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Full-Text Articles in Life Sciences
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
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
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
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
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 possesses cysteine S-conjugate β-lyase …
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
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
Dartmouth Scholarship
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 Cys …