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  - Eiring, Patrick
A1  - McLaughlin, Ryan
A1  - Matikonda, Siddharth S.
A1  - Han, Zhongying
A1  - Grabenhorst, Lennart
A1  - Helmerich, Dominic A.
A1  - Meub, Mara
A1  - Beliu, Gerti
A1  - Luciano, Michael
A1  - Bandi, Venu
A1  - Zijlstra, Niels
A1  - Shi, Zhen-Dan
A1  - Tarasov, Sergey G.
A1  - Swenson, Rolf
A1  - Tinnefeld, Philip
A1  - Glembockyte, Viktorija
A1  - Cordes, Thorben
A1  - Sauer, Markus
A1  - Schnermann, Martin J.
T1  - Targetable conformationally restricted cyanines enable photon-count-limited applications
JF  - Angewandte Chemie Internationale Edition
N2  - Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.
KW  - biology
KW  - super-resolution microscopy
KW  - conformational restriction
KW  - cyanine dyes
KW  - DNA nanotechnology
KW  - fluorescent dyes
KW  - single-molecule fluorescence spectroscopy
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256559
VL  - 60
IS  - 51
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  -