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  - Kienzler, Andrea
A1  - Flehr, Roman
A1  - Kramer, Rolf A.
A1  - Gehne, Soeren
A1  - Kumke, Michael Uwe
A1  - Bannwarth, Willi
T1  - Novel Three-Color FRET Tool Box for Advanced Protein and DNA Analysis
JF  - Bioconjugate chemistry
N2  - We report on a new three-color FRET system which we were able to verify in peptides as well as in synthetic DNA. All three chromophores could be introduced by a building block approach avoiding postsynthetic labeling. Additional features are robustness, matching spectroscopic properties, high-energy transfer, and sensitivity. The system was investigated in detail on a set of peptides as well as an array of tailored oligonucleotides. The detailed analysis of the experimental data and comparison with theoretical considerations were in excellent agreement. It is shown that in the case of polypeptides specific interaction with the fluorescence probes has to be considered. In contrast with DNA, the fluorescence probes did not show any indications of such interactions. The novel three-color FRET toolbox revealed the potential for applications studying fundamental processes of three interacting molecules in life science applications.
Y1  - 2011
U6  - https://doi.org/10.1021/bc2002659
SN  - 1043-1802
VL  - 22
IS  - 9
SP  - 1852
EP  - 1863
PB  - American Chemical Society
CY  - Washington
ER  -