols, TAG) and as transcriptional co-regulators that affect the expression of genes involved in lipid and fatty acid metabolism. Thus, they play central roles in metabolic control. Disruption of Lipin function has been implicated in lipodystrophy, obesity and insulin resistance. Using dLipin, the Drosophila homolog of Lipin, as a model, I aimed to elucidate the relationship between the two biochemical functions of Lipin and metabolic homeostasis. I discovered there is a …

Biochemical And Biophysical Studies Of Novel Features Of Ras-Related Protein Interactions, Kyla Marie Morinini Morris

Graduate Theses and Dissertations

The Ras superfamily of G-proteins are of great research interest for structure-function relationships among proteins as they act as molecular switches in the regulation of various biochemical reactions in the cell. They are regulated by protein-protein interactions targeted to the highly flexible switch regions. Mutations in G-proteins or their effectors may cause alterations in structure and/or function that can lead to overactivity.

The Ras-related protein Cell division cycle 42 (Cdc42) is important in regulating cell-signaling processes. The T35A mutation in Cdc42 leads to a decrease in flexibility of the Switch I region responsible for effector binding. The kinetics of the …

Design, Development, And Characterization Of Novel Antimicrobial Peptides For Pharmaceutical Applications, Yazan H. Akkam

Graduate Theses and Dissertations

Candida species are the fourth leading cause of nosocomial infection. The increased incidence of drug-resistant Candida species has emphasized the need for new antifungal drugs. Histatin 5 is a naturally occurring human salivary antifungal peptide and the first line of defense against infections of the oral cavity. This research has focused on understanding the activity of histatin 5, and subsequently designing novel peptides that may serve as models for the further development of therapeutics to treat fungal infection.

This objective has been achieved in three steps: studying the structural requirement of histatin 5 involved in antifungal activity, the identification of …

Effects Of Ergot Alkaloids And Bovine Bodily Fluids On Cytochrome P450 Activity, Nicholas Scott Ezell

Graduate Theses and Dissertations

This thesis evaluates the Promega^TM P450-Glo assay (Promega^TM V9800) as a tool for quantifying ergot alkaloid concentration. Current techniques used for detection of ergot alkaloids are slow and expensive, do not detect all ergot alkaloids, or are not effective on bovine bodily fluids. The first study was conducted to determine effects of commercial ergot alkaloids (n = 6; 0 - 400 μM) on the Promega^TM P450-Glo assay. Cytochrome P450 (CYP450) activity in assay had a differential response to each ergot alkaloid and concentration. As concentrations of ergotamine, dihydroergotamine, ergocornine, and ergocryptine increased CYP450 activity was inhibited (P …

An Analysis Of The Redox Properties And Stability Of Chlamydomonas Reinhardtii Cytochrome F, Cytochrome C6, And Mutants Thereof, Nicole Lynn Vanderbush

Graduate Theses and Dissertations

This body of work presents mutagenesis studies conducted on two c-type cytochromes from Chlamydomonas reinhardtii. Cytochrome f, a unique c-type cytochrome, is investigated in regards to its redox potential, the dependence of the redox potential, and the thermal stability of the protein. The mutations made were Y1F, Y9F, Y160F, Y160L, R156L, and R156K. The residues that were mutated surround the heme. It was found that, relative to the wild-type, only the Y160L and R156 mutants showed any difference in midpoint potential at pH 7. Wild-type and mutants both had a midpoint potential that was dependent upon pH indicating that none …

Investigations Of Protein-Lipid Interactions In Model Membranes: Influence Of Aromatic Anchoring Residues And Buried Polar Residues, Nick Gleason

Graduate Theses and Dissertations

To investigate in detail the interactions between transmembrane proteins and the lipid bilayers in which they are constituted, designed model peptides with selective isotopic labels were synthesized and analyzed by means of solid-state deuterium NMR spectroscopy. Starting from the well-characterized model peptide GWALP23, acetyl-GGALW(LA)6LWLAGA-amide, several Trp to Tyr mutations were compared to evaluate their respective interfacial anchoring abilities. It was found that Tyr, substituted on either or both termini, can effectively anchor the transmembrane alpha-helix, which then adopts a similar transmembrane topology in a range of bilayer thicknesses. Nevertheless, a consistent ~10° shift in tilt direction (helix rotation) is observed …

Characterization Of Novel Histone H2b Mutants Associated With Chromosome Segregation Defects In Saccharomyces Cerevisiae, Thiruchelvam Rajagopal

Graduate Theses and Dissertations

Histones are small basic proteins that associate with DNA to form the basic unit of chromatin, the nucleosome. Histones H3 and H4 form a tetramer that is bound by two H2A-H2B dimers to form the histone octamer, to which approximately 146 bp of DNA wrap around to form the nucleosome. High resolution structural information and recent advances in the understanding of histone post-translational modifications have illuminated the many regulatory functions chromatin exerts in the cell, from the transcriptional control of gene expression to chromosome segregation. However, the specific role that histones play in these processes is not well understood. Previous …

The Role Of Albino3 And The Lipid Environment In Chloroplast Signal Recognition Particle Targeting, Nathan Lewis

Graduate Theses and Dissertations

Signal recognition particles (SRPs) in pro- and eukaryotes function in cotranslational targeting of nascent poplypeptides to an SRP receptor at the target membrane. A unique chloroplast SRP (cpSRP) functions post-translationally to direct light-harvesting chlorophyll-binding proteins (LHCPs) to the receptor cpFtsY at the thylakoid membrane for LHCP insertion in a process involving the integral membrane protein Albino3 (Alb3) and requiring GTP. Work here focuses on understanding cpSRP targeting events at the thylakoid membrane, specifically those involving Alb3 and the lipid environment.

We show an interaction between the novel cpSRP subunit cpSRP43 and the soluble, stromal-exposed C terminus of Albino3 (Alb3-Cterm). We …

Tryptophan Anchored Peptides In Lipid Bilayer Membranes: Control Of Peptide Orientation And The Phase Behavior Of Cholesterol-Containing Ternary Lipid Mixtures, Johanna Maria Rankenberg

Graduate Theses and Dissertations

Model WALP peptides and "next generation" WALP-derived hydrophobic model peptides were employed to discover principles that govern protein-lipid interactions in biological membranes.

Ternary cholesterol-containing lipid mixtures were examined in the presence of WALP peptides of different lengths (acetyl-GWW(LA)_nLWWA-ethanolamide, with n between 3 and 8). Deuterium NMR spectra from labeled lipids reveal that WALP peptides may stabilize lipid ordered "raft" domains and therefore promote lipid phase separation, albeit to a minor extent. The results depend upon whether dioleolyl- or diphytanoyl-phosphatidylcholine is present as the fluid lipid component.

Several WALP peptides were modified to remove anchoring Trp residues from one …

Characterization And Functional Regulation Of Bioactive Peptides In Avian Macrophages And Heterophils, Lakshmi Kannan

Graduate Theses and Dissertations

Oligopeptides and low molecular weight polypeptides play central roles as effectors and signal transducers acting as hormones, neurotransmitters, growth factors, toxins, and antimicrobial factors that are important for the survival of the organism. Owing to the ubiquitous involvement of peptides in many key regulatory processes, we have been interested to identify native peptides in different cells and tissues and understand their functions. To conduct our studies, we used avian macrophages and heterophils as models of specialized cells which constitute central components of innate immunity. These studies involved (a) qualitative identification and characterization of the peptides associated with high intensity mass …