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Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two-component signalling cascades. Homologs of this sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the sensory rhodopsin II/transducer complex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the receptor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer. This mechanism might also be relevant for eubacterial chemoreceptor signalling.

– For the first time, we monitored time-resolved conformational changes inherent in bacterio-rhodopsin's reaction cycle as well as the H+-release events directly at the surface of bacteriorhodopsin (bR) at room temperature. Signals of optical pH-indicators in the aqueous bulk phase were compared with those of probes covalently linked to bR. The kinetics of H+-translocation and the correlation of the photocycle with the pumping cycle can only be determined with indicators bound to bR. The proton appears at the extracellular surface during the L550 to M412 transition. Upon short photo-excitation, diffraction patterns were obtained from both guanidine hydrochloride-treated wild type bR and an Asp96Asn mutant with millisecond time resolution by x-ray synchrotron radiation. The measured time course and location of the structural changes confirm and extend our previous static neutron diffraction study at -180°C. The temporal correlation of photocycle intermediates and proton translocation steps with structural changes in the protein under similar environmental conditions strongly contribute to the understanding of the pumping mechanism.

– The purple pigment bacteriorhodopsin (BR) acts as a light-driven proton pump in the cytoplasmic membrane of the archaebacterium Halobacterium halobium. The original gene encoding for the precursor of bacterio-opsin (bop-gene) and a shortened gene, where the first nucleotides encoding for the presequence are deleted, were introduced in yeast expression vectors. Northern blot analysis of transformed Schizosaccharomyces pombe cells revealed the strong transcription of the archaebacterial DNA directed by the constitutive alcohol dehydrogenase promoter of the fission yeast. The translated precursor and the second construction without the presequence were accumulated in the plasma membrane fraction of the eukaryote. Yeast cells containing the mature BR showed a weaker color than those harboring the precursor protein, if the cells were grown in minimal medium with 2% glucose. At higher glucose concentrations (7%) the expression of BR increased with and without presequence. The overexpressed precursor leads to coryneform fission yeast, whereas cells transformed with the vector containing the bop-gene without the presequence, producing the mature protein, retained the rod-shaped form.

– The location of the heavy-atom label of three different retinal analogues in the plane of the purple membrane was determined by x-ray diffraction. The three analogues, i.e. 9-bromoretinal and 13-bromoretinal labelled in the polyene chain as well as the ring-labelled HgCI-retinal, were incorporated into bacteriorhodopsin (BR) either biosynthetically using a retinal-deficient mutant strain of Halobacterium halobium or with photochemically bleached bacterioopsin. All BR samples regenerated with retinal analogues were functionally active as proton pumps. The diffraction data show that the cyclohexene ring of retinal is situated in the corner formed by helices 4E and 5D. the 13-methyl group adjacent to helix 6C and therefore the Schiff's base nitrogen about midway between helices 6C and 2G. The 9-bromo label is found slightly off the line connecting the two other labels, directed towards helix 3F, suggesting torsion of the polyene chain or slight incline of the ring. The position and orientation of retinal obtained in our experiments are in agreement with data from neutron diffraction and high-resolution electron microscopy. This indicates that the heavy-atom labeled chromophores, 9-bromo- and 13-bromo- as well as HgCI-retinal, are isomorphously incorporated into BR. These samples will allow kinetic investigation of light-induced structural changes of retinal during BR's pumping cycle using x-ray diffraction as well as extended x-ray absorption fine structure experiments probing changes in the retinal neighbourhood between different intermediaies of the photocycle.

Es wird ein Homodyngerat zur Messung der inelastischen Lichtstreuung beschrieben, das mit einem Argon-Ionenlaser ausgerustet ist und mit einem Autokorrelator arbeitet. Das Gerat wird benutzt, um das Verhalten der Fadenmolekule von Cellulosenitrat in verdunnter Losung zu untersuchen. Mit dieser Technik wird es moglich, sogar fur unabgebaute native Cellulose die Diffusionskoeffizienten der Translation D zu bestimmen, die ein sehr niedriges D (

Die Methode der inelastischen Lichtstreuung wird bis in den Bereich mittlerer Konzentration (8%) ausgedehnt. Gemessen wird ein Polystyrol enger Molekulargewichtsverteilung mit Mw = 670000 in Toluol bei 20°C sowie in Cyclohexan bei 35°C. Zur Kontrolle werden mit dem gleichen Gerat auch elastische Lichtstreuungsmessungen ausgefuhrt, die sich mit fruheren Untersuchungen in Ubereinstimmung bringen lassen. Die Diffusionskoeffizienten aus der inelastischen Lichtstreuung stimmen qualitativ mit denen aus klassischen Diffusionsmessungen uberein, die Rehage und Ernst an ahnlichen Systemen ausfuhrten. Bei verdunnten Losungen zeigte sich im betrachteten Winkelbereich ein linearer Zusammenhang zwischen 2Γ und sin2 ∂/2. Mit zunehmender Konzentration wurde in diesen Auftragungen eine Aufwartskrummung sichtbar, die vom Fremdkorrelationsanteil in der Paarverteilungsfunktion herruhren sollte. Die Konzentrationsabhangigkeit des Diffusionskoeffizienten bleibt im gesamten betrachten Konzentrationsbereich fur Toluol positiv und fur das Θ-Losungsmittel Cyclohexan negativ. Mit wachsender Konzentration nimmt diese Konzentrationsabhangigkeit ab. The method of inelastic light scattering is extended to the region of moderate concentrations (up to 8%). Measurements on a narow distributed polystyrene of Mw = 670000 in toluene at 20°C and in cyclohexane at 35°C are reported. For comparison the same photometer also is used for elastic light scattering measurements, which compare well with former results. The diffusion coefficients observed by inelastic light scattering are qualitatively comparable to those obtained by Rehage and Ernst on similar systems, who used classical

Besides the Coomassie-blue-stained band corresponding to mature bacterioopsin two additional bands of slightly higher apparent molecular masses were observed in purple membrane preparations from Halobacterium halobium by SDS-PAGE. The staining intensity within the triple band pattern varied with the age of the cell culture. For cells in the stationary growth phase the lower band, corresponding to mature bacterioopsin, is the predominant one. Immunodetection and site-specific proteolysis with papain identified the upper band as originating from the previously described precursor of bacterioopsin with its 13-amino-acid-long N-terminal presequence. Our results suggest that the intermediate band is due to a modified precursor of bacterioopsin with a truncated presequence of about eight amino acids. A two-step mechanism for the processing of pre-bacterioopsin to the mature protein in this archaebacterium is proposed.