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@ARTICLE{Rllen:906364,
      author       = {Röllen, Katrin and Granzin, Joachim and Remeeva, Alina and
                      Davari, Mehdi D. and Gensch, Thomas and Nazarenko, Vera V.
                      and Kovalev, Kirill and Bogorodskiy, Andrey and
                      Borshchevskiy, Valentin and Hemmer, Stefanie and
                      Schwaneberg, Ulrich and Gordeliy, Valentin and Jaeger,
                      Karl-Erich and Batra-Safferling, Renu and Gushchin, Ivan and
                      Krauss, Ulrich},
      title        = {{T}he molecular basis of spectral tuning in blue- and
                      red-shifted flavin-binding fluorescent proteins},
      journal      = {The journal of biological chemistry},
      volume       = {296},
      issn         = {0021-9258},
      address      = {Bethesda, Md.},
      publisher    = {Soc.},
      reportid     = {FZJ-2022-01393},
      pages        = {100662 -},
      year         = {2021},
      abstract     = {Photoactive biological systems modify the optical
                      properties of their chromophores, known as spectral tuning.
                      Determining the molecular origin of spectral tuning is
                      instrumental for understanding the function and developing
                      applications of these biomolecules. Spectral tuning in
                      flavin-binding fluorescent proteins (FbFPs), an emerging
                      class of fluorescent reporters, is limited by their
                      dependency on protein-bound flavins, whose structure and
                      hence electronic properties cannot be altered by mutation. A
                      blue-shifted variant of the plant-derived improved light,
                      oxygen, voltage FbFP has been created by introducing a
                      lysine within the flavin-binding pocket, but the molecular
                      basis of this shift remains unconfirmed. We here
                      structurally characterize the blue-shifted improved light,
                      oxygen, voltage variant and construct a new blue-shifted
                      CagFbFP protein by introducing an analogous mutation. X-ray
                      structures of both proteins reveal displacement of the
                      lysine away from the chromophore and opening up of the
                      structure as instrumental for the blue shift. Site
                      saturation mutagenesis and high-throughput screening yielded
                      a red-shifted variant, and structural analysis revealed that
                      the lysine side chain of the blue-shifted variant is
                      stabilized close to the flavin by a secondary mutation,
                      accounting for the red shift. Thus, a single additional
                      mutation in a blue-shifted variant is sufficient to generate
                      a red-shifted FbFP. Using spectroscopy, X-ray
                      crystallography, and quantum mechanics molecular mechanics
                      calculations, we provide a firm structural and functional
                      understanding of spectral tuning in FbFPs. We also show that
                      the identified blue- and red-shifted variants allow for
                      two-color microscopy based on spectral separation. In
                      summary, the generated blue- and red-shifted variants
                      represent promising new tools for application in life
                      sciences.},
      cin          = {IBI-1 / IBI-7 / IBG-1},
      ddc          = {540},
      cid          = {I:(DE-Juel1)IBI-1-20200312 / I:(DE-Juel1)IBI-7-20200312 /
                      I:(DE-Juel1)IBG-1-20101118},
      pnm          = {5241 - Molecular Information Processing in Cellular Systems
                      (POF4-524) / 2171 - Biological and environmental resources
                      for sustainable use (POF4-217)},
      pid          = {G:(DE-HGF)POF4-5241 / G:(DE-HGF)POF4-2171},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:33862085},
      UT           = {WOS:000672866400633},
      doi          = {10.1016/j.jbc.2021.100662},
      url          = {https://juser.fz-juelich.de/record/906364},
}