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Articles 1 - 14 of 14
Full-Text Articles in Biochemistry, Biophysics, and Structural Biology
Molecular Mechanism For Depolarization-Induced Modulation Of Kv Channel Closure, Alain J. Labro, Jerome J. Lacroix, Carlos A. Villalba-Galea, Dirk J. Snyders, Francisco Bezanilla
Molecular Mechanism For Depolarization-Induced Modulation Of Kv Channel Closure, Alain J. Labro, Jerome J. Lacroix, Carlos A. Villalba-Galea, Dirk J. Snyders, Francisco Bezanilla
School of Pharmacy Faculty Articles
Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K(+) permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K(+) conduction through constriction of the K(+) selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the …
Decomposing The Energetic Impact Of Drug-Resistant Mutations: The Example Of Hiv-1 Protease-Drv Binding, Yufeng Cai, Celia Schiffer
Decomposing The Energetic Impact Of Drug-Resistant Mutations: The Example Of Hiv-1 Protease-Drv Binding, Yufeng Cai, Celia Schiffer
Celia A. Schiffer
HIV-1 protease is a major drug target for AIDS therapy. With the appearance of drug-resistant HIV-1 protease variants, understanding the mechanism of drug resistance becomes critical for rational drug design. Computational methods can provide more details about inhibitor-protease binding than crystallography and isothermal titration calorimetry. The latest FDA-approved HIV-1 protease inhibitor is Darunavir (DRV). Herein, each DRV atom is evaluated by free energy component analysis for its contribution to the binding affinity with wild-type protease and ACT, a drug-resistant variant. This information can contribute to the rational design of new HIV-1 protease inhibitors.
Hydrophobic Core Flexibility Modulates Enzyme Activity In Hiv-1 Protease, Seema Mittal, Yufeng Cai, Madhavi Nalam, Daniel Bolon, Celia Schiffer
Hydrophobic Core Flexibility Modulates Enzyme Activity In Hiv-1 Protease, Seema Mittal, Yufeng Cai, Madhavi Nalam, Daniel Bolon, Celia Schiffer
Celia A. Schiffer
Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the …
36 Degrees Step Size Of Proton-Driven C-Ring Rotation In Fof1-Atp Synthase, Monika Düser, Nawid Zarrabi, Daniel Cipriano, Stefan Ernst, Gary Glick, Stanley Dunn, Michael Börsch
36 Degrees Step Size Of Proton-Driven C-Ring Rotation In Fof1-Atp Synthase, Monika Düser, Nawid Zarrabi, Daniel Cipriano, Stefan Ernst, Gary Glick, Stanley Dunn, Michael Börsch
Stanley D Dunn
Synthesis of adenosine triphosphate ATP, the 'biological energy currency', is accomplished by F(o)F(1)-ATP synthase. In the plasma membrane of Escherichia coli, proton-driven rotation of a ring of 10 c subunits in the F(o) motor powers catalysis in the F(1) motor. Although F(1) uses 120 degrees stepping during ATP synthesis, models of F(o) predict either an incremental rotation of c subunits in 36 degrees steps or larger step sizes comprising several fast substeps. Using single-molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36 degrees sequential stepping mode of the c-ring during ATP synthesis.
Molecular Characterization Of The Viab Locus Encoding The Biosynthetic Machinery For Vi Capsule Formation In Salmonella Typhi, Michael Wetter, David Goulding, Derek Pickard, Michael Kowarik, Charles J. Waechter, Gordon Dougan, Michael Wacker
Molecular Characterization Of The Viab Locus Encoding The Biosynthetic Machinery For Vi Capsule Formation In Salmonella Typhi, Michael Wetter, David Goulding, Derek Pickard, Michael Kowarik, Charles J. Waechter, Gordon Dougan, Michael Wacker
Molecular and Cellular Biochemistry Faculty Publications
The Vi capsular polysaccharide (CPS) of Salmonella enterica serovar Typhi, the cause of human typhoid, is important for infectivity and virulence. The Vi biosynthetic machinery is encoded within the viaB locus composed of 10 genes involved in regulation of expression (tviA), polymer synthesis (tviB-tviE), and cell surface localization of the CPS (vexA-vexE). We cloned the viaB locus from S. Typhi and transposon insertion mutants of individual viaB genes were characterized in Escherichia coli DH5α. Phenotype analysis of viaB mutants revealed that tviB, tviC, tviD and tviE are involved in Vi polymer synthesis. Furthermore, expression of tviB-tviE in E. coli DH5α …
Insights Into Mrnp Biogenesis Provided By New Genetic Interactions Among Export And Transcription Factors, Francisco Estruch, Christine Hodge, Natalia Gómez-Navarro, Lorena Peiró-Chova, Catherine V. Heath, Charles N. Cole
Insights Into Mrnp Biogenesis Provided By New Genetic Interactions Among Export And Transcription Factors, Francisco Estruch, Christine Hodge, Natalia Gómez-Navarro, Lorena Peiró-Chova, Catherine V. Heath, Charles N. Cole
Dartmouth Scholarship
The various steps of mRNP biogenesis (transcription, processing and export) are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation. We investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show …
Role Of Sequence And Structure Of The Hendra Fusion Protein Fusion Peptide In Membrane Fusion, Everett Clinton Smith, Sonia M. Gregory, Lukas K. Tamm, Trevor P. Creamer, Rebecca Ellis Dutch
Role Of Sequence And Structure Of The Hendra Fusion Protein Fusion Peptide In Membrane Fusion, Everett Clinton Smith, Sonia M. Gregory, Lukas K. Tamm, Trevor P. Creamer, Rebecca Ellis Dutch
Molecular and Cellular Biochemistry Faculty Publications
Viral fusion proteins are intriguing molecular machines that undergo drastic conformational changes to facilitate virus-cell membrane fusion. During fusion a hydrophobic region of the protein, termed the fusion peptide (FP), is inserted into the target host cell membrane, with subsequent conformational changes culminating in membrane merger. Class I fusion proteins contain FPs between 20 and 30 amino acids in length that are highly conserved within viral families but not between. To examine the sequence dependence of the Hendra virus (HeV) fusion (F) protein FP, the first eight amino acids were mutated first as double, then single, alanine mutants. Mutation of …
Membrane Fusion Proteins Are Required For Oskar Mrna Localization In The Drosophila Egg Chamber, Douglas Ruden, Vincent Sollars, Xiaoyan Wang, Daisuke Mori, Marina Alterman, Xiangyi Lu
Membrane Fusion Proteins Are Required For Oskar Mrna Localization In The Drosophila Egg Chamber, Douglas Ruden, Vincent Sollars, Xiaoyan Wang, Daisuke Mori, Marina Alterman, Xiangyi Lu
Vincent E Sollars
We used a genetic screen in Drosophila to identify mutations which disrupt the localization of oskar mRNA during oogenesis. Based on the hypothesis that some cytoskeletal components which are required during the mitotic divisions will also be required for oskar mRNA localization during oogenesis, we designed the following genetic screen. We screened for P-element insertions in genes which slow down the blastoderm mitotic divisions. A secondary genetic screen was to generate female germ-line clones of these potential cell division cycle genes and to identify those which cause the mislocalization of oskar mRNA. We identified mutations in ter94 which disrupt the …
Evidence For An Epigenetic Mechanism By Which Hsp90 Acts As A Capacitor For Morphological Evolution, Vincent E. Sollars, Xiangyi Lu, Li Xiao, Xiaoyan Wang, Mark D. Garfinkel, Douglas M. Ruden
Evidence For An Epigenetic Mechanism By Which Hsp90 Acts As A Capacitor For Morphological Evolution, Vincent E. Sollars, Xiangyi Lu, Li Xiao, Xiaoyan Wang, Mark D. Garfinkel, Douglas M. Ruden
Vincent E Sollars
Morphological alterations have been shown to occur in Drosophila melanogaster when function of Hsp90 (heat shock 0-kDa protein 1α, encoded by Hsp83) is compromised during development1. Genetic selection maintains the altered phenotypes in subsequent generations1. Recent experiments have shown, however, that phenotypic variation still occurs in nearly isogenic recombinant inbred strains of Arabidopsis thaliana2. Using a sensitized isogenic D. melanogaster strain, iso-KrIf-1, we confirm this finding and present evidence supporting an epigenetic mechanism for Hsp90’s capacitor function, whereby reduced activity of Hsp90 induces a heritably altered chromatin state. The altered chromatin state is evidenced by ectopic expression of the morphogen …
Mutation And Complementation Of A Cellulose Synthase (Cesa) Gene, Ahmed Y. El-Araby
Mutation And Complementation Of A Cellulose Synthase (Cesa) Gene, Ahmed Y. El-Araby
Senior Honors Projects
Cellulose is a carbohydrate polymer that is composed of repeating glucose subunits. Being the most abundant organic compound in the biosphere and comprising a large percentage of all plant biomass, cellulose is extremely plentiful and has a significant role in nature. Cellulose is present in plant cell walls, in commercial products such as those made from wood or cotton, and is of interest to the biofuel industry as a potential alternative fuel source. Although indigestible by humans, cellulose is nutritionally valuable, serving as a dietary fiber. Because of its ubiquity and importance in many areas, studying cellulose will prove to …
Characterization Of Novel Histone H2b Mutants Associated With Chromosome Segregation Defects In Saccharomyces Cerevisiae, Thiruchelvam Rajagopal
Characterization Of Novel Histone H2b Mutants Associated With Chromosome Segregation Defects In Saccharomyces Cerevisiae, Thiruchelvam Rajagopal
Graduate Theses and Dissertations
Histones are small basic proteins that associate with DNA to form the basic unit of chromatin, the nucleosome. Histones H3 and H4 form a tetramer that is bound by two H2A-H2B dimers to form the histone octamer, to which approximately 146 bp of DNA wrap around to form the nucleosome. High resolution structural information and recent advances in the understanding of histone post-translational modifications have illuminated the many regulatory functions chromatin exerts in the cell, from the transcriptional control of gene expression to chromosome segregation. However, the specific role that histones play in these processes is not well understood. Previous …
Active Site Mutations Change The Cleavage Specificity Of Neprilysin., Travis Sexton, Lisa J. Hitchcook, David W. Rodgers, Luke H. Bradley, Louis B. Hersh
Active Site Mutations Change The Cleavage Specificity Of Neprilysin., Travis Sexton, Lisa J. Hitchcook, David W. Rodgers, Luke H. Bradley, Louis B. Hersh
Molecular and Cellular Biochemistry Faculty Publications
Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe563 and Ser546. Among the mutants studied in detail we observed changes in their activity towards leucine5-enkephalin, insulin B chain, and amyloid β1-40. For example, NEPF563I displayed an increase in preference towards cleaving leucine5-enkephalin relative to insulin B chain, while mutant NEPS546E was less discriminating …
Guanosine Diphosphatase Is Required For Protein And Sphingolipid Glycosylation In The Golgi Lumen Of Saccharomyces Cerevisiae, Claudia Abeijon, Ken Yanagisawa, Elisabet Mandon, Alex Hausler, Kelley Moremen, Carlos Hirschberg, Phillips Robbins
Guanosine Diphosphatase Is Required For Protein And Sphingolipid Glycosylation In The Golgi Lumen Of Saccharomyces Cerevisiae, Claudia Abeijon, Ken Yanagisawa, Elisabet Mandon, Alex Hausler, Kelley Moremen, Carlos Hirschberg, Phillips Robbins
Elisabet Mandon
Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn …
Lesion-Specific Dna-Binding And Repair Activities Of Human O⁶-Alkylguanine Dna Alkyltransferase, Manana Melikishvili, Michael G. Fried
Lesion-Specific Dna-Binding And Repair Activities Of Human O⁶-Alkylguanine Dna Alkyltransferase, Manana Melikishvili, Michael G. Fried
Center for Structural Biology Faculty Publications
Binding experiments with alkyl-transfer-active and -inactive mutants of human O6-alkylguanine DNA alkyltransferase (AGT) show that it forms an O6-methylguanine (6mG)-specific complex on duplex DNA that is distinct from non-specific assemblies previously studied. Specific complexes with duplex DNA have a 2:1 stoichiometry that is formed without accumulation of a 1:1 intermediate. This establishes a role for cooperative interactions in lesion binding. Similar specific complexes could not be detected with single-stranded DNA. The small difference between specific and non-specific binding affinities strongly limits the roles that specific binding can play in the lesion search process. Alkyl-transfer kinetics with …