TY - JOUR A1 - De Michele, Roberto A1 - Ast, Cindy A1 - Loque, Dominique A1 - Ho, Cheng-Hsun A1 - Andrade, Susana L. A. A1 - Lanquar, Viviane A1 - Grossmann, Guido A1 - Gehne, Soeren A1 - Kumke, Michael Uwe A1 - Frommer, Wolf B. T1 - Fluorescent sensors reporting the activity of ammonium transceptors in live cells JF - ELIFE N2 - Ammonium serves as key nitrogen source and metabolic intermediate, yet excess causes toxicity. Ammonium uptake is mediated by ammonium transporters, whose regulation is poorly understood. While transport can easily be characterized in heterologous systems, measuring transporter activity in vivo remains challenging. Here we developed a simple assay for monitoring activity in vivo by inserting circularly-permutated GFP into conformation-sensitive positions of two plant and one yeast ammonium transceptors (’AmTrac and ’MepTrac’). Addition of ammonium to yeast cells expressing the sensors triggered concentration dependent fluorescence intensity (FI) changes that strictly correlated with the activity of the transporter. Fluorescence-based activity sensors present a novel technology for monitoring the interaction of the transporters with their substrates, the activity of transporters and their regulation in vivo, which is particularly valuable in the context of analytes for which no radiotracers exist, as well as for cell-specific and subcellular transport processes that are otherwise difficult to track. Y1 - 2013 U6 - https://doi.org/10.7554/eLife.00800 SN - 2050-084X VL - 2 IS - 3 PB - ELIFE SCIENCES PUBLICATIONS LTD CY - CAMBRIDGE ER - TY - JOUR A1 - Demetriou, Antri A1 - Pashalidis, Ioannis A1 - Nicolaides, Athanassios V. A1 - Kumke, Michael Uwe T1 - Surface mechanism of the boron adsorption on alumina in aqueous solutions JF - Desalination and water treatment : science and engineering N2 - The adsorption of boron (boric acid) from aqueous solutions on alumina has been investigated at pH 8.0, I=0.1M NaClO4, T=22 +/- 3 degrees C, and under normal atmospheric conditions. The characterization of the adsorbed species was performed by Raman spectroscopy and the spectroscopic speciation was assisted by theoretical DFT calculations. Evaluation of the spectroscopic data points to the formation of inner-sphere surface complexes and indicates the formation of two different types of adsorbed boron species. The theoretical calculations corroborate the spectroscopic data and indicate that at low boron concentration the monodentate surface species dominates, whereas increased boron concentration favors the formation of a bidentate surface species. Assuming low coverage, the conditional formation constant for the monodentate surface species has been evaluated to be log=4.1 +/- 0.1. KW - Boric acid KW - Alumina KW - Raman spectroscopy KW - DFT calculations KW - Surface complexes KW - Formation constant Y1 - 2013 U6 - https://doi.org/10.1080/19443994.2013.764354 SN - 1944-3994 SN - 1944-3986 VL - 51 IS - 31-33 SP - 6130 EP - 6136 PB - Taylor & Francis Group CY - Philadelphia ER - TY - JOUR A1 - Techen, Anne A1 - Czapla, Sylvia A1 - Möllnitz, Kristian A1 - Budach, Dennis B. A1 - Wessig, Pablo A1 - Kumke, Michael Uwe T1 - Synthesis and spectroscopic characterization of fluorophore-labeled oligospiroketal rods JF - Helvetica chimica acta N2 - Fluorescence probes consisting of well-established fluorophores in combination with rigid molecular rods based on spirane-type structures were investigated with respect to their fluorescence properties under different solvent conditions. The attachment of the dyes was accomplished by 1,3-dipolar cycloaddition between alkynes and azides (click' reaction) and is a prime example for a novel class of sensor constructs. Especially, the attachment of two (different) fluorophores on opposite sides of the molecular rods paves the way to new sensor systems with less bulky (compared to the conventional DNA- or protein-based concepts), nevertheless rigid spacer constructs, e.g., for FRET-based sensing applications. A detailed photophysical characterization was performed in MeOH (and in basic H2O/MeOH mixtures) for i) rod constructs containing carboxyfluorescein, ii) rod constructs containing carboxyrhodamine, iii) rod constructs containing both carboxyfluorescein and carboxyrhodamine, and iv) rod constructs containing both pyrene and perylene parts. For each dye (pair), two rod lengths with different numbers of spirane units were synthesized and investigated. The rod constructs were characterized in ensemble as well as single-molecule fluorescence experiments with respect to i) specific roddye and ii) dyedye interactions. In addition to MeOH and MeOH/NaOH, the rod constructs were also investigated in micellar systems, which were chosen as a simplified model for membranes. KW - Molecular rods KW - Forster resonance energy transfer (FRET) KW - Carboxyfluorescein KW - Carboxyrhodamine KW - Pyrene KW - Perylene KW - Fluorescence Y1 - 2013 U6 - https://doi.org/10.1002/hlca.201200616 SN - 0018-019X SN - 1522-2675 VL - 96 IS - 11 SP - 2046 EP - 2067 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Gehne, Sören A1 - Sydow, Karl A1 - Dathe, Margitta A1 - Kumke, Michael Uwe T1 - Characterization of cell-penetrating lipopeptide micelles by spectroscopic methods JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry N2 - The transport of bioactive compounds to the site of action is a great challenge. A promising approach to overcome application-related problems is the development of targeting colloidal transport systems, such as micelles which are equipped with uptake mediating moieties. Here, we investigated a set of novel lipopeptides which exhibit a surfactant-like structure due to attachment of two palmitoyl chains to the Nterminus of cationic or anionic amino acid sequences. We analyzed the association behavior of these lipopeptides by using 5(6)-carboxyfluorescein (CF)-labeled derivatives as a fluorescent probe and different spectroscopic methods such as fluorescence anisotropy and fluorescence correlation spectroscopy (FCS). The photophysical properties as well as the diffusion and rotational movements of the CF-labeled lipopeptides were exploited to determine the cmc and the size of the micelles consisting of lipopeptides. We could distinguish cationic and anionic lipopeptides by their association behavior and by studying the interactions with mouse brain capillary endothelial cells (b.end3). The cationic derivatives turned out to be very strong surfactants with a very low cmc in the micromolar range (0.5-14 mu M). The unique combination of micelle-forming property and cell-penetrating ability can pave the road for the development of a novel class of efficient drug carrier systems. Y1 - 2013 U6 - https://doi.org/10.1021/jp406053g SN - 1520-6106 VL - 117 IS - 46 SP - 14215 EP - 14225 PB - American Chemical Society CY - Washington ER -