Biochemistry, Biophysics, and Structural Biology Commons^™

Titus Thesis Figures.Docx, Titus Hou

Titus Hou

No abstract provided.

Titus Thesis.Docx, Titus Hou

Titus Hou

No abstract provided.

Pot1‐Independent Single‐Strand Telomeric Dna Binding Activities In Brassicaceae, Eugene V. Shakirov, Thomas D. Mcknight, Dorothy E. Shippen

Yevgeniy (Eugene) Shakirov

Telomeres define the ends of linear eukaryotic chromosomes and are required for genome maintenance and continued cell proliferation. The extreme ends of telomeres terminate in a single‐strand protrusion, termed the G‐overhang, which, in vertebrates and fission yeast, is bound by evolutionarily conserved members of the POT1 (protection of telomeres) protein family. Unlike most other model organisms, the flowering plant Arabidopsis thaliana encodes two divergent POT1‐like proteins. Here we show that the single‐strand telomeric DNA binding activity present in A. thaliana nuclear extracts is not dependent on POT1a or POT1b proteins. Furthermore, in contrast to POT1 proteins from yeast and vertebrates, …

Selaginella Moellendorffii Telomeres: Conserved And Unique Features In An Ancient Land Plant Lineage, Eugene V. Shakirov, Dorothy E. Shippen

Yevgeniy (Eugene) Shakirov

Telomeres, the essential terminal regions of linear eukaryotic chromosomes, consist of G-rich DNA repeats bound by a plethora of associated proteins. While the general pathways of telomere maintenance are evolutionarily conserved, individual telomere complex components show remarkable variation between eukaryotic lineages and even within closely related species. The recent genome sequencing of the lycophyte Selaginella moellendorffii and the availability of an ever-increasing number of flowering plant genomes provides a unique opportunity to evaluate the molecular and functional evolution of telomere components from the early evolving non-seed plants to the more developmentally advanced angiosperms. Here we analyzed telomere sequence in S. …

Pot1 Proteins In Green Algae And Land Plants: Dna-Binding Properties And Evidence Of Co-Evolution With Telomeric Dna, Eugene V. Shakirov, Xiangyu Song, Jessica A. Joseph, Dorothy E. Shippen

Yevgeniy (Eugene) Shakirov

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins …

Heterologous Expression Of Pantoea Agglomerans Phytase Gene Optimized For Plant-Host Expression, N. N. Khabipova, L. R. Valeeva, I. B. Chastukhina, M. R. Sharipova, Eugene V. Shakirov

Yevgeniy (Eugene) Shakirov

Here we report expression and characterization of recombinant bacterial phytase PaPhyC from Pantoea sp. Codon-optimized phytase gene was expressed E.coli BL21 pLysS and protein expression was confirmed by Western blotting. Recombinant protein expressed in E.coli has high phytase activity. We show that PaPhyC recombinant phytase has different molecular masses when expressed in bacteria and plants, suggesting that possible protein glycosylation in plants may influence its overall size.

Promises And Challenges Of Eco-Physiological Genomics In The Field: Tests Of Drought Responses In Switchgrass. Plant Physiology, John T. Lovell, Eugene V. Shakirov, Scott Schwartz, David B. Lowry, Michael J. Aspinwall, Samuel H. Taylor, Jason Bonnette, Juan Diego Palacio-Mejia, Christine V. Hawkes, Philip A. Fay, Thomas E. Juenger

Yevgeniy (Eugene) Shakirov

Identifying the physiological and genetic basis of stress tolerance in plants has proven to be critical to understanding adaptation in both agricultural and natural systems. However, many discoveries were initially made in the controlled conditions of greenhouses or laboratories, not in the field. To test the comparability of drought responses across field and greenhouse environments, we undertook three independent experiments using the switchgrass reference genotype Alamo AP13. We analyzed physiological and gene expression variation across four locations, two sampling times, and three years. Relatively similar physiological responses and expression coefficients of variation across experiments masked highly dissimilar gene expression responses …

Drought Responsive Gene Expression Regulatory Divergence Between Upland And Lowland Ecotypes Of A Perennial C4 Grass, John T. Lovell, Scott Schwartz, David B. Lowry, Eugene V. Shakirov, Jason E. Bonnette, Xiaoyu Weng, Mei Wang, Jenifer Johnson, Avinash Sreedasyam, Christopher Plott, Jerry Jenkins, Jeremy Schmutz, Thomas E. Juenger

Yevgeniy (Eugene) Shakirov

Climatic adaptation is an example of a genotype-by-environment interaction (G×E) of fitness. Selection upon gene expression regulatory variation can contribute to adaptive phenotypic diversity; however, surprisingly few studies have examined how genome-wide patterns of gene expression G×E are manifested in response to environmental stress and other selective agents that cause climatic adaptation. Here, we characterize drought-responsive expression divergence between upland (drought-adapted) and lowland (mesic) ecotypes of the perennial C₄ grass, Panicum hallii, in natural field conditions. Overall, we find that cis-regulatory elements contributed to gene expression divergence across 47% of genes, 7.2% of which exhibit drought-responsive G×E. …

Heterologous Expression Of Secreted Bacterial Bpp And Hap Phytases In Plants Stimulates Arabidopsis Thaliana Growth On Phytate., Lia R. Valeeva, Chuluuntsetseg Nyamsuren, Margarita R. Sharipova, Eugene V. Shakirov

Yevgeniy (Eugene) Shakirov

Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family …

Microbial Phytases And Phytate: Exploring Opportunities For Sustainable Phosphorus Management In Agriculture, Nelly P. Balaban, Aliya D. Suleimanova, Lia R. Valeeva, Inna B. Chastukhina, Natalia L. Rudakova, Margarita R. Sharipova, Eugene V. Shakirov

Yevgeniy (Eugene) Shakirov

Myo-inositol phosphates (phytates) are important biological molecules produced largely by plants to store phosphorus. Phytate is very abundant in many different soils making up a large portion of all soil phosphorus. This review assesses current phytase science from the perspective of its substrate, phytate, by examining the intricate relationship between the phytate-hydrolyzing enzymes and phytate as their substrate. Specifically, we examine available data on phytate’s structural features, distribution in nature and functional roles. The role of phytases and their localization in soil and plant tissues are evaluated. We provide a summary of the current biotechnological advances in using industrial …

Synthesis And Evaluation Of Novel Silica Hydride-Based Stationary Phases For Bioanalytical Applications

Seiichiro Watanabe

Janani Subramaniam Thesis.Pdf, Janani Subramaniam

Janani Subramaniam

Arca Controls Metabolism, Chemotaxis, And Motility Contributing To The Pathogenicity Of Avian Pathogenic Escherichia Coli, Fengwei Jiang, Chunxia An, Yinli Bao, Xuefeng Zhao, Robert L. Jernigan, Andrew Lithio, Dan Nettleton, Ling Li, Eve S. Wurtele, Lisa K. Nolan, Chengping Lu, Ganwu Li

Dan Nettleton

Avian pathogenic Escherichia coli (APEC) strains cause one of the three most significant infectious diseases in the poultry industry and are also potential food-borne pathogens threating human health. In this study, we showed that ArcA (aerobic respiratory control), a global regulator important for E. coli's adaptation from anaerobic to aerobic conditions and control of that bacterium's enzymatic defenses against reactive oxygen species (ROS), is involved in the virulence of APEC. Deletion of arcA significantly attenuates the virulence of APEC in the duck model. Transcriptome sequencing (RNA-Seq) analyses comparing the APEC wild type and the arcA mutant indicate that ArcA regulates …

Structure-Based Design Of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses To A Conserved Epitope, Brian G. Pierce, Elisabeth N. Boucher, Kurt H. Piepenbrink, Ejemel Monir, Chelsea A. Rapp, William D. Thomas Jr., Eric J. Sundberg, Zhiping Weng, Yan Wang

Kurt Piepenbrink

Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, as …

Some Of The Most Interesting Casp11 Targets Through The Eyes Of Their Authors, Andriy Kryshtafovych, John Moult, Arnaud Basle, Alex Burgin, Timonthy K. Craig, Robert A. Edwards, Deborah Fass, Marcus D. Hartmann, Mateusz Korycinski, Richard J. Lewis, Donald Lorimer, Andrei N. Lupas, Janet Newman, Thomas S. Peat, Kurt H. Piepenbrink, Janani Prahlad, Mark J. Van Raaij, Forest Rohwer, Anca M. Segall, Victor Seguritan, Eric J. Sundberg, Abhimanyu K. Singh, Mark A. Wilson, Torsten Schwede

Kurt Piepenbrink

The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features, and assess whether these features were correctly reproduced in the predictions submitted to CASP11.

Dna Uptake By Type Iv Filaments, Kurt H. Piepenbrink

Kurt Piepenbrink

Bacterial uptake of DNA through type IV filaments is an essential component of natural competence in numerous gram-positive and gram-negative species. Recent advances in the field have broadened our understanding of the structures used to take up extracellular DNA. Here, we review seminal experiments in the literature describing DNA binding by type IV pili, competence pili and the flp pili of Micrococcus luteus; collectively referred to here as type IV filaments. We compare the current state of the field on mechanisms of DNA uptake for these three appendage systems and describe the current mechanistic understanding of both DNA-binding and …

Regulation Of The Drosophila Imd Pathway By Signaling Amyloids, Anni Kleino, Neal S. Silverman

Neal Silverman

Fruit flies elicit effective defense responses against numerous microbes. The responses against Gram-negative bacteria are mediated by the Imd pathway, an evolutionarily conserved NF-kappaB pathway recognizing meso-diaminopimelic acid (DAP)-type peptidoglycan from bacterial cell walls. Several reviews already provide a detailed view of ligand recognition and signal transduction during Imd signaling, but the formation and regulation of the signaling complex immediately downstream of the peptidoglycan-sensing receptors is still elusive. In this review, we focus on the formation of the Imd amyloidal signaling center and post-translational modifications in the assembly and disassembly of the Imd signaling complex.

Bridging From Intramuscular To Limb Perfusion Delivery Of Raav: Optimization In A Non-Human Primate Study, Alisha Gruntman, Gwladys Gernoux, Qiushi Tang, Guo-Jie Ye, Dave R. Knop, Gensheng Wang, Janet Benson, Kristen E. Coleman, Allison M. Keeler, Christian Mueller, Louis G. Chicoine, Jeffrey D. Chulay, Terence R. Flotte

Christian Mueller

Phase 1 and phase 2 gene therapy trials using intramuscular (IM) administration of a recombinant adeno-associated virus serotype 1 (rAAV1) for replacement of serum alpha-1 antitrypsin (AAT) deficiency have shown long-term (5-year) stable transgene expression at approximately 2% to 3% of therapeutic levels, arguing for the long-term viability of this approach to gene replacement of secreted serum protein deficiencies. However, achieving these levels required 100 IM injections to deliver 135 mL of vector, and further dose escalation is limited by the scalability of direct IM injection. To further advance the dose escalation, we sought to bridge the rAAV-AAT clinical development …

Synthesis And Evaluation Of Novel Silica Hydride-Based Stationary Phases For Bioanalytical Application, Seiichiro Watanabe

Seiichiro Watanabe

Circular Dichroism And Molecular Modeling Yield A Structure For The Complex Of Human Immunodeficiency Virus Type 1 Trans-Activation Response Rna And The Binding Region Of Tat, The Trans-Acting Transcriptional Activator, Erwann P. Loret, Philippe T. Georgel, W. Curtis Johnson Jr., Pui Shing Ho

Philippe T. Georgel

Transcription in the human immunodeficiency virus type 1 (HIV-1) retrovirus is regulated by binding the viral Tat protein (trans-acting transcriptional activator) to the trans-activation response (TAR) RNA sequence. Here, vacuum UV circular dichroism (VUV-CD) is used to study the structure of TAR and its complex with two peptide fragments that are important for Tat binding to TAR. The VUV-CD spectrum of TAR is typical of A-form RNA and is minimally perturbed when bound to either the short or the long Tat peptide. The CD spectra ofthe complexes indicate an extended structure in the argnine-rich region of Tat from amino acid …

Biotransformation Of Praziquantel By Cunninghamella Elegans, Umbelopsis Ramanniana, And Yarrowia Lipolytica Metabolism, Samuel Chivers

Samuel Chivers

No abstract provided.

The Worlds Of Splicing And Chromatin Collide, J. Adam Hall, Philippe T. Georgel

Philippe T. Georgel

Both transcription and splicing take place in a nuclear environment which, at face value, may seem refractory to the efficiency afforded by the coupling of both processes. This environment, chromatin, was once viewed as only a passive packaging system for genetic material, with very little contribution to the variety of nuclear activities occurring within and around it. However, overwhelming evidence now points to the chromatin environment as being highly dynamic, and an active player in nuclear activities.

Sir3-Dependent Assembly Of Supramolecular Chromatin Structures In Vitro, Philippe T. Georgel, Madeleine A. Palacios Debeer, Gregory Pietz, Catherine A. Fox, Jeffrey C. Hansen

Philippe T. Georgel

Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterized in vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p–chromatin interactions. Together these data indicate …

Promoter Library Designed For Fine-Tuned Gene Expression In Pichia Pastoris, Franz S. Hartner, Claudia Ruth, David Langenegger, Sabrina N. Johnson, Petr Hyka, Geoff P. Lin-Cereghino, Joan Lin-Cereghino, Karin Kovar, James Cregg, Anton Glieder

Joan Lin-Cereghino

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P_AOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing …

Condensed Protocol For Competent Cell Preparation And Transformation Of The Methylotrophic Yeast Pichia Pastoris, Joan Lin-Cereghino, William Wong, See Xiong, William Giang, Linda Luong, Jane Vu, Sabrina Johnson, Geoff P. Lin-Cereghino

Joan Lin-Cereghino

No abstract provided.

Endosome To Golgi Retrieval Of The Vacuolar Protein Sorting Receptor, Vps10p, Requires The Function Of The Vps29, Vps30, And Vps35 Gene Products, Matthew N. J. Seaman, Eric G. Marcusson, Joan Lin-Cereghino, Scott D. Emr

Joan Lin-Cereghino

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The …

An Automated Bayesian Pipeline For Rapid Analysis Of Single-Molecule Binding Data, Carlas Smith, Karina Jouravleva, Maximiliaan Huisman, Samson M. Jolly, Phillip D. Zamore, David Grünwald

David Grünwald

Single-molecule binding assays enable the study of how molecular machines assemble and function. Current algorithms can identify and locate individual molecules, but require tedious manual validation of each spot. Moreover, no solution for high-throughput analysis of single-molecule binding data exists. Here, we describe an automated pipeline to analyze single-molecule data over a wide range of experimental conditions. In addition, our method enables state estimation on multivariate Gaussian signals. We validate our approach using simulated data, and benchmark the pipeline by measuring the binding properties of the well-studied, DNA-guided DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We …

The Cytoplasmic Tail Domain Of The Vacuolar Protein Sorting Receptor Vps10p And A Subset Of Vps Gene Products Regulate Receptor Stability, Function, And Localization, Joan Lin-Cereghino, Eric G. Marcusson, Scott D. Emr

Joan Lin-Cereghino

VPS10 of Saccharomyces cerevisiae encodes a type I transmembrane receptor protein required for the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY). To characterize the essential structural features and intercompartmental transport itinerary of the CPY receptor, we have constructed mutant forms of Vps10p that alter the carboxyterminal cytoplasmic tail of the protein. In addition, we have analyzed the effect these mutations as well as mutations in several VPS genes have on the function, stability, and localization of Vps10p. Although wild-type Vps10p is very stable over a 3-h chase period, overproduction of Vps10p results in PEP4-dependent degradation of the receptor. …

Scale Up Isolation Of Aaptamine For In Vivo Evaluation Indicates Its Neurobiological Activity Is Linked To The Delta Opioid Receptor, Nicole L. Mcintosh, Eptisam Lambo, Laura Millan-Lobo, Fei Li, Li He, Phillip Crews, Jennifer L. Whistler, Tyler Johnson

Tyler Johnson

Opioid receptors belong to the large superfamily of seven transmembrane-spanning (7TM) G protein-coupled receptors (GPCRs). As a class, GPCRs are of fundamental physiological importance mediating the actions of the majority of known neurotransmitters and hormones. The Mu, Delta, and Kappa (MOP, DOP, KOP) opioid receptors are particularly intriguing members of this receptor family as they are the targets involved in many neurobiological diseases such as addiction, pain, stress, anxiety, and depression. Recently we discovered that the aaptamine class of marine sponge derived natural products exhibit selective agonist activity in vitro for the DOP versus MOP receptor. Our findings may explain …

Development And Validation Of A Rapid Method For The Detection Of Latrunculol A In Plasma., Jiajiu Shaw, Frederick A. Valeriote, Joseph Media, Tyler A. Johnson, Taro Amagata, Karen Tenney, Phillip Crews

Tyler Johnson

Latrunculol A is a recently discovered 6,7-dihydroxy analog of the potent actin inhibitor latrunculin A. Latrunculol A has exhibited greater cytotoxicity than latrunculin A against both murine and human colon tumor cell lines in vitro. Currently, there are no reports regarding the bioavailability of latrunculol A in vivo. This study was undertaken as a prelude to pharmacokinetic assessments and it is the first work where bioavailability of latrunculol A was studied. In the present work, a simple plasma preparation and a rapid HPLC method have been developed. Mouse plasma containing latrunculol A was first treated by acetonitrile and then centrifuged …