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@PHDTHESIS{Helten:55205,
      author       = {Helten, Andreas},
      title        = {{D}ynamik der c{GMP}-{S}ynthese in {S}ehzellen -
                      {R}egulation membranständiger {G}uanzylatzkyklasen durch
                      modifizierte {K}alzium-{S}ensor-{P}roteine},
      volume       = {4213},
      issn         = {0944-2952},
      school       = {Univ. Köln},
      type         = {Dr. (Univ.)},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {PreJuSER-55205, Juel-4213},
      series       = {Berichte des Forschungszentrums Jülich},
      pages        = {IX, 105 p.},
      year         = {2006},
      note         = {Record converted from VDB: 12.11.2012; Köln, Univ., Diss.,
                      2006},
      abstract     = {Membrane bound guanylate cyclases GC1 and GC2 are important
                      for the phototransduction in photoreceptors. They regulate,
                      in an interplay with a phosphodiesterase, the concentration
                      of the intracellular messenger cyclic guanosine
                      monophosphate (cGMP). At low calciumconcentrations both GCs
                      are activated by guanylate cyclase activating proteins
                      (GCAP1 and GCAP2). The cGMP-concentration increases and the
                      photoreceptor adapts. If GCs become activated a GC-dimer
                      forms a complex with an unidentified number of GCAPs. The
                      stoichiometry and calcium dependent conformational changes
                      within this complex are unknown. GCAP2 has three cysteine
                      residues, interestingly one in the first and one in the
                      third calcium-binding-motif (EF-hand-motif). In this work
                      cysteine mutants of GCAP2 were generated, heterologously
                      expressed, and purified. All cysteine mutants exhibited
                      EC50- and IC$_{50}$-values comparable to GCAP2-wildtype.
                      However, mutants with no cysteine residue in the first
                      EF-Hand-motif activated GCs weaker compared to
                      GCAP2-wildtype. This indicates an important function of this
                      cysteine residue in GC-regulation. In further experiments I
                      investigated the accessibility of cysteine residues for the
                      thiolreactive substance 5,5'-Dithiobis(2-nitrobenzoic acid).
                      The cysteine residues within the first and third
                      EF-hand-motif were only accessible at low calcium
                      concentrations. This was surprising because the first
                      EF-hand-motif is assumed to bind calcium with only very low
                      affinity. Thus no calcium induced conformational change was
                      expected. By determining the calcium sensitivities of the
                      DTNB-reaction, which could be interpreted as apparent
                      calcium affinities of the EF-hand-motifs, I was able to
                      develop following model: calcium dissociation from the third
                      EF-hand-motif in GCAP2 induces a conformational change that
                      causes GC-activation. Furthermore I demonstrated that
                      magnesium increases the apparent calcium affinities of the
                      first EF-hand-motif in GCAP2 and of the first and third
                      EF-hand-motif in GCAP1. I coupled thiolreactive dyes to
                      cysteine mutants of GCAP2. Thereby I wanted to detect
                      conformational changes in the vicinity of the dye.
                      Furthermore, by recording Förster Resonance Energy Transfer
                      between dye labeled GCAP1- and GCAP2-mutants I wanted to
                      show the simultaneous binding of both GCAP isoforms to GC1.
                      Both experimental approaches gave negative results. Using a
                      soluble, enzymatic active GC1-construct instead of wildtype
                      GC1 produced also not the expected results.},
      cin          = {IBI-1},
      cid          = {I:(DE-Juel1)VDB57},
      pnm          = {Funktion und Dysfunktion des Nervensystems},
      pid          = {G:(DE-Juel1)FUEK409},
      typ          = {PUB:(DE-HGF)11 / PUB:(DE-HGF)3},
      url          = {https://juser.fz-juelich.de/record/55205},
}