Biochemistry, Biophysics, and Structural Biology Commons^™

Hemi-Methylated Dna Regulates Dna Methylation Inheritance Through Allosteric Activation Of H3 Ubiquitylation By Uhrf1, Joseph S. Harrison, Evan M. Cornett, Dennis Goldfarb, Paul A. Darosa, Zimeng M. Li, Feng Yan, Bradley M. Dickson, Angela H. Guo, Daniel V. Cantu, Lilia Kaustov, Peter J. Brown, Cheryl H. Arrowsmith, Dorothy A. Erie, Michael B. Major, Rachel E. Klevit, Krzysztof Krajewski, Brian Kuhlman, Brian D. Strahl, Scott B. Rothbart

College of the Pacific Faculty Articles

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the …

A Simplified Direct Lipid Mixing Lipoplex Preparation: Comparison Of Liposomal-, Dimethylsulfoxide-, And Ethanol-Based Methods, Joseph Meisel, George Gokel

Chemistry & Biochemistry Faculty Works

Established transfection methodology often uses commercial reagents, which must be formed into liposomes in a sequence of about half a dozen steps. The simplified method reported here is a direct lipid mixing approach that requires fewer steps, less manipulation, and is less time-consuming. Results are comparable to those obtained with more commonly used methods, as judged by a variety of analytical techniques and by comparisons of transfection results. The method reported here may be applied to non-liposome-forming compounds, thereby greatly expanding the range of structures that can be tested for transfection ability.

Interactions Of Nucleic Acid Bases With Temozolomide. Stacked, Perpendicular, And Coplanar Heterodimers, Okuma Emile Kasende, Steve Scheiner

Chemistry and Biochemistry Faculty Publications

Temozolomide (TMZ) was paired with each of the five nucleic acid bases, and the potential energy surface searched for all minima, in the context of dispersion-corrected density functional theory and MP2 methods. Three types of arrangements were observed, with competitive stabilities. Coplanar H-bonding structures, reminiscent of Watson–Crick base pairs were typically the lowest in energy, albeit by a small amount. Also very stable were perpendicular arrangements that included one or more H-bonds. The two monomers were stacked approximately parallel to one another in the third category, some of which contained weak and distorted H-bonds. Dispersion was found to be a …

App Regulates Microglial Phenotype In A Mouse Model Of Alzheimer's Disease, Gunjan Manocha, Angela Floden, Keiko Rausch, Joshua Kulas, Brett Mcgregor, Lalida Rojanathammanee, Kelley Puig, Kendra Puig, Sanjib Karki, Michael Nichols, Diane Darland, James Porter, Colin Combs

Chemistry & Biochemistry Faculty Works

Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP−/−) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP−/− cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP−/− mice compared with wild-type mice. The mAPP−/− mice crossed to an APP/PS1 transgenic mouse line …

Monitoring The Charge Distribution During Proton And Sodium Ion Conduction Along Chains Of Water Molecules And Protein Residues, Steve Scheiner

Chemistry and Biochemistry Faculty Publications

Quantum calculations are used to determine the level of delocalization of the charge of a cation as it translates along a chain of water molecules or glycine residues. Charge dispersal is monitored via the molecular electrostatic potential and the dipole moment of the entire system. The positive charge is largely localized on the water molecule on which the proton is situated, but becomes more intense and extended as the proton moves along the chain. The positive charge is more delocalized in protonated polyglycine, where it extends over at least an entire residue. Displacement of the proton along the chain intensifies …

Comparative Analysis Of In Situ Fibronectin Using Tof-Sims, Spi-Ms, And Dropdesi-Ms In A Microfluidic Reactor, Shannon Fasing, Xiao-Ying Yu, Juan Yao, Jiachao Yu

STAR Program Research Presentations

Fibronectin is an important biomolecule due to its role in cell differentiation, growth, kinesis, and adhesion. Such biological responses are mediated through membrane recognition and signaling; where fibronectin is found. Studying the outer molecular surface of fibronectin allows deeper insight into the microbiological reactions that occur during these processes. In situ mass spectrometry analysis in aqueous solution accurately represents fibronectin’s chemical components, made possible by a vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface). SALVI was paired with the analytical tools: time-of-flight secondary ion mass spectrometer (ToF-SIMS), single photon ionization mass spectrometer (SPI-MS) and drop …

Parameterization Of Single-Scattering Albedo (Ssa) And Absorption Ångström Exponent (Aae) With Ec/Oc For Aerosol Emissions From Biomass Burning, Rudra P. Pokhrel, Nick L. Wagner, Justin M. Langridge, Daniel A. Lack, Thilina Jayarathne, Elizabeth A. Stone, Chelsea E. Stockwell, Robert J. Yokelson, Shane M. Murphy

Chemistry and Biochemistry Faculty Publications

Single-scattering albedo (SSA) and absorption Ångström exponent (AAE) are two critical parameters in determining the impact of absorbing aerosol on the Earth's radiative balance. Aerosol emitted by biomass burning represent a significant fraction of absorbing aerosol globally, but it remains difficult to accurately predict SSA and AAE for biomass burning aerosol. Black carbon (BC), brown carbon (BrC), and non-absorbing coatings all make substantial contributions to the absorption coefficient of biomass burning aerosol. SSA and AAE cannot be directly predicted based on fuel type because they depend strongly on burn conditions. It has been suggested that SSA can be effectively parameterized …

Enhancing Silymarin Fractionation Via Molecular Modeling Using The Conductor-Like Screening Model For Real Solvents, Emma C. Brace

Open Access Theses

The market for bio-based products from plant sources is on the rise. There is a global challenge to implement environmentally clean practices for the production of fuels and pharmaceuticals from sustainable resources. A significant hurdle for discovery of comparable plant-derived products is the extensive volume of trial-and-error experimentation required. To alleviate the experimental burden, a quantum mechanics based molecular modeling approach known as the COnductor-like Screening Model for Real Solvents (COSMO-RS) was used to predict the best biphasic solvent system to purify silymarins from an aqueous mixture. Silymarins are a class of flavonolignans present in milk thistle ( Silybum marianum …

Investigation Of Cellular Microenvironments And Heterogeneity With Biodynamic Imaging, Daniel Alexander Merrill

Open Access Dissertations

Imaging of biological tissue in a relevant environment is critical to accurately assessing the effectiveness of chemotherapeutic agents in combatting cancer. Though many three-dimensional (3D) culture models exist, conventional in vitro assays continue to use two-dimensional (2D) cultures because of the difficulty in imaging through deep tissue. 3D tomographic imaging techniques exist and are being used in the development of 3D efficacy assays. However, most of these assays look at therapy endpoint (dead or living cancer cell count) and do not capture the dynamics of tissue response.

Biodynamic imaging (BDI) is a 3D tomographic imaging and assay technique that uses …

Heparin-Peptide Interactions, Jacqueline Anastasia Morris

Graduate Theses and Dissertations

Heparin is a polydispersed sulfated molecule that is part of the family called glycosaminoglycans found in the extracellular matrix and cell surfaces. This molecule is extremely important for the activation of proteins and protein-receptor interactions that are responsible for downstream cell signaling pathways. Heparin has been isolated from porcine intestine and used as an anticoagulant for the prevention of embolisms, heart thrombosis, and clotting during heart surgeries. This so-called miracle drug was in use until 2008, when isolated batches were found to be contaminated with other glycosaminoglycans similar to heparin. From 2008, there has been a dire need for a …

Analysis Of The Intricacies Of Substrate Recognition Of High Mobility Group Proteins And Aminoacyl-Trna Synthetases Using Non-Cognate Substrates, Douglas Van Iverson Ii

Dissertations

The studies presented in section 1 (Chapters I-IV) focus on the design and development of nucleic acid four-way junctions (4WJs) to target a member of the high mobility group (HMG) proteins, the proinflammatory cytokine high mobility group box 1 protein (HMGB1). In the present study, hybrid PNA-DNA 4WJs based on a model DNA 4WJ were constructed to improve the thermal stability of 4WJs while maintaining strong binding affinity toward HMGB1. An electrophoretic mobility shift assay (EMSA) was used to examine the binding affinity of an isolated DNA binding domain of HMGB1, the HMGB1 b-box (HMGB1b), toward a set of PNA-DNA …

Incremental Phylogenetics By Repeated Insertions: An Evolutionary Tree Algorithm, Peter Revesz, Zhiqiang Li

School of Computing: Faculty Publications

We introduce the idea of constructing hypothetical evolutionary trees using an incremental algorithm that inserts species one-by-one into the current evolutionary tree. The method of incremental phylogenetics by repeated insertions lead to an algorithm that can be used on DNA, RNA and amino acid sequences. According to experimental results on both synthetic and biological data, the new algorithm generates more accurate evolutionary trees than the UPGMA and the Neighbor Joining algorithms.

Investigation Of The Interaction Of Dimeric Ruthenium Complexes With Cytochrome B5, Christopher Dain Rupar

Graduate Theses and Dissertations

Photoreactive complexes to study the kinetics of electron transfer of proteins have been in use for a long time. It has always been speculated that complexes bind near the heme or the electron transfer reaction would not occur. But it is unkown exactly how the complex interacts with the protein. The structural, thermodynamic, and kinetic properties of rat liver microsomal cytochrome b5 were investigated when bound to ruthenium dimer complexes. Heteronuclear Single Quantum Coherence studies support a dynamic binding model of a dimer Ru complex bound near the protein’s heme involving residues H39, E44, G42, V61, G62, and H63. The …

Mechanism Of Rapid Electron Transfer Reactions Involving Cytochrome Bc1, Cytochrome C And Cytochrome Oxidase, Jeremy Erik Durchman

Graduate Theses and Dissertations

Electron transfer between mitochondrial proteins complexes represents the primary means by which living things acquire the requisite energy for survival. The coupling of electron transfer to proton translocation creates an electrochemical gradient that drives the synthesis of highly energetic compounds such as ATP. The purpose of these studies is to measure rates of electron transfer and elucidate the important governing factors in the redox events involving cytochrome bc1, cytochrome c and cytochrome oxidase. Using rapid initiation of redox events triggered by laser flash excitation of ruthenium compounds, and strategically monitoring unique spectral properties of these proteins in the visible region …

The San1 Ubiquitin Ligase Functions Preferentially With Ubiquitin-Conjugating Enzyme Ubc1 During Protein Quality Control, Rebeca Lea Ibarra

UNLV Theses, Dissertations, Professional Papers, and Capstones

Protein quality control (PQC) is a critical process wherein misfolded or damaged proteins are cleared from the cell to maintain protein homeostasis. In eukaryotic cells, the removal of misfolded proteins is primarily accomplished by the ubiquitin-proteasome system (UPS). In the UPS, ubiquitin-conjugating enzymes and ubiquitin ligases append poly-ubiquitin chains onto misfolded protein substrates signaling for their degradation. The kinetics of protein ubiquitylation are paramount since a balance must be achieved between the rapid removal of misfolded proteins versus providing sufficient time for protein chaperones to attempt refolding. To uncover the molecular basis for how PQC substrate ubiquitylation rates are controlled, …

Use Of Clustering Techniques For Protein Domain Analysis, Eric Rodene

School of Computing: Dissertations, Theses, and Student Research

Next-generation sequencing has allowed many new protein sequences to be identified. However, this expansion of sequence data limits the ability to determine the structure and function of most of these newly-identified proteins. Inferring the function and relationships between proteins is possible with traditional alignment-based phylogeny. However, this requires at least one shared subsequence. Without such a subsequence, no meaningful alignments between the protein sequences are possible. The entire protein set (or proteome) of an organism contains many unrelated proteins. At this level, the necessary similarity does not occur. Therefore, an alternative method of understanding relationships within diverse sets of proteins …

Fulleretic Well-Defined Scaffolds: Donor–Fullerene Alignment Through Metal Coordination And Its Effect On Photophysics, Derek E. Williams, Ekaterina A. Dolgopolova, Danielle C. Godfrey, Evgeniya D. Ermolaeva, Perry J. Pellechia Dr., Andrew B. Greytak Prof. Dr., Mark Smith Dr., Stanislav M. Avdoshenko, Dr., Alexey A. Popov Dr., Natalia B. Shustova Prof. Dr.

Faculty Publications

Herein, we report the first example of a crystalline metal–donor–fullerene framework, in which control of the donor–fullerene mutual orientation was achieved through chemical bond formation, in particular, by metal coordination. The 13C cross-polarization magic-angle spinning NMR spectroscopy, X-ray diffraction, and time-resolved fluorescence spectroscopy were performed for comprehensive structural analysis and energy-transfer (ET) studies of the fulleretic donor–acceptor scaffold. Furthermore, in combination with photoluminescence measurements, the theoretical calculations of the spectral overlap function, Förster radius, excitation energies, and band structure were employed to elucidate the photophysical and ET processes in the prepared fulleretic material. We envision that the well-defined fulleretic donor–acceptor …

Long-Range Acoustic Interactions In Insect Swarms: An Adaptive Gravity Model, Dan Gorbonos, Reuven Ianconescu, James G. Puckett, Rui Ni, Nicholas T. Ouellette, Nir S. Gov

Physics and Astronomy Faculty Publications

The collective motion of groups of animals emerges from the net effect of the interactions between individual members of the group. In many cases, such as birds, fish, or ungulates, these interactions are mediated by sensory stimuli that predominantly arise from nearby neighbors. But not all stimuli in animal groups are short range. Here, we consider mating swarms of midges, which are thought to interact primarily via long-range acoustic stimuli. We exploit the similarity in form between the decay of acoustic and gravitational sources to build a model for swarm behavior. By accounting for the adaptive nature of the midges' …

In Vitro Monitoring Of Time And Dose Dependent Cytotoxicity Of Aminated Nanoparticles Using Raman Spectroscopy, Esen Efeoglu, Alan Casey, Hugh Byrne

Articles

No abstract provided.

Spectroscopic Studies Of Anthracyclines: Structural Characterization And In Vitro Tracking, Zeineb Farhane, Hugh Byrne, Malgorzata Baranska

Articles

A broad spectroscopic characterization, using ultraviolet-visible (UV-vis) and Fourier transform infrared absorption as well as Raman scattering, of two commonly used anthracyclines antibiotics (DOX) daunorubicin (DNR), their epimers (EDOX, EDNR) and ten selected analogs is presented. The paper serves as a comprehensive spectral library of UV-vis, IR and Raman spectra of anthracyclines in the solid state and in solution. The particular advantage of Raman spectroscopy for the measurement and analysis of individual antibiotics is demonstrated. Raman spectroscopy can be used to monitor the in vitro uptake and distribution of the drug in cells, using both 488 nm and 785 nm …

Multiplexed Detection Of Double-Stranded Pathogenic Dna With Engineered Zinc Finger Proteins, Juhwa Kim

Masters Theses & Specialist Projects

The development of a new technology for the detection of doublestranded (ds) DNA enables multiple biomedical applications including identifying multiple pathogens simultaneously. We previously employed colorimetric SEquence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-LAC) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. Biotin was simply conjugated to …

Using Zinc Finger Proteins As A Diagnostic Tool For The Detection Of A Cancer Biomarker, Anu Kini

Masters Theses & Specialist Projects

RASSF1A is a tumor suppressor gene which loses its function due to methylation of CpG islands on its promoter region. Detection of methylation leads to early diagnosis of cancer.

Zinc finger proteins are capable of detecting a specific DNA sequence and Methyl binding domain can bind to the methyl group on the CpG, using this idea mCpG SEER- Lac system makes use of a split protein, β-lactamase. Lac A attached to the ZFP and Lac B attached to the MBD protein. On binding to the DNA, the Lac A and Lac B come in close proximity with each other causing …

Synthetic Investigation On The Biomimetic Metal-Catalyzed Sulfoxidations And Photochemical Generation Of A Highly Reactive Ruthenium(V)-Oxo Porphyrin, Weilong Luo

Masters Theses & Specialist Projects

Catalytic oxidation plays a crucial role in current chemical and pharmaceutical industries which is also a leading technology for green chemical processes. In Nature, the ubiquitous cytochrome P450 enzymes can catalyze a wide variety of oxidation reactions with high efficiency and selectivity. Many transition metal catalysts are designed as the biomimetic model of cytochrome P450 enzymes. In this work, series of metalloporphyrins and metallocorroles have been successfully synthesized to investigate and develop catalytic selective oxidation of sulfides to sulfoxides.

Manganese(III) porphyrin complexes (2) and manganese(III) corrole complexes (6) with iodobenzene diacetate [PhI(OAc)2] as a mild oxygen source exhibited remarkable catalytic …

Inquiry Of Lipid Membranes Interacting With Functional Peptides And Polyphenol Drug Molecules, Chian Sing Ho

USF Tampa Graduate Theses and Dissertations

Cellular membranes are important targets for many membrane-active peptides and drug compounds. Here we are interested in deciphering how lipid membranes are perturbed by several membrane-active molecules, including the transmembrane domain of the influenza M2 protein (M2TM), aggregates formed by a synthetic polyglutamine peptide, and three polyphenol compounds (i.e., tamoxifen, genistein, and verapamil). We employ phase-separated ternary lipid model membranes in the form of giant unilamellar vesicles (GUVs) to simulate raft-like structures that have been proposed to govern many important processes in plasma membranes (e.g., intracellular singling and trafficking). Specifically, we use fluorescent microscopy to interrogate how those membrane additives …

Required Biochem Class Live-Streamed This Summer: Collaboration With Union College Makes Required Credits Easier To Obtain, Chris Mclean-Wheeler

Andrews Agenda: Campus News

No abstract provided.

Interaction Of Spliceosomal U2 Snrnp Protein P14 With Its Branch Site Rna Target, William Perea Vargas

Dissertations, Theses, and Capstone Projects

Newly transcribed precursor messenger RNA (pre-mRNA) molecules contain coding sequences (exons) interspersed with non-coding intervening sequences (introns). These introns must be removed in order to generate a continuous coding sequence prior to translation of the message into protein. The mechanism through which these introns are removed is known as pre-mRNA splicing, a two-step reaction catalyzed be a large macromolecular machine, the spliceosome, located in the nucleus of eukaryotic cells. The spliceosome is a protein-directed ribozyme composed of small nuclear RNAs (snRNA) and hundreds of proteins that assemble in a very dynamic process. One of these snRNAs, the U2 snRNA, is …

A Comparison Of Catabolic Pathways Induced In Primary Macrophages By Pristine Single Walled Carbon Nanotubes And Pristine Graphene, Caroline More, Jennifer Mcintyre, Luke O'Neill, Hugh Byrne

Articles

Understanding the correlation between the physico-chemical properties of carbonaceous nanomaterials and how these properties impact on cells and subcelluar mechanisms is critical to their risk assessment and safe translation into newly engineered devices. Here the toxicity, uptake and catabolic response of primary human macrophages to pristine graphene (PG) and pristine single walled carbon nanotubes (pSWCNT) are explored, compared and contrasted. The nanomaterial toxicity was assessed using three complementary techniques (live-dead assay, real time impedance technique and confocal microscopic analysis), all of which indicated no signs of acute cytotoxicity in response to PG or pSWCNT. Transmission electron microscopy (TEM) demonstrated that …

Mechanism Of Lysine 48 Selectivity During Polyubiquitin Chain Formation By The Ube2r1/2 Ubiquitin-Conjugating Enzyme, Spencer Hill, Joseph S. Harrison, Steven M. Lewis, Brian Kuhlman, Gary Kleiger

College of the Pacific Faculty Articles

Lysine selectivity is of critical importance during polyubiquitin chain formation because the identity of the lysine controls the biological outcome. Ubiquitins are covalently linked in polyubiquitin chains through one of seven lysine residues on its surface and the C terminus of adjacent protomers. Lys 48-linked polyubiquitin chains signal for protein degradation; however, the structural basis for Lys 48 selectivity remains largely unknown. The ubiquitin-conjugating enzyme Ube2R1/2 has exquisite specificity for Lys 48, and computational docking of Ube2R1/2 and ubiquitin predicts that Lys 48 is guided to the active site through a key electrostatic interaction between Arg 54 on ubiquitin and …

A Tonb-Dependent Receptor Regulates Antifungal Hsaf Biosynthesis In Lysobacter, Ruping Wang, Huiyong Xu, Liangcheng Du, Shan-Ho Chou, Hongxia Liu, Youzhou Liu, Fengquan Liu, Guoliang Qian

Liangcheng Du Publications

Lysobacter species are Gram-negative bacteria that are emerging as new sources of antibiotics, including HSAF (Heat Stable Antifungal Factor), which was identified from L. enzymogenes with a new mode of action. LesR, a LuxR solo, was recently shown to regulate the HSAF biosynthesis via an unidentified mechanism in L. enzymogenes OH11. Here, we used a comparative proteomic approach to identify the LesR targets and found that LesR influenced the expression of 33 proteins belonging to 10 functional groups, with 9 proteins belonging to the TBDR (TonB-Dependent Receptor) family. The fundamental role of bacterial TBDR in nutrient uptake motivates us to …

Analysis Of Gompertzian Growth In Aggregating Multicellular Tumor Nodules, Gwendolyn A. Deger

Graduate Masters Theses

Past studies have shown that tumor growth generally follows an exponential growth function or, with a limiting growth constraint, the sigmoid Gompertzian function, where a terminal tumor size is reached at late times. The classical Gompertzian description of tumor growth applies in the case of two-dimensional (2D) in vitro cell studies due to the effect of physical limitations on possible growth area. This project asked whether Gompertzian form applies to the in vitro growth of multifocal 3D tumor nodules, whose size is determined by aggregation events as well as cell proliferation. Previous reports have indicated that these three-dimensional (3D) spheroids …