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@PHDTHESIS{Hfig:885908,
      author       = {Höfig, Henning},
      title        = {{S}ingle-{M}olecule {C}haracterization of {FRET}-based
                      {B}iosensors and {D}evelopment of {T}wo-{C}olor
                      {C}oincidence {D}etection},
      volume       = {225},
      school       = {RWTH Aachen},
      type         = {Dissertation},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {FZJ-2020-04172},
      isbn         = {978-3-95806-502-4},
      series       = {Schriften des Forschungszentrums Jülich. Reihe
                      Schlüsseltechnologien / Key Technologies},
      pages        = {XVIII, 160 S.},
      year         = {2020},
      note         = {Dissertation, RWTH Aachen, 2020},
      abstract     = {Biomolecules often exhibit heterogeneous properties, e. g.
                      they can exist in different conformational states. Ensemble
                      measurements provide only averaged values and, hence, cannot
                      resolve the underlying heterogeneity. In contrast,
                      single-molecule techniques are based on a
                      molecule-by-molecule analysis which allows to reveal the
                      heterogeneity. In this work, two single-molecule methods
                      based on the confocal detection of fluorescent biomolecules
                      are presented. First, genetically-encoded FRET-based
                      biosensors are investigated that utilize Förster resonance
                      energy transfer (FRET) between two fluorescent proteins as
                      the sensor signal. FRETbased biosensors have great potential
                      for applications in biotechnology or diagnostics. Yet, the
                      development of highly-sensitive sensor constructs is
                      complicated by the complexity of the sensor design and the
                      limited information content of the ensemble
                      characterization. This work presents the experimental
                      requirements for the single-molecule detection of FRET-based
                      biosensors followed by a comprehensive single-molecule study
                      of a set of FRET-based biosensors for the determination of
                      glucose concentrations. The single-molecule characterization
                      allows to dissect different parameters that contribute to
                      the sensor performance and, hence, facilitates a more
                      targeted sensor design. Furthermore, the effect of
                      macromolecular crowding on the glucose sensor and two
                      specific crowding sensor constructs is investigated on the
                      single-molecule level. In order to elucidate the role of the
                      fluorescent proteins for the sensor performance, a
                      dye-labeled analogue of the glucose biosensor is
                      investigated on the single-molecule level. The small changes
                      of the FRET signal upon glucose addition in comparison to
                      the biosensor equipped with fluorescent proteins indicate
                      the importance of the relative fluorophore orientation for
                      the FRET signal, that is unique for the employed fluorescent
                      proteins. The second part of this thesis deals with
                      two-color coincidence detection (TCCD) which enables to
                      investigate the binding of two biomolecules that exhibit
                      fluorescence of two differentcolors. Existing methods
                      underestimate the coincidence due to an imperfect overlap of
                      the confocal volumes of the different colors. The
                      introduction of a brightness gating (BTCCD) for the
                      single-molecule fluorescence intensity facilitates the
                      selection of molecule trajectories that transverse both
                      confocal volumes and, hence, enables quantitative analyses.
                      The capability ofthe brightness gating is demonstrated by
                      reproducing the coincidence of various,
                      dual-labeledreference samples. After a rigorous testing of
                      the limits of BTCCD, it is applied to FRET-based biosensors
                      to reveal the donor-only and acceptor-only fractions.
                      Finally, BTCCD is used tocharacterize protein synthesis in a
                      cell-free protein synthesis system and to reveal a
                      previously unrecognized mode of protein translation
                      initiation in bacteria. The ability of BTCCDto provide
                      quantitative results in combination with the versatility of
                      fluorescence assays turns BTCCD into a helpful tool for
                      further studies of the interaction of biomolecules.},
      cin          = {IBI-6},
      cid          = {I:(DE-Juel1)IBI-6-20200312},
      pnm          = {899 - ohne Topic (POF3-899)},
      pid          = {G:(DE-HGF)POF3-899},
      typ          = {PUB:(DE-HGF)3 / PUB:(DE-HGF)11},
      urn          = {urn:nbn:de:0001-2020120902},
      url          = {https://juser.fz-juelich.de/record/885908},
}