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2015 | 2 | 1 |

Article title

Monitoring the diffusion behavior of Na,K-ATPase
by fluorescence correlation spectroscopy (FCS)
upon fluorescence labelling with eGFP or


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Measurement of lateral mobility of membraneembedded
proteins in living cells with high spatial and
temporal precision is a challenging task of optofluidics.
Biological membranes are complex structures, whose
physico-chemical properties depend on the local lipid
composition, cholesterol content and the presence of integral
or peripheral membrane proteins, which may be involved
in supramolecular complexes or are linked to cellular
matrix proteins or the cytoskeleton. The high proteinto-
lipid ratios in biomembranes indicate that membrane
proteins are particularly subject to molecular crowding,
making it difficult to follow the track of individual
molecules carrying a fluorescence label. Novel switchable
fluorescence proteins such as Dreiklang [1], are, in principle,
promising tools to study the diffusion behavior of individual
molecules in situations of molecular crowding due
to excellent spectral control of the ON- and OFF-switching
process. In this work, we expressed an integral membrane
transport protein, the Na,K-ATPase comprising the human
α2-subunit carrying an N-terminal eGFP or Dreiklang tag
and human β1-subunit, in HEK293T cells and measured
autocorrelation curves by fluorescence correlation spectroscopy
(FCS). Furthermore,we measured diffusion times
and diffusion constants of eGFP and Dreiklang by FCS,
first, in aqueous solution after purification of the proteins
upon expression in E. coli, and, second, upon expression
as soluble proteins in the cytoplasm of HEK293T cells. Our
data show that the diffusion behavior of the purified eGFP
and Dreiklang in solution as well as the properties of the
proteins expressed in the cytoplasm are very similar. However,
the autocorrelation curves of eGFP- and Dreiklanglabeled
Na,K-ATPase measured in the plasma membrane
exhibit marked differences, with the Dreiklang-labeled
construct showing shorter diffusion times. This may be related
to an additional, as yet unrecognized quenching process
that occurs on the same time scale as the diffusion of the labeled complexes through the detection volume (1–
100 ms). Since the origin of this quenching process is currently
unclear, care has to be taken when the Dreiklang label
is intended to be used in FCS applications.







Physical description


20 - 10 - 2015
31 - 12 - 2015
6 - 10 - 2015


  • Technical University of
    Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623
    Berlin, Germany
  • Technical University of
    Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623
    Berlin, Germany
  • Karolinska Institutet, Department of Clinical
    Neuroscience, Center for Molecular Medicine CMM L8:01, 17176
    Stockholm, Sweden
  • Technical University of
    Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623
    Berlin, Germany


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