Biochemistry Commons^™

Disorder Levels Of C-Myb Transactivation Domain Regulate Its Binding Affinity To The Kix Domain Of Creb Binding Protein, Anusha Poosapati

USF Tampa Graduate Theses and Dissertations

Intrinsically disordered proteins (IDPs) do not form stable tertiary structures like their ordered partners. They exist as heterogeneous ensembles that fluctuate over a time scale. Intrinsically disordered regions and proteins are found across different phyla and exert crucial biological functions. They exhibit transient secondary structures in their free state and become folded upon binding to their protein partners via a mechanism called coupled folding and binding. Some IDPs form alpha helices when bound to their protein partners. We observed a set of cancer associated IDPs where the helical binding segments of IDPs are flanked by prolines on both the sides. …

Dissecting Molecular Pathways That Ensure Proper Chromosome Segregation And Cell Division, Anna Ye

Doctoral Dissertations

Equal segregation of the genome is a prerequisite for cell survival. During cell division the duplicated DNA is compacted into chromosomes and a multi-protein macrostructure, known as the kinetochore (Kt), is assembled on each copy of compacted DNA. Simultaneously, the mitotic spindle, which is made up of microtubules (MTs), is built to facilitate the equal distribution of the chromosomes between the resulting daughter cells. Kinetochores mediate the interaction between the MTs and the chromosomes, properly positioning them for segregation. To ensure that the DNA is equally divided in every cell division, cells have built a surveillance system to detect any …

Pore Forming Protein Assembly And The Use In Nanopore Sensing: A Study On E. Coli Proteins Clya And Ompg, Monifa Fahie

Doctoral Dissertations

Pore forming proteins are typically the proteins that form channels in membranes. They have several roles ranging from molecule transport to triggering the death of a cell. This work focuses on two E. coli pore forming proteins that have vastly differing roles in nature. Outer membrane protein G (OmpG) is an innocuous β-barrel porin while Cytolysin A (ClyA) is an α-helical pore forming toxin. For OmpG we probed its potential to be a nanopore sensor for protein detection and quantification. A small high affinity ligand, biotin, was covalently attached to loop 6 of OmpG and used to capture biotin-binding proteins. …

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 …

Sequence Determinants Of The Individual And Collective Behaviour Of Intrinsically Disordered Proteins, Alexander S. Holehouse

Arts & Sciences Electronic Theses and Dissertations

Intrinsically disordered proteins and protein regions (IDPs) represent around thirty percent of the eukaryotic proteome. IDPs do not fold into a set three dimensional structure, but instead exist in an ensemble of inter-converting states. Despite being disordered, IDPs are decidedly not random; well-defined - albeit transient - local and long-range interactions give rise to an ensemble with distinct statistical biases over many length-scales. Among a variety of cellular roles, IDPs drive and modulate the formation of phase separated intracellular condensates, non-stoichiometric assemblies of protein and nucleic acid that serve many functions. In this work, we have explored how the amino …

Elucidating Mechanisms Of Protein Aggregation In Alzheimer’S Disease Using Antibody-Based Strategies., Benjamin A. Colvin

Dissertations

Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder. There are two characteristic histopathological hallmarks in the brain: senile plaques and neurofibrillary tangles, composed of insoluble aggregates of the amyloids Amyloid-β (Aβ) and tau protein, respectively. These diagnostic markers, though distinctive, are not apparent effectors of AD pathology. Evidence has mounted suggesting smaller soluble aggregates (oligomers) of Aβ or tau are the true drivers of disease progression. This dissertation presents several amyloid biophysics projects. Aggregate biophysical parameters such as weight, shape, and conformation were measured using a range of methodologies, including Multiangle Light Scattering, Dynamic Light Scattering, UV-Circular Dichroism, UV-Fluorescence, Scanning …

The Mechanistic Requirements Of Passive H+ Import Through The Na, K-Atpase, Kevin S. Stanley

Theses and Dissertations

This work focuses on the elucidation of the mechanism of passive proton import through the Na,K-ATPase. This enzyme uses the energy in ATP hydrolysis to exchange three intracellular Na+ for two extracellular K+ to maintain ion gradients within the cell, and while in the absence of physiological external Na+ and K+, the phosphorylated externally open (E2P) conformation passively imports protons, generating an inward current (IH). Chapter one reports on the effects of intracellular cations and nucleotides to shift the Na,K-ATPase into the E2P conformation. We identified that a combination of either internal Na+ and ATP or K+ and Pi. In …

Structural, Biophysical, And Functional Studies Of Trem2 In Neurodegenerative Disease, Daniel L. Kober

Arts & Sciences Electronic Theses and Dissertations

Alzheimer's disease (AD) and other neurodegenerative diseases present a large and growing challenge to global health. The immune system, particularly the innate immune system, is increasingly recognized as having a major role in these pathologies. The innate immune system is responsible to contain disease and promote healing. However, immune misregulation exacerbates disease. The innate immunomodulatory receptor Triggering receptor expressed on myeloid cells-2 (TREM2) is expressed on myeloid cells such as dendritic cells, macrophages, and in the brain, on microglia. TREM2 is a single-pass transmembrane receptor with an extracellular Ig domain that mediates ligand binding. This protein regulates inflammation in vitro …

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 …

Fret-Based Investigations Of The Structure-Function Relationships In The Nmda Receptor, Drew M. Dolino

Dissertations & Theses (Open Access)

The N-methyl-D-aspartate (NMDA) receptor is one member of a class of proteins known as the ionotropic glutamate receptors. Ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the central nervous system, with the NMDA receptor standing out among these receptors for its requirement of a co-agonist, its magnesium-block-based coincidence detection, its slow kinetics, its calcium permeability, its allosteric modulation, and its especially important functional roles in synaptic plasticity, excitotoxicity, and more. In recent years, a wealth of structural information has come about describing endpoint structures to high resolution, but such structures are unable to fully resolve the movements …

Disorder In Cysteine-Rich Granulin-3 And Its Implication In Alzheimer Disease, Gaurav Ghag

Dissertations

Granulins (GRNs) are a family of small, cysteine-rich proteins that are generated upon proteolytic cleavage of their precursor, progranulin (PGRN) during inflammation. All seven GRNs (1 – 7 or A – G) contain twelve conserved cysteines that form six intramolecular disulfide bonds, rendering this family of proteins unique. GRNs play multiple roles and are involved in a myriad of physiological as well as pathological processes. They are known to a play role in growth and embryonic development, wound healing, and signaling cascades as well as in tumorigenesis. They are also implicated in neurodegenerative diseases like frontotemporal dementia (FTD), Alzheimer disease …

Biophysical Studies Of Hairpin Polyamides With Broad-Spectrum Activity Against High-Risk Human Papillomaviruses, Carlos H. Castaneda

Dissertations

Human papillomavirus is a small dsDNA virus that infects mucosal and cutaneous epithelial tissues. Persistent infection with high-risk HPV is the main etiological agent in the development of cervical cancer worldwide. Although prophylactic vaccines against HPV are available, these preventative measures are type-specific and are ineffective against existing infections. Thus, there is a pressing need for antiviral drugs with a broad-spectrum activity against HPV to eradicate existing infections, no matter the subtype.

Our group and collaborators have synthesized an extensive library of novel N-methylpyrrole/N-methylimidazole (Py/Im) hairpin polyamides (PAs) with broad-spectrum activities against three prevalent oncogenic-HPV types (HPV16, …

Chloride And Proton Binding In The E. Coli 2cl¯:1h+ Clc Exchanger, Catherine Chenal

Dissertations, Theses, and Capstone Projects

The CLC family of membrane proteins is a ubiquitously expressed class of proton and usually voltage-activated chloride transporters involved in a myriad of physiological functions. Crystallographic structures identify up to three chloride binding sites: external, central and intracellular located in the inner half of the trans-membrane domain. The CLC proteins, except for the kidney isoforms, are gated by the extracellular side-facing gating Glutamate (Ex, E148 in CLC-ec1, the E. coli exchanger), which is thought to undergo a conformational change upon protonation.

To sort out how the thermodynamic paths to H+ coupled Cl¯ binding and conformational change in CLC-ec1 at the …

Neuronal Gq Structures In Neurodegeneration, Damian S. Mcaninch

Electronic Theses and Dissertations

This study investigates protein nucleic acid interactions between various proteins and G quadruplex (GQ) forming messenger RNAs (mRNAs) in human neurological disorders. GQ structures are formed in DNA/RNA, when four guanine residues form planar tetrads stabilized by Hoogsteen base pairing, that stack forming a GQ structure stabilized by potassium ions. These GQ structures are targeted by the arginine-glycine-glycine (RGG) repeat domain containing RNA-binding domain.

Three RGG domain containing RNA-binding proteins, all of which have been implicated in neurological disorders, and their interactions with GQ forming mRNAs, were investigated in this study: fused in sarcoma (FUS), fragile X mental retardation protein …

Micro-Spectroscopy Of Bio-Assemblies At The Single Cell Level, Jeslin Kera

Honors Undergraduate Theses

In this thesis, we investigate biological molecules on a micron scale in the ultraviolet spectral region through the non-destructive confocal absorption microscopy. The setup involves a combination of confocal microscope with a UV light excitation beam to measure the optical absorption spectra with spatial resolution of 1.4 μm in the lateral and 3.6 μm in the axial direction. Confocal absorption microscopy has the benefits of requiring no labels and only low light intensity for excitation while providing a strong signal from the contrast generated by the attenuation of propagating light due to absorption. This enables spatially resolved measurements of single …

Evolution Of The Kinetics And Dynamics Of Heme-Crevice Loop Regulating Chemistry In Human Cytochrome C, Shiloh M. Nold

Graduate Student Theses, Dissertations, & Professional Papers

Cytochrome c, cytc, is a metalloprotein that plays primary roles in electron transport and intrinsic apoptotic pathways. Much of the chemistry that cytc is involved with is regulated by a highly conserved region known as the heme crevice loop, consisting of residues 70-85. Only three of these residues (those at positions 81, 83 and 85) are not universally conserved within the evolutionary timeline. Here I look to elucidate possible evolutionary roles for several of the key residues known to be important in regulating heme chemistry of cytc.

I first address the role that lysine …

Multifunctional Nanomaterials Utilizing Hybridization Chain Reaction For Molecular Diagnostics And Bioanalytical Applications, Md Muhit Rana

Legacy Theses & Dissertations (2009 - 2024)

DNA nanotechnology has shown great promise in molecular diagnostic, bioanalytical and biomedical applications. The great challenge of detecting target analytes, biomarkers and small molecules, in molecular diagnostics is low yield sensitivity. To address this challenge, different nanomaterials have been used for a long time and to date there is no such cost-effective bioanalytical technique which can detect these target biomarkers (DNA, RNA, circulating DNA/miRNA) or environmental heavy metal ions (Hg2+ and Ag+) in a cost-effective and efficient manner.