TY  - JOUR
A1  - Balakrishnan, Ashwin
A1  - Hemmen, Katherina
A1  - Choudhury, Susobhan
A1  - Krohn, Jan-Hagen
A1  - Jansen, Kerstin
A1  - Friedrich, Mike
A1  - Beliu, Gerti
A1  - Sauer, Markus
A1  - Lohse, Martin J.
A1  - Heinze, Katrin G.
T1  - Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence
JF  - Communications Biology
N2  - G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β\(_{2}\)-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm\(^{2}\)/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbrück model.
KW  - G-protein-coupled receptors
KW  - molecular mobility
KW  - temporal range
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301140
VL  - 5
IS  - 1
ER  - 
TY  - JOUR
A1  - Kuhlemann, Alexander
A1  - Beliu, Gerti
A1  - Janzen, Dieter
A1  - Petrini, Enrica Maria
A1  - Taban, Danush
A1  - Helmerich, Dominic A.
A1  - Doose, Sören
A1  - Bruno, Martina
A1  - Barberis, Andrea
A1  - Villmann, Carmen
A1  - Sauer, Markus
A1  - Werner, Christian
T1  - Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
JF  - Frontiers in Synaptic Neuroscience
N2  - Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.
KW  - super-resolution microscopy (SRM)
KW  - click-chemistry
KW  - dSTORM
KW  - GABA-A receptor
KW  - genetic code expansion
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251035
SN  - 1663-3563
VL  - 13
ER  -