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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. |
