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Articles 1 - 30 of 77

Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Discovery Of Platelet-Type 12-Human Lipoxygenase Selective Inhibitors By High-Throughput Screening Of Structurally Diverse Libraries., Joshua D. Deschamps, Jeffrey T. Gautschi, Stephanie Whitman, Tyler A. Johnson, Nadine C. Gassner, Phillip Crews, Theodore R. Holman Feb 2019

Discovery Of Platelet-Type 12-Human Lipoxygenase Selective Inhibitors By High-Throughput Screening Of Structurally Diverse Libraries., Joshua D. Deschamps, Jeffrey T. Gautschi, Stephanie Whitman, Tyler A. Johnson, Nadine C. Gassner, Phillip Crews, Theodore R. Holman

Tyler Johnson

Human lipoxygenases (hLO) have been implicated in a variety of diseases and cancers and each hLO isozyme appears to have distinct roles in cellular biology. This fact emphasizes the need for discovering selective hLO inhibitors for both understanding the role of specific lipoxygenases in the cell and developing pharmaceutical therapeutics. To this end, we have modified a known lipoxygenase assay for high-throughput (HTP) screening of both the National Cancer Institute (NCI) and the UC Santa Cruz marine extract library (UCSC-MEL) in search of platelet-type 12-hLO (12-hLO) selective inhibitors. The HTP screen led to the characterization of five novel 12-hLO inhibitors ...


Modeling And Analyzing An Optogenetic System For Photoactivatable Protein Dissociation, Anvin Thomas, James Schaff May 2018

Modeling And Analyzing An Optogenetic System For Photoactivatable Protein Dissociation, Anvin Thomas, James Schaff

Honors Scholar Theses

Computational modeling of cell-cell interactions can grant clues and can answer questions about an experiment, especially for observations about binding interactions and kinetics. This approach was used to investigate an interaction between a light-oxygen-voltage (LOV) domain and an engineered protein called Zdark (Zdk). The LOV domain is membrane-bound while Zdk is cytosolic. The LOV domain and Zdk bind strongly in dark (Kd 26.2 nM), and weakly upon exposure to blue light (Kd > 4 μM). Total internal reflection fluorescence (TIRF) images are acquired of Zdk, the fluorescent species bound to a mCherry tag, and the loss of fluorescence ...


Understanding Human Erythrocyte Glucose Transporter (Glut1) Mediated Glucose Transport Phenomena Through Structural Analysis, Kenneth P. Lloyd Feb 2018

Understanding Human Erythrocyte Glucose Transporter (Glut1) Mediated Glucose Transport Phenomena Through Structural Analysis, Kenneth P. Lloyd

GSBS Dissertations and Theses

GLUT1-mediated, facilitated sugar transport is proposed to be an example of transport by a carrier that alternately presents exofacial (e2) and endofacial (e1) substrate binding sites, commonly referred to as the alternating access carrier model. This hypothesis is incompatible with observations of co-existent exo- and endofacial ligand binding sites, transport allostery, and e1 ligand (e.g. cytochalasin B) induced GLUT1 sugar occlusion. The fixed-site carrier model proposes co-existent, interacting e2 and e1 ligand binding sites but involves sugar translocation by geminate exchange through internal cavities. Demonstrations of membrane-resident dimeric and tetrameric GLUT1 and of e2, e1 and occluded GLUT conformations ...


Using Competition Assays To Quantitatively Model Cooperative Binding By Transcription Factors And Other Ligands., Jacob Peacock, James B Jaynes Nov 2017

Using Competition Assays To Quantitatively Model Cooperative Binding By Transcription Factors And Other Ligands., Jacob Peacock, James B Jaynes

Department of Biochemistry and Molecular Biology Faculty Papers

BACKGROUND: The affinities of DNA binding proteins for target sites can be used to model the regulation of gene expression. These proteins can bind to DNA cooperatively, strongly impacting their affinity and specificity. However, current methods for measuring cooperativity do not provide the means to accurately predict binding behavior over a wide range of concentrations.

METHODS: We use standard computational and mathematical methods, and develop novel methods as described in Results.

RESULTS: We explore some complexities of cooperative binding, and develop an improved method for relating in vitro measurements to in vivo function, based on ternary complex formation. We derive ...


Quaternary Interactions And Supercoiling Modulate The Cooperative Dna Binding Of Agt, Manana Melikishvili, Michael G. Fried Jul 2017

Quaternary Interactions And Supercoiling Modulate The Cooperative Dna Binding Of Agt, Manana Melikishvili, Michael G. Fried

Center for Structural Biology Faculty Publications

Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein–protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ωave. We found that ...


Statistical Biophysics Blog: What I Have Against (Most) Pmf Calculations, Daniel M. Zuckerman May 2017

Statistical Biophysics Blog: What I Have Against (Most) Pmf Calculations, Daniel M. Zuckerman

Scholar Archive

The potential of mean force (PMF) is one of the most widely used characterizations of a biomolecular system computed from simulation, but calculating and interpreting a PMF both present challenges.


The Large Terminase Dna Packaging Motor Grips Dna With Its Atpase Domain For Cleavage By The Flexible Nuclease Domain, Brendan J. Hilbert, Janelle A. Hayes, Nicholas P. Stone, Rui-Gang Xu, Brian A. Kelch Apr 2017

The Large Terminase Dna Packaging Motor Grips Dna With Its Atpase Domain For Cleavage By The Flexible Nuclease Domain, Brendan J. Hilbert, Janelle A. Hayes, Nicholas P. Stone, Rui-Gang Xu, Brian A. Kelch

Open Access Articles

Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding ...


Synthesis, Kinetic And Catalytic Studies Of Manganese Complexes With Corrole And Porphyrin Ligands, Haleh Jeddi Apr 2017

Synthesis, Kinetic And Catalytic Studies Of Manganese Complexes With Corrole And Porphyrin Ligands, Haleh Jeddi

Masters Theses & Specialist Projects

High-valent transition metal-oxo intermediates play a significant role in the catalytic cycle of the ubiquitous cytochrome P450 enzymes and in biomimetic catalytic systems. In this work, manganese(III) porphyrin and corrole systems (2) were synthesized and characterized by UV-vis absorbance and 1H-NMR, matching literaturereported spectroscopic data. Manganese(V)-oxo corroles (3) and a manganese(IV)-oxo porphyrin (4) were successfully generated by chemical oxidation using mchloroperoxybenzoic acid (m-CPBA), and their oxidation reactions with organic reductants were comparatively investigated. Results from single-turnover kinetic studies indicate that in the tris(pentafluorophenyl)corrole system (3a), the active oxidizing intermediate differs in different solvents ...


Mechanism Of Rapid Electron Transfer Reactions Involving Cytochrome Bc1, Cytochrome C And Cytochrome Oxidase, Jeremy Erik Durchman Aug 2016

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

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 ...


Steroid Binding To Autotaxin Links Bile Salts And Lysophosphatidic Acid Signalling, Willem-Jan Keune, Jens Hausmann, Ruth Bolier, Dagmar Tolenaars, Andreas Kremer, Tatjana Heidebrecht, Robbie P. Joosten, Manjula Sunkara, Andrew J. Morris, Elisa Matas-Rico, Wouter H. Moolenaar, Ronald P. Oude Elferink, Anastassis Perrakis Apr 2016

Steroid Binding To Autotaxin Links Bile Salts And Lysophosphatidic Acid Signalling, Willem-Jan Keune, Jens Hausmann, Ruth Bolier, Dagmar Tolenaars, Andreas Kremer, Tatjana Heidebrecht, Robbie P. Joosten, Manjula Sunkara, Andrew J. Morris, Elisa Matas-Rico, Wouter H. Moolenaar, Ronald P. Oude Elferink, Anastassis Perrakis

Gill Heart & Vascular Institute Faculty Publications

Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors ...


Frontal Plane Comparison Between Drop Jump And Vertical Jump: Implications For The Assessment Of Acl Risk Of Injury, Guilherme Manna Cesar, Curtis L. Tomasevicz, Judith M. Burnfield Jan 2016

Frontal Plane Comparison Between Drop Jump And Vertical Jump: Implications For The Assessment Of Acl Risk Of Injury, Guilherme Manna Cesar, Curtis L. Tomasevicz, Judith M. Burnfield

Athletic Performance Research

The potential to use the vertical jump (VJ) to assess both athletic performance and risk of anterior cruciate ligament (ACL) injury could have widespread clinical implications since VJ is broadly used in high school, university, and professional sport settings. Although drop jump (DJ) and VJ observationally exhibit similar lower extremity mechanics, the extent to which VJ can also be used as screening tool for ACL injury risk has not been assessed. This study evaluated whether individuals exhibit similar knee joint frontal plane kinematic and kinetic patterns when performing VJs compared with DJs. Twenty-eight female collegiate athletes performed DJs and VJs ...


Thermodynamics And Kinetics Of The Three-Way Junction Of Phi29 Motor Prna And Its Assembly Into Nanoparticles For Therapeutic Delivery To Prostate Cancer, Daniel W. Binzel Jan 2016

Thermodynamics And Kinetics Of The Three-Way Junction Of Phi29 Motor Prna And Its Assembly Into Nanoparticles For Therapeutic Delivery To Prostate Cancer, Daniel W. Binzel

Theses and Dissertations--Pharmacy

The emerging field of RNA nanotechnology necessitates creation of functional RNA nanoparticles, but has been limited by particle instability. Previously, it was found the three-way junction (3WJ) of the Phi29 DNA packaging motor pRNA was found to be ultra-stable and assemble in solution without the presence of metal ions. The three-way junction is composed of three short oligo RNA strands and proven to be thermodynamically stable. Here the assembly mechanism, thermodynamic and enzymatic stabilities, and kinetics are examined in order to understand the stability behind this unique motif. Thermodynamic and kinetics studies found that the pRNA 3WJ formed out of ...


A Conserved Three-Nucleotide Core Motif Defines Musashi Rna Binding Specificity, Nancy Zearfoss, Laura Deveau, Carina Clingman, Eric Schmidt, Emily Johnson, Francesca Massi, Sean Ryder Sep 2015

A Conserved Three-Nucleotide Core Motif Defines Musashi Rna Binding Specificity, Nancy Zearfoss, Laura Deveau, Carina Clingman, Eric Schmidt, Emily Johnson, Francesca Massi, Sean Ryder

Sean P. Ryder

Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside ...


Intra-Domain Cross-Talk Regulates Serine-Arginine Protein Kinase 1-Dependent Phosphorylation And Splicing Function Of Transformer 2Β1, Michael A. Jamros, Brandon E. Aubol, Malik M. Keshwani, Zhaiyi Zhang, Stefan Stamm, Joseph A. Adams Jul 2015

Intra-Domain Cross-Talk Regulates Serine-Arginine Protein Kinase 1-Dependent Phosphorylation And Splicing Function Of Transformer 2Β1, Michael A. Jamros, Brandon E. Aubol, Malik M. Keshwani, Zhaiyi Zhang, Stefan Stamm, Joseph A. Adams

Molecular and Cellular Biochemistry Faculty Publications

Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a ...


Molecular Effects Of Cancer-Associated Somatic Mutations On The Structural And Target Recognition Properties Of Keap1., Halema Khan, Ryan C Killoran, Anne Brickenden, Jingsong Fan, Daiwen Yang, Wing-Yiu Choy Apr 2015

Molecular Effects Of Cancer-Associated Somatic Mutations On The Structural And Target Recognition Properties Of Keap1., Halema Khan, Ryan C Killoran, Anne Brickenden, Jingsong Fan, Daiwen Yang, Wing-Yiu Choy

Biochemistry Publications

Kelch-like ECH-associated protein 1 (Keap1) plays an important regulatory role in the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent oxidative stress response pathway. It functions as a repressor of Nrf2, a key transcription factor that initiates the expression of cytoprotective enzymes during oxidative stress to protect cells from damage caused by reactive oxygen species. Recent studies show that mutations of Keap1 can lead to aberrant activation of the antioxidant pathway, which is associated with different types of cancers. To gain a mechanistic understanding of the links between Keap1 mutations and cancer pathogenesis, we have investigated the molecular effects of ...


Substrate Envelope-Designed Potent Hiv-1 Protease Inhibitors To Avoid Drug Resistance, Madhavi Nalam, Akbar Ali, G. S. Kiran Kumar Reddy, Hong Cao, Saima Anjum, Michael Altman, Nese Yilmaz, Bruce Tidor, Tariq Rana, Celia Schiffer Jan 2015

Substrate Envelope-Designed Potent Hiv-1 Protease Inhibitors To Avoid Drug Resistance, Madhavi Nalam, Akbar Ali, G. S. Kiran Kumar Reddy, Hong Cao, Saima Anjum, Michael Altman, Nese Yilmaz, Bruce Tidor, Tariq Rana, Celia Schiffer

Celia A. Schiffer

The rapid evolution of HIV under selective drug pressure has led to multidrug resistant (MDR) strains that evade standard therapies. We designed highly potent HIV-1 protease inhibitors (PIs) using the substrate envelope model, which confines inhibitors within the consensus volume of natural substrates, providing inhibitors less susceptible to resistance because a mutation affecting such inhibitors will simultaneously affect viral substrate processing. The designed PIs share a common chemical scaffold but utilize various moieties that optimally fill the substrate envelope, as confirmed by crystal structures. The designed PIs retain robust binding to MDR protease variants and display exceptional antiviral potencies against ...


Ph Dependence Of Cyanide And Imidazole Binding To The Heme Domains Of Sinorhizobium Meliloti And Bradyrhizobium Japonicum Fixl, Anil K. Bidwai, Angela J. Ahrendt, John S. Sullivan, Lidia B. Vitello, James E. Erman Jan 2015

Ph Dependence Of Cyanide And Imidazole Binding To The Heme Domains Of Sinorhizobium Meliloti And Bradyrhizobium Japonicum Fixl, Anil K. Bidwai, Angela J. Ahrendt, John S. Sullivan, Lidia B. Vitello, James E. Erman

Faculty Publications & Research

Equilibrium and kinetic properties of cyanide and imidazole binding to the heme domains of Sinorhizobium meliloti and Bradyrhizobium japonicum FixL (SmFixLH and BjFixLH) have been investigated between pH 5 and 11. KD determinations were made at integral pH values, with the strongest binding at pH 9 for both ligands. KD for the cyanide complexes of BjFixLH and SmFixLH is 0.15 ± 0.09 and 0.50 ± 0.20 μM, respectively, and 0.70 ± 0.01 mM for imido-BjFixLH. The association rate constants are pH dependent with maximum values of 443 ± 8 and 252 ± 61 ...


Ef-G:Trna Dynamics During The Elongation Cycle Of Protein Synthesis, Rong Shen Jan 2015

Ef-G:Trna Dynamics During The Elongation Cycle Of Protein Synthesis, Rong Shen

Publicly Accessible Penn Dissertations

During polypeptide elongation cycle, prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs. Continued progress has been achieved in understanding this key process, including results of structural, ensemble kinetic and single-molecule studies. However, most of work has been focused on the pre-equilibrium states of this fast process, leaving the real time dynamics, especially how EF-G interacts with the A-site tRNA in the pretranslocation complex, not fully elucidated.

In this thesis, the kinetics of EF-G catalyzed translocation is investigated by both ensemble and single molecule fluorescence resonance energy transfer studies ...


Platination Kinetics: Insight Into Rna-Cisplatin Interactions As A Probe For Rna Microenvironments, Gayani Dedduwa-Mudalige Jan 2015

Platination Kinetics: Insight Into Rna-Cisplatin Interactions As A Probe For Rna Microenvironments, Gayani Dedduwa-Mudalige

Wayne State University Dissertations

RNAs are crucial for many cellular functions. Thus, studying ligand-RNA interactions and their dynamics in response to changes in the surrounding environment is important. In spite of the well-known DNA coordination, current research also indicates cisplatin binding to RNA. Kinetic studies of rRNA platination reactions are largely unexplored. This research was conducted to achieve two objectives. First, a broad kinetic study was carried out to investigate the cisplatin-rRNA interactions. The structure, function, and ligand interactions depend on RNA microenvironments. Second, the application of platination kinetics as a tool to interrogate RNA electrostatic environments was explored.

Three model rRNA hairpins from ...


Nuclear Transport Of Single Molecules: Dwell Times At The Nuclear Pore Complex, Ulrich Kubitscheck, David Grunwald, Andreas Hoekstra, Daniel Rohleder, Thorsten Kues, Jan Peter Siebrasse, Reiner Peters Nov 2014

Nuclear Transport Of Single Molecules: Dwell Times At The Nuclear Pore Complex, Ulrich Kubitscheck, David Grunwald, Andreas Hoekstra, Daniel Rohleder, Thorsten Kues, Jan Peter Siebrasse, Reiner Peters

David Grünwald

The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of ...


Intranuclear Binding Kinetics And Mobility Of Single Native U1 Snrnp Particles In Living Cells, David Grunwald, Beatrice Spottke, Volker Buschmann, Ulrich Kubitscheck Nov 2014

Intranuclear Binding Kinetics And Mobility Of Single Native U1 Snrnp Particles In Living Cells, David Grunwald, Beatrice Spottke, Volker Buschmann, Ulrich Kubitscheck

David Grünwald

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay ...


Autonomy And Robustness Of Translocation Through The Nuclear Pore Complex: A Single-Molecule Study, Thomas Dange, David Grunwald, Antje Grunwald, Reiner Peters, Ulrich Kubitscheck Nov 2014

Autonomy And Robustness Of Translocation Through The Nuclear Pore Complex: A Single-Molecule Study, Thomas Dange, David Grunwald, Antje Grunwald, Reiner Peters, Ulrich Kubitscheck

David Grünwald

All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapalpha2, kapbeta1, kapbeta1DeltaN44, and kapbeta2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy ...


Kynurenine Aminotransferase Iii And Glutamine Transaminase L Are Identical Enzymes That Have Cysteine S-Conjugate Beta-Lyase Activity And Can Transaminate L-Selenomethionine, John T. Pinto, Boris F. Krasnikov, Steven Alcutt, Melanie E. Jones, Thambi Dorai, Arthur J L Cooper Nov 2014

Kynurenine Aminotransferase Iii And Glutamine Transaminase L Are Identical Enzymes That Have Cysteine S-Conjugate Beta-Lyase Activity And Can Transaminate L-Selenomethionine, John T. Pinto, Boris F. Krasnikov, Steven Alcutt, Melanie E. Jones, Thambi Dorai, Arthur J L Cooper

NYMC Faculty Publications

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 ...


Catalytic Mechanism Of Bacteriophage T4 Rad50 Atp Hydrolysis, Timothy J. Herdendorf, Scott W. Nelson Aug 2014

Catalytic Mechanism Of Bacteriophage T4 Rad50 Atp Hydrolysis, Timothy J. Herdendorf, Scott W. Nelson

Biochemistry, Biophysics and Molecular Biology Publications

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a ...


Single-Molecule Kinetic And Thermodynamic Studies Of Cosolute-Influenced Nucleic Acid Conformational Transitions, Erik Dylan Holmstrom Jul 2014

Single-Molecule Kinetic And Thermodynamic Studies Of Cosolute-Influenced Nucleic Acid Conformational Transitions, Erik Dylan Holmstrom

Chemistry & Biochemistry Graduate Theses & Dissertations (1986-2018)

Over the last 40 years the number of biochemical functionalities attributed to nucleic acids has increased tremendously. This diverse array of chemical functionality is intimately coupled to the spatial arrangement of atoms associated with these molecules. The three-dimensional structures and functions of nucleic acids are known to be dependent on the concentration and identity of solutes in solution. These nucleic acid cosolutes can be as simple and universal as atomic metal cations that favorably interact with the negatively charged phosphate backbone of nucleic acids, resulting in stabilization of electronegatively dense conformations. Alternatively, they may be complex organic molecules that are ...


Identification Of Molecular Determinants That Shift Co- And Post-Translational N-Glycosylation Kinetics In Type I Transmembrane Peptides: A Dissertation, Heidi L. H. Malaby Apr 2014

Identification Of Molecular Determinants That Shift Co- And Post-Translational N-Glycosylation Kinetics In Type I Transmembrane Peptides: A Dissertation, Heidi L. H. Malaby

GSBS Dissertations and Theses

Asparagine (N)-linked glycosylation occurs on 90% of membrane and secretory proteins and drives folding and trafficking along the secretory pathway. The N-glycan can be attached to an N-X-T/S-Y (X,Y ≠ P) consensus site by one of two oligosaccharyltransferase (OST) STT3 enzymatic isoforms either during protein translation (co-translational) or after protein translation has completed (post-translational). While co-translational N-glycosylation is both rapid and efficient, post-translational N-glycosylation occurs on a much slower time scale and, due to competition with protein degradation and forward trafficking, could be detrimental to the success of a peptide heavily reliant on post-translational N-glycosylation. In evidence, mutations ...


Redox-Dependent Stability, Protonation, And Reactivity Of Cysteine-Bound Heme Proteins, Fangfang Zhong, George P. Lisi, Daniel P. Collins, John H. Dawson, Ekaterina V. Pletneva Jan 2014

Redox-Dependent Stability, Protonation, And Reactivity Of Cysteine-Bound Heme Proteins, Fangfang Zhong, George P. Lisi, Daniel P. Collins, John H. Dawson, Ekaterina V. Pletneva

Open Dartmouth: Faculty Open Access Articles

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to ...


Translocation Channel Gating Kinetics Balances Protein Translocation Efficiency With Signal Sequence Recognition Fidelity, Steven Trueman, Elisabet Mandon, Reid Gilmore Jul 2013

Translocation Channel Gating Kinetics Balances Protein Translocation Efficiency With Signal Sequence Recognition Fidelity, Steven Trueman, Elisabet Mandon, Reid Gilmore

Elisabet Mandon

The transition between the closed and open conformations of the Sec61 complex permits nascent protein insertion into the translocation channel. A critical event in this structural transition is the opening of the lateral translocon gate that is formed by four transmembrane (TM) spans (TM2, TM3, TM7, and TM8 in Sec61p) to expose the signal sequence-binding site. To gain mechanistic insight into lateral gate opening, mutations were introduced into a lumenal loop (L7) that connects TM7 and TM8. The sec61 L7 mutants were found to have defects in both the posttranslational and cotranslational translocation pathways due to a kinetic delay in ...


Structural And Thermodynamic Basis Of Amprenavir/Darunavir And Atazanavir Resistance In Hiv-1 Protease With Mutations At Residue 50, Seema Mittal, Rajintha Bandaranayake, Nancy King, Moses Prabu-Jeyabalan, Madhavi Nalam, Ellen Nalivaika, Nese Yilmaz, Celia Schiffer Jul 2013

Structural And Thermodynamic Basis Of Amprenavir/Darunavir And Atazanavir Resistance In Hiv-1 Protease With Mutations At Residue 50, Seema Mittal, Rajintha Bandaranayake, Nancy King, Moses Prabu-Jeyabalan, Madhavi Nalam, Ellen Nalivaika, Nese Yilmaz, Celia Schiffer

Celia A. Schiffer

Drug resistance occurs through a series of subtle changes that maintain substrate recognition but no longer permit inhibitor binding. In HIV-1 protease, mutations at I50 are associated with such subtle changes that confer differential resistance to specific inhibitors. Residue I50 is located at the protease flap tips, closing the active site upon ligand binding. Under selective drug pressure, I50V/L substitutions emerge in patients, compromising drug susceptibility and leading to treatment failure. The I50V substitution is often associated with amprenavir (APV) and darunavir (DRV) resistance, while the I50L substitution is observed in patients failing atazanavir (ATV) therapy. To explain how ...


Characterization Of The Product Specificity And Kinetic Mechanism Of Protein Arginine Methyltransferase 1, Shanying Gui May 2013

Characterization Of The Product Specificity And Kinetic Mechanism Of Protein Arginine Methyltransferase 1, Shanying Gui

All Graduate Theses and Dissertations

Protein arginine methylation is an essential post-translational modification catalyzed by protein arginine methyltransferases (PRMTs). Type I PRMTs transfer the methyl group from S-adenosyl-L-methionine (AdoMet) to the arginine residues and catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA). Type II PRMTs generate MMA and symmetric dimethylarginine (SDMA). PRMT-catalyzed methylation is involved in many biological processes and human diseases when dysregulated. As the predominant PRMT, PRMT1 catalyzes an estimated 85% of all protein arginine methylation in vivo. Nevertheless, the product specificity of PRMT1 remains poorly understood. A few articles have been published regarding the kinetic mechanism of PRMT1, yet with ...