This title appears in the Scientific Report : 2001 

Zeitauflösende Schwingungsspektroskopie an Bacteriorhodopsin und Halorhodopsin
Bolwien, Carsten (Corresponding author)
Biologische Strukturforschung; IBI-2
Jülich Forschungszentrum Jülch GmbH Zentralbibliothek, Verlag 2002
II, 125 p.
Düsseldorf, Univ., Diss., 2001
Dissertation / PhD Thesis
Biologische Strukturforschung
Berichte des Forschungszentrums Jülich 3945
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The Proteins Bacteriorhodopsin (BR) and Halorhodopsin (HR) can be found in the cell membrane of the halophilic archaebacterium Halobacterium salinarum. Under extreme circumstances, BR as a light-driven proton pump provides an alternative to the respiratory chain in order to establish a proton gradient. HR as a light-driven chloride pump is used to maintain the Chloride concentration in the cytoplasm under varying conditions. The previously published ground state x-ray structures of both Proteins-in the case of BR additional structures of photocycle intermediates are known-form an important basis for the understanding of the functional mechanism, although they fail to provide a dynamic picture of the process. However, this picture is provided by time-resolving vibrational spectroscopy, which is able to detect transient changes in the structure of the Proteins. Fourier-Transform Infrared (FTIR)-spectroscopy on BR with polarized measuring light presents an additional parameter, the linear dichroism, to tracé the structural changes during the photocycle. These measurements are used not only for improved separation of the photocycle intermediates, but also to calculate orientations of transition dipole moments and thereby of structural elements . In addition, it is possible to resolve transient protonations of amino acid side chains, an important part of the mechanism of a proton pump and also invisible with the x-ray technique . In contrast to BR not much is known about the photocycle of the Chloride pump HR. In this work, the influence of the Chloride concentration on the kinetics is investigated with time-resolving FTIR-spectroscopy. The use of the attenuated total reflection ATR-technique, here with a new diamond ATR cell, allows reproducible adjustment of the sait concentration in infrared experiments in the range between 0 to 5 mol/L. An alternative photocycle model has been formulated based on the respective influence on the formation and decay of the intermediates . Resonance Raman measurements on both proteins give additional information on the assignment of vibrational modes and demonstrate the potential of the spectroscopic setup as a micro-Raman-spectrometer for the investigation of protein crystals .