Biochemistry, Biophysics, and Structural Biology Commons^™

Exploring The Structure And Biochemistry Of Oxidation-Mediated Inhibitation Of The Peptidyl-Prolyl Isomerase Pin1, Brendan T. Innes

Electronic Thesis and Dissertation Repository

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that has been shown to be neuroprotective in aging-related neurodegenerative diseases such as Alzheimer's disease (AD). However, it is not active in AD brain, and a recent proteomic screen of Mild Cognitive Impairment (MCI) brain samples revealed that Pin1 is oxidized in the brains of these pre-AD patients. This suggests that this oxidation may be the cause of the loss of the neuroprotective Pin1 function in AD. The Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pKa, making it highly susceptible to oxidation. We hypothesize that Pin1 is …

Intrinsic Disorder Within An Akap-Protein Kinase A Complex Guides Local Substrate Phosphorylation, F. Donelson Smith, Steve L. Reichow, Jessica L. Esseltine, Dan Shi, Lorene K. Langeberg, John D. Scott, Tamir Gonen

Chemistry Faculty Publications and Presentations

Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme …

Study Of Coronavirus Protease Using Cfp-Yfp Fluorescent Assay, Caitlin E. Specht, Andrew Mesecar Ph.D., Katrina Molland Ph.D.

The Summer Undergraduate Research Fellowship (SURF) Symposium

Middle Eastern Respiratory Syndrome (MERS) is an emerging viral disease originating in the Arabian Peninsula with a current mortality rate of nearly fifty percent throughout Europe and Asia according to the World Health Organization. Characterization of this disease is being done to understand the basis of viral replication. One target for viral inhibition are replication proteases. Replication proteases are enzymes that cleave proteins specific to cell growth and reproduction that form the viral replicase complex making them an ideal target for viral replication inhibition. First, replication proteases were characterized using a fluorescence resonance energy transfer (FRET) construct by measuring the …

Pathoscope: Species Identification And Strain Attribution With Unassembled Sequencing Data., Owen E Francis, Matthew Bendall, Solaiappan Manimaran, Changjin Hong, Nathan L Clement, Eduardo Castro-Nallar, Quinn Snell, G Bruce Schaalje, Mark J Clement, Keith A Crandall, W Evan Johnson

Computational Biology Institute

Emerging next-generation sequencing technologies have revolutionized the collection of genomic data for applications in bioforensics, biosurveillance, and for use in clinical settings. However, to make the most of these new data, new methodology needs to be developed that can accommodate large volumes of genetic data in a computationally efficient manner. We present a statistical framework to analyze raw next-generation sequence reads from purified or mixed environmental or targeted infected tissue samples for rapid species identification and strain attribution against a robust database of known biological agents. Our method, Pathoscope, capitalizes on a Bayesian statistical framework that accommodates information on sequence …

Allosteric Mechanism Of Water Channel Gating By Ca2+–Calmodulin, Steve Reichow, Daniel M. Clemens, J. Alfredo Freites, Karin L. Németh-Cahalan, Matthias Heyden, Douglas J. Tobias, James E. Hall, Tamir Gonen

Chemistry Faculty Publications and Presentations

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 …

The N-Terminus Of The Saccharomyces Cerevisiae G Protein-Coupled Receptor Ste2p: Formation Of Dimer Interfaces And Negative Regulation, Mohammad Seraj Uddin

Doctoral Dissertations

G protein-coupled receptors (GPCRs), the largest family of membrane proteins on the cell surface, play essential roles in signal transduction in all eukaryotic organisms. These proteins are responsible for sensing and detecting a wide range of extracellular stimuli and translating them to intracellular responses. This signaling requires a tight control for receptor activation without which abnormal signal leads to diseases. In fact, malfunctions of these receptors are associated with numerous pathological conditions and currently an estimated 40-50% of therapeutic drugs are designed to target these receptors suggesting that further increases in understanding of GPCRs and the signaling pathways they initiate …

Bioengineering The Expression Of Active Recombinant Human Cathepsin G, Enteropeptidase, Neutrophil Elastase, And C-Reactive Protein In Yeast, Eliot T. Smith

Electronic Theses and Dissertations

The yeasts Pichia pastoris and Kluyveromyces lactis were used to express several recombinant human proteins for further biochemical characterization. Two substitution variants of recombinant human enteropeptidase light chain (rhEPL) were engineered to modify the extended substrate specificity of this serine protease. Both were secreted as active enzymes in excess of 1.7 mg/L in P. pastoris fermentation broth. The substitution variant rhEPL R96Q showed significantly reduced specificities for the preferred substrate sequences DDDDK and DDDDR; however, the rhEPL Y174R variant displayed improved specificities for these substrate sequences relative to all other reported variants of this enzyme. The neutrophil serine proteases human …

Structural Characterization Of The Redox-Dependent Differences In The Cytochrome P450cam-Putidaredoxin Complex Using Solution Nmr Spectroscopy, Nicholas John Lopes

Masters Theses

Complexation between proteins as part of biological electron transfer reactions is driven by precise interactions that are often characterized by short lifetimes, weak affinities and high turnover rates. These complex interactions are difficult to study structurally in physiologically relevant oxidation states due to their transient nature and/or large molecular sizes. One such protein complex in the cytochrome P450 family of enzymes that is of great interest to researchers due to its prototypical nature is the Putidaredoxin (Pdx)- cytochrome P450cam (CYP101) electron transfer complex that is involved in hydroxylation of D-camphor in the bacterium Pseudomonas putida. While the individual protein structures …

Function And X-Ray Crystal Structure Of Escherichia Coli Yfde, Elwood A. Mullins, Kelly L. Sullivan, T. Joseph Kappock

T. Joseph Kappock

Many food plants accumulate oxalate, which humans absorb but do not metabolize, leading to the formation of urinary stones. The commensal bacterium Oxalobacter formigenes consumes oxalate by converting it to oxalyl-CoA, which is decarboxylated by oxalyl-CoA decarboxylase (OXC). OXC and the class III CoA-transferase formyl-CoA:oxalate CoA-transferase (FCOCT) are widespread among bacteria, including many that have no apparent ability to degrade or to resist external oxalate. The EvgA acid response regulator activates transcription of the Escherichia coli yfdXWUVE operon encoding YfdW (FCOCT), YfdU (OXC), and YfdE, a class III CoA-transferase that is ~30% identical to YfdW. YfdW and YfdU are necessary …

Phage Cluster Relationships Identified Through Single Gene Analysis., Kyle C Smith, Eduardo Castro-Nallar, Joshua Nb Fisher, Donald P Breakwell, Julianne H Grose, Sandra H Burnett

Computational Biology Institute

BACKGROUND: Phylogenetic comparison of bacteriophages requires whole genome approaches such as dotplot analysis, genome pairwise maps, and gene content analysis. Currently mycobacteriophages, a highly studied phage group, are categorized into related clusters based on the comparative analysis of whole genome sequences. With the recent explosion of phage isolation, a simple method for phage cluster prediction would facilitate analysis of crude or complex samples without whole genome isolation and sequencing. The hypothesis of this study was that mycobacteriophage-cluster prediction is possible using comparison of a single, ubiquitous, semi-conserved gene. Tape Measure Protein (TMP) was selected to test the hypothesis because it …

Germline Transgenic Methods For Tracking Cells And Testing Gene Function During Regeneration In The Axolotl, Shahryar Khattak, Maritta Schuez, Tobias Richter, Dunja Knapp, Saori L. Haigo, Tatiana Sandoval-Guzmán, Kristyna Hradlikova, Annett Duemmler, Ryan R. Kerney, Elly M. Tanaka

Biology Faculty Publications

The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum(axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly …

Systematic Analysis Of Residues In Conserved Region 3 Of The Human Papillomavirus 16 E7 Oncoprotein, Biljana Todorovic

Electronic Thesis and Dissertation Repository

Although remarkable biological diversity is exhibited by viruses, as obligate intracellular parasites, they rely on host cell functions. As such, viruses typically must overcome a set of host barriers that prevent infection. For human papillomaviruses (HPV) one of these barriers is the state of terminal differentiation of the host cell. For that purpose HPVs encode two major oncoproteins, E6 and E7, which combine their efforts to effectively uncouple cellular differentiation from the cell cycle arrest. The E7 proteins have no intrinsic enzymatic activity or DNA binding ability, but they bind and manipulate numerous host proteins. E7 is a modular oncoprotein …

Understanding The Origins Of Product Specificity Of Protein Methyltransferases From Qm/Mm Md And Free Energy Simulations, Yuzhuo Chu

Doctoral Dissertations

Protein lysine methyltransferases (PKMTs) catalyze the methylation of certain lysine residues on histone tails using S-adenosyl-L-methionine (AdoMet) as the methyl donor. Regulation of chromatin structure and gene expression through histone lysine methylation depends on the degree of methylation. Therefore, it is of importance to understand the features of PKMTs that control how many methyl groups would be added to the target lysine (product specificity). In my dissertation, I have applied quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations to understand the origin of the product specificities of SET8, GLP and their mutants. The relative free energy barriers …

Biophysical Studies Of Styrene-Maleic Acid Copolymer Stabilized Membrane Mimetics, Diane Yu

University Scholar Projects

Traditional methods of isolating membrane proteins involve the use of detergents that destabilize the membrane environment. Biophysical studies of membrane proteins are challenged by the requirement of a stable membrane platform. Lipodisq® nanoparticles are novel membrane mimetics that consist of 3:1 styrene-maleic acid copolymer encircling phospholipids to form discoidal species on the order of 10 nm in diameter. Bacteriorhodopsin was previously incorporated into Lipodisq® nano-particles from its native purple membrane without the addition of detergent. In this study, dynamic light scattering was used to investigate the stability of empty Lipodisq® nano-particles over time. These data show that Lipodisq® nano-particles are …

Thermodynamics Of Binding By Calmodulin Correlates With Target Peptide Α-Helical Propensity, Tori B. Dunlap, Jessime M. Kirk, Emily A. Pena, Meghan S. Yoder, Trevor P. Creamer

Center for Structural Biology Faculty Publications

In this work, we have examined contributions to the thermodynamics of calmodulin (CaM) binding from the intrinsic propensity for target peptides to adopt an α-helical conformation. CaM target sequences are thought to commonly reside in disordered regions within proteins. Using the ability of TFE to induce α-helical structure as a proxy, the six peptides studied range from having almost no propensity to adopt α-helical structure through to a very high propensity. This despite all six peptides having similar CaM-binding affinities. Our data indicate there is some correlation between the deduced propensities and the thermodynamics of CaM binding. This finding implies …

Bayesian Reconstruction Of P(R) Directly From Two-Dimensional Detector Images Via A Markov Chain Monte Carlo Method, Sudeshna Paul, Alan M. Friedman, Chris Bailey-Kellogg, Bruce Craig

Dartmouth Scholarship

The interatomic distance distribution, P(r), is a valuable tool for evaluating the structure of a molecule in solution and represents the maximum structural information that can be derived from solution scattering data without further assumptions. Most current instrumentation for scattering experiments (typically CCD detectors) generates a finely pixelated two-dimensional image. In contin­uation of the standard practice with earlier one-dimensional detectors, these images are typically reduced to a one-dimensional profile of scattering inten­sities, I(q), by circular averaging of the two-dimensional image. Indirect Fourier transformation methods are then used to reconstruct P(r) from …

Fuzzy Complex Formation Between The Intrinsically Disordered Prothymosin Α And The Kelch Domain Of Keap1 Involved In The Oxidative Stress Response., Halema Khan, Elio A Cino, Anne Brickenden, Jingsong Fan, Daiwen Yang, Wing-Yiu Choy

Biochemistry Publications

Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription. Prothymosin α (ProTα), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus …

Nuclear Magnetic Resonance Spectroscopic And Computational Investigations Of Chloroperoxidase Catalyzed Regio- And Enantio-Selective Transformations, Rui Zhang

FIU Electronic Theses and Dissertations

Chloroperoxidase (CPO) is the most versatile heme-containing enzyme that catalyzes a broad spectrum of reactions. The remarkable feature of this enzyme is the high regio- and enantio-selectivity exhibited in CPO-catalyzed oxidation reactions. The aim of this dissertation is to elucidate the structural basis for regio- and enantio-selective transformations and investigate the application of CPO in biodegradation of synthetic dyes.

To unravel the mechanism of CPO-catalyzed regioselective oxidation of indole, the dissertation explored the structure of CPO-indole complex using paramagnetic relaxation and molecular modeling. The distances between the protons of indole and the heme iron revealed that the pyrrole ring of …

Isoflurane Increases Neuronal Cell Death Vulnerability By Downregulating Mir-214, Hailiang Yan, Tao Xu, Hongfeng Zhao, Kuo-Chieh Lee, Hoau-Yan Wang, Yan Zhang

Publications and Research

Since accumulating evidence suggests the application of anesthetics may increase the risk of Alzheimer’s disease (AD), we investigated the cytotoxicity of inhaled general anesthesia in neurons and its underlying mechanism. Using primary cultured rat hippocampal neurons as the study model, here we show that isoflurane increases vulnerability to intracellular or extracellular amyloid b with or without serum deprivation. This isoflurane-induced effect is mediated by the downregulation of miR-214 level that lead to an elevated expression of Bax, a prominent target for miR-214. We conclude that isoflurane increases cell death in the presence of amyloid b by increasing Bax level through …

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 …

The Disordered Regulation Of Calcineurin: How Calmodulin-Induced Regulatory Domain Structural Changes Lead To The Activation Of Calcineurin, Victoria B. Dunlap

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has …

Dna-Binding Site Recognition By Bhlh And Mads-Domain Transcription Factors, Joshua R. Werkman

Theses and Dissertations--Plant and Soil Sciences

Herewithin, two transcription factor (TF) regulatory complexes were investigated. A bHLH–MYB–WDR (BMW) DNA-binding complex from maize was the first complex to be studied. R, a maize bHLH involved in the activation of genes in the anthocyanin pathway, had been characterized to indirectly bind DNA despite the presence of a functional DNA-binding domain. Findings presented here reveal that this is only partially correct. Direct DNA-binding by R was found to be dependent upon two distinct dimerization domains that function as a switch. This switch-like mechanism allows R to be repurposed for the activation of promoters of differing cis-element structure.

The …

Investigating Therapeutic Options For Lafora Disease Using Structural Biology And Translational Methods, Amanda R. Sherwood

Theses and Dissertations--Molecular and Cellular Biochemistry

Lafora disease (LD) is a rare yet invariably fatal form of epilepsy characterized by progressive degeneration of the central nervous and motor systems and accumulation of insoluble glucans within cells. LD results from mutation of either the phosphatase laforin, an enzyme that dephosphorylates cellular glycogen, or the E3 ubiquitin ligase malin, the binding partner of laforin. Currently, there are no therapeutic options for LD, or reported methods by which the specific activity of glucan phosphatases such as laforin can be easily measured. To facilitate our translational studies, we developed an assay with which the glucan phosphatase activity of laforin as …

The Effect Of Visual Wulst Lesions And Trigeminal Nerve Sectioning On The Discrimination Of Magnetic Inclination In The Homing Pigeon (Columba Livia), Merissa Acerbi

Honors Projects

The ability of homing pigeons to return to their loft from unknown places has fascinated scientists for centuries. It is well established that homing pigeons, like migratory birds, posses an innate magnetic inclination compass to determine direction by measuring the angle between the magnetic field vector and the Earth's surface. Recent work has indicated that the avian magnetic compass is light mediated and appears to mediate magnetic information to the brain. This occurs via a visual pathway with processing in the visual Wulst area of the forebrain. There is, however, also evidence from other avian species that magnetic direction may …