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@PHDTHESIS{Fastermann:35976,
      author       = {Fastermann, Dirk},
      title        = {{H}alorhodopsin : {K}lonierung, {E}xpression,
                      {K}ristallisation und spektroskopische {U}ntersuchungen zum
                      {M}echanismus dieser {A}nionenpumpe},
      volume       = {3934},
      issn         = {0944-2952},
      school       = {Univ. Düsseldorf},
      type         = {Dr. (Univ.)},
      address      = {Jülich},
      publisher    = {Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag},
      reportid     = {PreJuSER-35976, Juel-3934},
      series       = {Berichte des Forschungszentrums Jülich},
      pages        = {III, 111 p.},
      year         = {2002},
      note         = {Record converted from VDB: 12.11.2012; Düsseldorf, Univ.,
                      Diss. 2002},
      abstract     = {Four retinal proteins have been discovered in Halobacterium
                      salinarum : The sensory rhodopsins I and II which function
                      as photoreceptors and bacteriorhodopsin (bR) and
                      halorhodopsin (hR) which function as ion pumps. In this
                      work, functional investigations of thé anion pump hR were
                      undertaken, with thé main focus on thé mechanistic
                      analysis of thé light driven anion transport . In order to
                      overexpress hR, thé hR gene was first linked to thé
                      promotor of bR. The expression took place in thé bR
                      negative strain L33 from H. salinarum . The expression level
                      of this fusion construct was so high that hR crystallized in
                      vivo in a two-dimensional lattice. Due to this, it was
                      possible to isolate and to purify hR in a membrane-bound
                      state. For thé crystallization and spectroscopic
                      measurements a purification method was established which
                      preserves native structural and functional integrity.
                      Crystallization trials with solubilized hR using sitting
                      drop and cubic lipidic phase method were performed. With
                      thé cubic lipidic phase, it was possible to get three
                      dimensional crystals, but they showed no diffraction. The
                      chloride dependency of thé hR photocycle was studied with
                      spectroscopic measurements employing a non-time resolved
                      chloride titration of hR membrane patches . The différence
                      absorbance spectra showed three différent states (at 540
                      nm, 580 nm and 650 mn) which depend on thé chloride
                      concentration . The chloride progression of thé states
                      showed three potential binding constants for chloride to hR.
                      Two photocycle models, one in thé absence and one in thé
                      présence of chloride, were formulated from thé time
                      resolved visible measurements. Specifically, thé photocycle
                      model without chloride suggests two states: hR54o and
                      hR-y.650, whereas thé photocycle model with chloride
                      suggests three states: hR5Bo, hR-K65o and hRL52o. The
                      photocycle without chloride is much faster. Chloride
                      dependent investigations were also done with time-resolved
                      FTIR spectroscopy in combination with thé ATR technique.
                      With increasing chloride concentration, thé formation of
                      thé hRLintermediate was accelerated. On thé other hand,
                      thé decay of thé L intermediate is showed down with
                      increasing chloride concentration . At very high chloride
                      concentrations, e.g. 5 M, différences in thé amide I
                      region between early and late hR-L différence spectra were
                      detected. This implies structural changes in thé protein
                      backbone. This was corroborated with linear dichroism. The
                      chloride dependent formation of thé hR-Ll and thé decay of
                      thé hR-L2 intermediate implies that thé chloride uptake
                      takes place during thé transition from thé hR-K
                      intermediate to thé hR-Ll intermediate . In addition thé
                      transfer of thé chloride to thé cytoplasm occurs between
                      thé hRL2 intermediate and thé ground state.},
      cin          = {IBI-2},
      cid          = {I:(DE-Juel1)VDB58},
      pnm          = {Biologische Strukturforschung},
      pid          = {G:(DE-Juel1)FUEK96},
      typ          = {PUB:(DE-HGF)11 / PUB:(DE-HGF)3},
      url          = {https://juser.fz-juelich.de/record/35976},
}