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Full-Text Articles in Molecular Biology

Conversion Of Red Fluorescent Protein Into A Bright Blue Probe, Oksana M. Subach, Illia S. Gundorov, Masami Yoshimura, Fedor V. Subach, Jinghang Zhang, David Grunwald, Ekaterina A. Souslova, Dmitriy M. Chudakov, Vladislav V. Verkhusha Nov 2014

Conversion Of Red Fluorescent Protein Into A Bright Blue Probe, Oksana M. Subach, Illia S. Gundorov, Masami Yoshimura, Fedor V. Subach, Jinghang Zhang, David Grunwald, Ekaterina A. Souslova, Dmitriy M. Chudakov, Vladislav V. Verkhusha

David Grünwald

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins ...


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 ...