@article{BalakrishnanHemmenChoudhuryetal.2022,
  author    = {Balakrishnan, Ashwin and Hemmen, Katherina and Choudhury, Susobhan and Krohn, Jan-Hagen and Jansen, Kerstin and Friedrich, Mike and Beliu, Gerti and Sauer, Markus and Lohse, Martin J. and Heinze, Katrin G.},
  title     = {Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence},
  series = {Communications Biology},
  volume    = {5},
  journal   = {Communications Biology},
  number    = {1},
  doi       = {10.1038/s42003-022-03106-4},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-301140},
  year      = {2022},
  abstract  = {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-Delbr{\"u}ck model.},
  language  = {en}
}
@phdthesis{Balakrishnan2021,
  author    = {Balakrishnan, Ashwin},
  title     = {Fast molecular mobility of β\(_2\)-adrenergic receptor revealed by time-resolved fluorescence spectroscopy},
  doi       = {10.25972/OPUS-25085},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250856},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {G-protein- coupled receptors (GPCRs) are the largest family of membrane confined receptors and they transduce ligand binding to downstream effects. Almost 40\% of the drugs in the world target GPCRs due to their function, albeit knowing less about their activation. Understanding their dynamic behaviour in basal and activated state could prove key to drug development in the future. GPCRs are known to exhibit complex molecular mobility patterns. A plethora of studies have been and are being conducted to understand the mobility of GPCRs. Due to limitations of imaging and spectroscopic techniques commonly used, the relevant timescales are hard to access. The most commonly used techniques are electron paramagnetic resonance or double electronelectron resonance, nuclear magnetic resonance, time-resolved fluorescence, single particle tracking and fluorescence recovery after photobleaching. Among these techniques only fluorescence has the potential to probe live cells. In this thesis, I use different time-resolved fluorescence spectroscopic techniques to quantify diffusion dynamics / molecular mobility of β2-adrenergic receptor (β2-AR) in live cells. The thesis shows that β2-AR exhibits mobility over an exceptionally broad temporal range (nanosecond to second) that can be linked to its respective physiological scenario. I explain how β2-AR possesses surprisingly fast lateral mobility (~10 μm²/s) associated with vesicular transport in contrast to the prior reports of it originating from fluorophore photophysics and free fluorophores in the cytosol. In addition, β2-AR has rotational mobility (~100 μs) that makes it conform to the Saffman-Delbr{\"u}ck model of membrane diffusion unlike earlier studies. These contrasts are due to the limitations of the methodologies used. The limitations are overcome in this thesis by using different time-resolved fluorescence techniques of fluorescence correlation spectroscopy (FCS), time-resolved anisotropy (TRA) and polarisation resolved fullFCS (fullFCS). FCS is limited to microsecond to the second range and TRA is limited to the nanosecond range. fullFCS complements the two techniques by covering the blind spot of FCS and TRA in the microsecond range. Finally, I show how ligand stimulation causes a decrease in lateral mobility which could be a hint at cluster formation due to internalisation and how β2-AR possesses a basal oligomerisation that does not change on activation. Thus, through this thesis, I show how different complementary fluorescence techniques are necessary to overcome limitations of each technique and to thereby elucidate functional dynamics of GPCR activation and how it orchestrates downstream signalling.},
  language  = {en}
}