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Articles 1 - 30 of 352

Full-Text Articles in Molecular Biology

Ribosome Inhibition By C9orf72-Als/Ftd-Associated Poly-Pr And Poly-Gr Proteins Revealed By Cryo-Em [Preprint], Anna B. Loveland, Egor Svidritskiy, Denis Susorov, Soojin Lee, Alexander Park, Gabriel Demo, Fen-Biao Gao, Andrei A. Korostelev Aug 2020

Ribosome Inhibition By C9orf72-Als/Ftd-Associated Poly-Pr And Poly-Gr Proteins Revealed By Cryo-Em [Preprint], Anna B. Loveland, Egor Svidritskiy, Denis Susorov, Soojin Lee, Alexander Park, Gabriel Demo, Fen-Biao Gao, Andrei A. Korostelev

University of Massachusetts Medical School Faculty Publications

Toxic dipeptide repeat (DPR) proteins are produced from expanded G4C2 hexanucleotide repeats in the C9ORF72 gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥ 20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel ...


The Chemical Structure And Phosphorothioate Content Of Hydrophobically Modified Sirnas Impact Extrahepatic Distribution And Efficacy, Annabelle Biscans, Jillian Caiazzi, Sarah M. Davis, Nicholas Mchugh, Jacquelyn Sousa, Anastasia Khvorova Aug 2020

The Chemical Structure And Phosphorothioate Content Of Hydrophobically Modified Sirnas Impact Extrahepatic Distribution And Efficacy, Annabelle Biscans, Jillian Caiazzi, Sarah M. Davis, Nicholas Mchugh, Jacquelyn Sousa, Anastasia Khvorova

Open Access Articles

Small interfering RNAs (siRNAs) have revolutionized the treatment of liver diseases. However, robust siRNA delivery to other tissues represents a major technological need. Conjugating lipids (e.g. docosanoic acid, DCA) to siRNA supports extrahepatic delivery, but tissue accumulation and gene silencing efficacy are lower than that achieved in liver by clinical-stage compounds. The chemical structure of conjugated siRNA may significantly impact invivo efficacy, particularly in tissues with lower compound accumulation. Here, we report the first systematic evaluation of the impact of siRNA scaffold-i.e. structure, phosphorothioate (PS) content, linker composition-on DCA-conjugated siRNA delivery and efficacy in vivo. We found that ...


Deciphering Complex Mechanisms Of Resistance And Loss Of Potency Through Coupled Molecular Dynamics And Machine Learning [Preprint], Florian Leidner, Nese Kurt Yilmaz, Celia A. Schiffer Jun 2020

Deciphering Complex Mechanisms Of Resistance And Loss Of Potency Through Coupled Molecular Dynamics And Machine Learning [Preprint], Florian Leidner, Nese Kurt Yilmaz, Celia A. Schiffer

University of Massachusetts Medical School Faculty Publications

Drug resistance threatens many critical therapeutics through mutations in the drug target. The molecular mechanisms by which combinations of mutations, especially involving those distal from the active site, alter drug binding to confer resistance are poorly understood and thus difficult to counteract. A strategy coupling parallel molecular dynamics simulations and machine learning to identify conserved mechanisms underlying resistance was developed. A series of 28 HIV-1 protease variants with up to 24 varied substitutions were used as a rigorous model of this strategy. Many of the mutations were distal from the active site and the potency to darunavir varied from low ...


Dynamics Of The 4d Genome During In Vivo Lineage Specification And Differentiation, A. Marieke Oudelaar, Daniel Hidalgo, Merav Socolovsky, Douglas R. Higgs, Jim R. Hughes Jun 2020

Dynamics Of The 4d Genome During In Vivo Lineage Specification And Differentiation, A. Marieke Oudelaar, Daniel Hidalgo, Merav Socolovsky, Douglas R. Higgs, Jim R. Hughes

Open Access Articles

Mammalian gene expression patterns are controlled by regulatory elements, which interact within topologically associating domains (TADs). The relationship between activation of regulatory elements, formation of structural chromatin interactions and gene expression during development is unclear. Here, we present Tiled-C, a low-input chromosome conformation capture (3C) technique. We use this approach to study chromatin architecture at high spatial and temporal resolution through in vivo mouse erythroid differentiation. Integrated analysis of chromatin accessibility and single-cell expression data shows that regulatory elements gradually become accessible within pre-existing TADs during early differentiation. This is followed by structural re-organization within the TAD and formation of ...


Mrna Stem-Loops Can Pause The Ribosome By Hindering A-Site Trna Binding, Chen Bao, Sarah Loerch, Clarence Ling, Andrei A. Korostelev, Nikolaus Grigorieff, Dmitri N. Ermolenko May 2020

Mrna Stem-Loops Can Pause The Ribosome By Hindering A-Site Trna Binding, Chen Bao, Sarah Loerch, Clarence Ling, Andrei A. Korostelev, Nikolaus Grigorieff, Dmitri N. Ermolenko

Open Access Articles

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Forster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of ...


Cloning And Expression Of Hydra Innexin 2, A Gap Junction Protein Required For Coordinated Contraction Of The Body Column, Ashley O'Brien May 2020

Cloning And Expression Of Hydra Innexin 2, A Gap Junction Protein Required For Coordinated Contraction Of The Body Column, Ashley O'Brien

Biology Theses

In invertebrates gap junctions are formed by the innexin family of proteins. Remarkably, the genome of Hydra magnipapillata contains 17 innexin genes. This study focused on Hydra innexin-2 (h-Inx2) which is expressed in nerve cells and plays a role in contraction of the body column. The gene sequence of H-Inx2 was obtained from the National Center for Biotechnology Information (NCBI), the gene was synthesized externally and transferred to a vector suitable for expression in Xenopus oocytes (pcDNA3.1 CT-GFP TOPO). The TOPO CT-GFP vector includes a priming site for RNA polymerase which allows in vitro preparation of RNA. Another advantage ...


Conformational Changes In The Ebola Virus Membrane Fusion Machine Induced By Ph, Ca2+, And Receptor Binding, Dibyendu Kumar Das, Uriel Bulow, William E. Diehl, Natasha D. Durham, Fernando Senjobe, Kartik Chandran, Jeremy Luban, James B. Munro Feb 2020

Conformational Changes In The Ebola Virus Membrane Fusion Machine Induced By Ph, Ca2+, And Receptor Binding, Dibyendu Kumar Das, Uriel Bulow, William E. Diehl, Natasha D. Durham, Fernando Senjobe, Kartik Chandran, Jeremy Luban, James B. Munro

Open Access Articles

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Forster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational ...


Skp, Cullin, F-Box (Scf)-Met30 And Scf-Cdc4-Mediated Proteolysis Of Cenp-A Prevents Mislocalization Of Cenp-A For Chromosomal Stability In Budding Yeast, Wei-Chun Au, Richard E. Baker, Munira A. Basrai Feb 2020

Skp, Cullin, F-Box (Scf)-Met30 And Scf-Cdc4-Mediated Proteolysis Of Cenp-A Prevents Mislocalization Of Cenp-A For Chromosomal Stability In Budding Yeast, Wei-Chun Au, Richard E. Baker, Munira A. Basrai

Open Access Articles

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two ...


A Thermophilic Phage Uses A Small Terminase Protein With A Fixed Helix-Turn-Helix Geometry, Janelle A. Hayes, Brendan J. Hilbert, Christl Gaubitz, Nicholas P. Stone, Brian A. Kelch Feb 2020

A Thermophilic Phage Uses A Small Terminase Protein With A Fixed Helix-Turn-Helix Geometry, Janelle A. Hayes, Brendan J. Hilbert, Christl Gaubitz, Nicholas P. Stone, Brian A. Kelch

Open Access Articles

Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here, we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerS(P76-26) oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerS(P76-26) revealed that it forms a ring ...


Non-Invasive Method For Leptin Supplementation In Zebrafish (Danio Rerio), Regan Mcnamara Jan 2020

Non-Invasive Method For Leptin Supplementation In Zebrafish (Danio Rerio), Regan Mcnamara

Williams Honors College, Honors Research Projects

I tested the hypothesis that recombinant leptin protein can be introduced to zebrafish in vivo through non-invasive soaking in a solution containing the protein. One way to study various molecules’ effects in vivo is through intraperitoneal or intracerebroventricular injections during the embryonic or larval stage, which is invasive, difficult to administer, and can have a high mortality rate. 48 hours post fertilization (hpf) zebrafish were soaked in a His-tagged recombinant leptin protein solution at 10 nM and 100 nM concentrations (produced by Genscript). After soaking, zebrafish larvae were washed extensively to remove all recombinant protein on their exterior before homogenization ...


The Exploration Of Nanotoxicological Copper And Interspecific Saccharomyces Hybrids, Matthew Joseph Winans Phd Jan 2020

The Exploration Of Nanotoxicological Copper And Interspecific Saccharomyces Hybrids, Matthew Joseph Winans Phd

Graduate Theses, Dissertations, and Problem Reports

Nanotechnology takes advantage of cellular biology’s natural nanoscale operations by interacting with biomolecules differently than soluble or bulk materials, often altering normal cellular processes such as metabolism or growth. To gain a better understanding of how copper nanoparticles hybridized on cellulose fibers called carboxymethyl cellulose (CMC) affected growth of Saccharomyces cerevisiae, the mechanisms of toxicity were explored. Multiple methodologies covering genetics, proteomics, metallomics, and metabolomics were used during this investigation. The work that lead to this dissertation discovered that these cellulosic copper nanoparticles had a unique toxicity compared to copper. Further investigation suggested a possible ionic or molecular mimicry ...


Archaeosine Modification Of Archaeal Trna - A Role In Structural Stabilization, Ben Turner, Brett W. Burkhart, Katrin Weidenbach, Robert Ross, Patrick A. Limbach, Ruth A. Schmitz, Valérie De Crécy-Lagard, Kenneth M. Stedman, Thomas J. Santangelo, Dirk Iwata-Reuyl Jan 2020

Archaeosine Modification Of Archaeal Trna - A Role In Structural Stabilization, Ben Turner, Brett W. Burkhart, Katrin Weidenbach, Robert Ross, Patrick A. Limbach, Ruth A. Schmitz, Valérie De Crécy-Lagard, Kenneth M. Stedman, Thomas J. Santangelo, Dirk Iwata-Reuyl

Biology Faculty Publications and Presentations

Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside of the Archaea. G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted ...


An Allosteric Pocket For Inhibition Of Bacterial Enzyme I Identified By Nmr-Based Fragment Screening, Trang T. Nguyen, Vincenzo Venditti Jan 2020

An Allosteric Pocket For Inhibition Of Bacterial Enzyme I Identified By Nmr-Based Fragment Screening, Trang T. Nguyen, Vincenzo Venditti

Chemistry Publications

Enzyme I (EI), which is the key enzyme to activate the bacterial phosphotransferase system, plays an important role in the regulation of several metabolic pathways and controls the biology of bacterial cells at multiple levels. The conservation and ubiquity of EI among different types of bacteria makes the enzyme a potential target for antimicrobial research. Here, we use NMR-based fragment screening to identify novel inhibitors of EI. We identify three molecular fragments that allosterically inhibit the phosphoryl transfer reaction catalyzed by EI by interacting with the enzyme at a surface pocket located more than 10 Å away from the substrate ...


The Structure Of The Endogenous Esx-3 Secretion System, Nicole Poweleit, Nadine Czudnochowski, Rachel Nakagawa, Donovan Trinidad, Kenan C. Murphy, Christopher M. Sassetti, Oren S. Rosenberg Dec 2019

The Structure Of The Endogenous Esx-3 Secretion System, Nicole Poweleit, Nadine Czudnochowski, Rachel Nakagawa, Donovan Trinidad, Kenan C. Murphy, Christopher M. Sassetti, Oren S. Rosenberg

Open Access Articles

The ESX (or Type VII) secretion systems are protein export systems in mycobacteria and many Gram-positive bacteria that mediate a broad range of functions including virulence, conjugation, and metabolic regulation. These systems translocate folded dimers of WXG100-superfamily protein substrates across the cytoplasmic membrane. We report the cryo-electron microscopy structure of an ESX-3 system, purified using an epitope tag inserted with recombineering into the chromosome of the model organism Mycobacterium smegmatis. The structure reveals a stacked architecture that extends above and below the inner membrane of the bacterium. The ESX-3 protomer complex is assembled from a single copy of the EccB3 ...


Characterization Of The Nuclear Pore Complex In Red Alga, Cyanidioschyzon Merolae, Michelle Veronin Dec 2019

Characterization Of The Nuclear Pore Complex In Red Alga, Cyanidioschyzon Merolae, Michelle Veronin

Health and Kinesiology Theses

Cyanidioschyzon merolae (C. merolae) is a primitive, unicellular species of red alga that is considered to be one of the simplest self-sustaining eukaryotes. The highly elementary nature of C. merolae makes it an excellent model organism for studying evolution as well as cell function and organelle communication. In our study, we hypothesize that C. merolae contains the minimal assembly of proteins to make up their Nuclear Pore Complexes (NPCs), and hence are the first ancestral NPCs. NPCs are essential for basic nuclear transport in the cell. They are embedded in the double membrane of the nucleus, the nuclear envelope (NE ...


Structural Organization And Dynamics Of Homodimeric Cytohesin Family Arf Gtpase Exchange Factors In Solution And On Membranes, Sanchaita Das, Andrew W. Malaby, Agata Nawrotek, Wenhua Zhang, Mahel Zeghouf, Sarah Maslen, Mark Skehel, Srinivas Chakravarthy, Thomas C. Irving, Osman Bilsel, Jacqueline Cherfils, David G. Lambright Dec 2019

Structural Organization And Dynamics Of Homodimeric Cytohesin Family Arf Gtpase Exchange Factors In Solution And On Membranes, Sanchaita Das, Andrew W. Malaby, Agata Nawrotek, Wenhua Zhang, Mahel Zeghouf, Sarah Maslen, Mark Skehel, Srinivas Chakravarthy, Thomas C. Irving, Osman Bilsel, Jacqueline Cherfils, David G. Lambright

Open Access Articles

Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains ...


Natural Variation In Yeast Stress Signaling Reveals Multiple Paths To Similar Phenotypes, Amanda N. Scholes Dec 2019

Natural Variation In Yeast Stress Signaling Reveals Multiple Paths To Similar Phenotypes, Amanda N. Scholes

Theses and Dissertations

Natural environments are dynamic, and organisms must sense and respond to changing conditions. One common way organisms deal with stressful environments is through gene expression changes, allowing for stress acclimation and resistance. Variation in stress sensing and signaling can potentially play a large role in how individuals with different genetic backgrounds are more or less resilient to stress. However, the mechanisms underlying how gene expression variation affects organismal fitness is often obscure.

To understand connections between gene expression variation and stress defense phenotypes, we have been exploiting natural variation in Saccharomyces cerevisiae stress responses using a unique phenotype called acquired ...


Structure And Assembly Of Calcium Homeostasis Modulator Proteins [Preprint], Johanna L. Syrjanen, Kevin Michalski, Tsung-Han Chou, Timothy Grant, Shanlin Rao, Noriko Simorowski, Stephen J. Tucker, Nikolaus Grigorieff, Hiro Furukawa Nov 2019

Structure And Assembly Of Calcium Homeostasis Modulator Proteins [Preprint], Johanna L. Syrjanen, Kevin Michalski, Tsung-Han Chou, Timothy Grant, Shanlin Rao, Noriko Simorowski, Stephen J. Tucker, Nikolaus Grigorieff, Hiro Furukawa

University of Massachusetts Medical School Faculty Publications

Biological membranes of many tissues and organs contain large-pore channels designed to permeate a wide variety of ions and metabolites. Examples include connexin, innexin, and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), which permeate ions and ATP in a voltage-dependent manner to control neuronal excitability, taste signaling, and pathologies of depression and Alzheimer’s disease. Despite such critical biological roles, the structures and patterns of oligomeric assembly remain unclear. Here, we reveal the first structures of two CALHMs, CALHM1 and CALHM2, by single ...


Physical Models Of Living Systems Chapter 12: Single Particle Reconstruction In Cryo-Electron Microscopy, Philip C. Nelson Nov 2019

Physical Models Of Living Systems Chapter 12: Single Particle Reconstruction In Cryo-Electron Microscopy, Philip C. Nelson

Department of Physics Papers

This chapter extends Part III of the book Physical Models of Living Systems (WH Freeman 2015). This preliminary version is made freely available as-is in the hope that it will be useful.


Discovery Of An Egfr Inhibitor For The Treatment Of Lung And Other Cancers, Jodie Meng '20 Nov 2019

Discovery Of An Egfr Inhibitor For The Treatment Of Lung And Other Cancers, Jodie Meng '20

Student Publications & Research

The Epidermal Growth Factor Receptor (EGFR), a transmembrane protein involved in the regulation of signaling pathways, is frequently overexpressed in epithelial tumors. First generation EGFR TKIs, such as erlotinib and gefitinib, traditionally improved outcomes for non-small-cell lung carcinoma and pancreatic cancer patients by attaching competitively and reversibly to the ATP binding domain of EGFR. Second-generation EGFR TKIs have been developed to combat resistance among patients, despite demonstrating toxic side effects. In the present study, 1400 selective inhibitors were designed based on the molecular scaffolds of first and second generation EGFR TKIs. Results were refined by parameters outlined in Lipinski’s ...


Structural Organization Of The C1a-E-C Supercomplex Within The Ciliary Central Apparatus, Gang Fu, Lei Zhao, Erin Dymek, Yuqing Hou, Kangkang Song, Nhan Phan, Zhiguo Shang, Elizabeth F. Smith, George B. Witman, Daniela Nicastro Oct 2019

Structural Organization Of The C1a-E-C Supercomplex Within The Ciliary Central Apparatus, Gang Fu, Lei Zhao, Erin Dymek, Yuqing Hou, Kangkang Song, Nhan Phan, Zhiguo Shang, Elizabeth F. Smith, George B. Witman, Daniela Nicastro

Radiology Publications

Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type Chlamydomonas with CA mutants. We identified a large ( > 2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76 ...


Highly Structured Homolog Pairing Reflects Functional Organization Of The Drosophila Genome, Jumana Alhaj Abed, Jelena Erceg, Anton Goloborodko, Son C. Nguyen, Ruth B. Mccole, Wren Saylor, Geoffrey Fudenberg, Bryan R. Lajoie, Job Dekker, Leonid A. Mirny, C-Ting Wu Oct 2019

Highly Structured Homolog Pairing Reflects Functional Organization Of The Drosophila Genome, Jumana Alhaj Abed, Jelena Erceg, Anton Goloborodko, Son C. Nguyen, Ruth B. Mccole, Wren Saylor, Geoffrey Fudenberg, Bryan R. Lajoie, Job Dekker, Leonid A. Mirny, C-Ting Wu

Program in Systems Biology Publications

Trans-homolog interactions have been studied extensively in Drosophila, where homologs are paired in somatic cells and transvection is prevalent. Nevertheless, the detailed structure of pairing and its functional impact have not been thoroughly investigated. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, showing that homologs pair with varying precision genome-wide, in addition to establishing trans-homolog domains and compartments. We also elucidate the structure of pairing with unprecedented detail, observing significant variation across the genome and revealing at least two forms of pairing: tight pairing, spanning contiguous small domains, and loose pairing, consisting of single ...


Principles For Enhancing Virus Capsid Capacity And Stability From A Thermophilic Virus Capsid Structure, Nicholas P. Stone, Gabriel Demo, Emily Agnello, Brian A. Kelch Oct 2019

Principles For Enhancing Virus Capsid Capacity And Stability From A Thermophilic Virus Capsid Structure, Nicholas P. Stone, Gabriel Demo, Emily Agnello, Brian A. Kelch

Open Access Articles

The capsids of double-stranded DNA viruses protect the viral genome from the harsh extracellular environment, while maintaining stability against the high internal pressure of packaged DNA. To elucidate how capsids maintain stability in an extreme environment, we use cryoelectron microscopy to determine the capsid structure of thermostable phage P74-26 to 2.8-A resolution. We find P74-26 capsids exhibit an overall architecture very similar to those of other tailed bacteriophages, allowing us to directly compare structures to derive the structural basis for enhanced stability. Our structure reveals lasso-like interactions that appear to function like catch bonds. This architecture allows the capsid ...


Excess No Stabilizes The Luminal Domain Of Stim2 In A Cys-Specific Manner Thereby Regulating Basal Calcium Homeostasis And Store-Operated Calcium Entry, Matthew Novello Sep 2019

Excess No Stabilizes The Luminal Domain Of Stim2 In A Cys-Specific Manner Thereby Regulating Basal Calcium Homeostasis And Store-Operated Calcium Entry, Matthew Novello

Electronic Thesis and Dissertation Repository

Stromal-interaction molecule 2 (STIM2) is an endoplasmic reticulum (ER) membrane-inserted Ca2+-sensing protein which, together with the plasma membrane Ca2+ channel Orai1, regulates basal Ca2+ homeostasis and store-operated Ca2+ entry (SOCE). Recent evidence suggests that S-nitrosylation, which is the covalent attachment of a nitric oxide (NO) moiety to a cysteine thiol, can attenuate the function of the paralog STIM1 protein. Compared to STIM1, STIM2 also functions as a basal Ca2+ homeostatic feedback regulator. Therefore, the objective of my study was to evaluate the susceptibility of STIM2 to S-nitrosylation and the effects that this ...


The Mechanism And Regulation Of Bacteriophage Dna Packaging Motors, Janelle A. Hayes Sep 2019

The Mechanism And Regulation Of Bacteriophage Dna Packaging Motors, Janelle A. Hayes

GSBS Dissertations and Theses

Many double-stranded DNA viruses use a packaging motor during maturation to recognize and transport genetic material into the capsid. In terminase motors, the TerS complex recognizes DNA, while the TerL motor packages the DNA into the capsid shell. Although there are several models for DNA recognition and translocation, how the motor components assemble and power DNA translocation is unknown.

Using the thermophilic P74-26 bacteriophage model system, we discover that TerL uses a trans-activated ATP hydrolysis mechanism. Additionally, we identify critical residues for TerL ATP hydrolysis and DNA binding. With a combination of x-ray crystallography, SAXS, and molecular docking, we ...


The Central Role Of The Tail In Switching Off 10s Myosin Ii Activity, Shixin Yang, Kyounghwan Lee, John L. Woodhead, Osamu Sato, Mitsuo Ikebe, Roger Craig Sep 2019

The Central Role Of The Tail In Switching Off 10s Myosin Ii Activity, Shixin Yang, Kyounghwan Lee, John L. Woodhead, Osamu Sato, Mitsuo Ikebe, Roger Craig

Radiology Publications

Myosin II is a motor protein with two heads and an extended tail that plays an essential role in cell motility. Its active form is a polymer (myosin filament) that pulls on actin to generate motion. Its inactive form is a monomer with a compact structure (10S sedimentation coefficient), in which the tail is folded and the two heads interact with each other, inhibiting activity. This conformation is thought to function in cells as an energy-conserving form of the molecule suitable for storage as well as transport to sites of filament assembly. The mechanism of inhibition of the compact molecule ...


Mechanism For Apobec3g Catalytic Exclusion Of Rna And Non-Substrate Dna, William C. Solomon, Wazo Myint, Shurong Hou, Tapan Kanai, Rashmi Tripathi, Nese Kurt Yilmaz, Celia A. Schiffer, Hiroshi Matsuo Aug 2019

Mechanism For Apobec3g Catalytic Exclusion Of Rna And Non-Substrate Dna, William C. Solomon, Wazo Myint, Shurong Hou, Tapan Kanai, Rashmi Tripathi, Nese Kurt Yilmaz, Celia A. Schiffer, Hiroshi Matsuo

Schiffer Lab Publications

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of ...


Promotion Of Adipogenesis By Jmjd6 Requires The At Hook-Like Domain And Is Independent Of Its Catalytic Function, Pablo Reyes-Gutierrez, Jake W. Carrasquillo-Rodriguez, Anthony N. Imbalzano Aug 2019

Promotion Of Adipogenesis By Jmjd6 Requires The At Hook-Like Domain And Is Independent Of Its Catalytic Function, Pablo Reyes-Gutierrez, Jake W. Carrasquillo-Rodriguez, Anthony N. Imbalzano

Open Access Articles

JMJD6 is a member of the Jumonji C domain containing enzymes that demethylate and/or hydroxylate substrate proteins. It is a multi-functional protein that has been implicated in disparate aspects of transcriptional and post-transcriptional control of gene expression, including but not limited to enhancer and promoter binding, release of paused RNA polymerase II, control of splicing, and interaction with the translation machinery. JMJD6 contributes to multiple aspects of animal development, including adipogenesis modeled in culture. We mutated proposed or characterized domains in the JMJD6 protein to better understand the requirement for JMJD6 in adipogenic differentiation. Mutation of JMJD6 amino acids ...


Nonnative Structure In A Peptide Model Of The Unfolded State Of Sod1: Implications For Als-Linked Aggregation, Noah R. Cohen, Jill A. Zitzewitz, Osman Bilsel, C. Robert Matthews Jul 2019

Nonnative Structure In A Peptide Model Of The Unfolded State Of Sod1: Implications For Als-Linked Aggregation, Noah R. Cohen, Jill A. Zitzewitz, Osman Bilsel, C. Robert Matthews

Open Access Articles

Dozens of mutations throughout the sequence of the gene encoding superoxide dismutase 1 (SOD1) have been linked to toxic protein aggregation in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A parsimonious explanation for numerous genotypes resulting in a common phenotype would be mutation-induced perturbation of the folding free-energy surface that increases the populations of high-energy states prone to aggregation. The absence of intermediates in the folding of monomeric SOD1 suggests that the unfolded ensemble is a potential source of aggregation. To test this hypothesis, here we dissected SOD1 into a set of peptides end-labeled with FRET probes to model the ...


Rapid Irreversible Transcriptional Reprogramming In Human Stem Cells Accompanied By Discordance Between Replication Timing And Chromatin Compartment, Vishnu Dileep, Rachel Patton Mccord, Job Dekker, David M. Gilbert Jul 2019

Rapid Irreversible Transcriptional Reprogramming In Human Stem Cells Accompanied By Discordance Between Replication Timing And Chromatin Compartment, Vishnu Dileep, Rachel Patton Mccord, Job Dekker, David M. Gilbert

Open Access Articles

The temporal order of DNA replication is regulated during development and is highly correlated with gene expression, histone modifications and 3D genome architecture. We tracked changes in replication timing, gene expression, and chromatin conformation capture (Hi-C) A/B compartments over the first two cell cycles during differentiation of human embryonic stem cells to definitive endoderm. Remarkably, transcriptional programs were irreversibly reprogrammed within the first cell cycle and were largely but not universally coordinated with replication timing changes. Moreover, changes in A/B compartment and several histone modifications that normally correlate strongly with replication timing showed weak correlation during the early ...