ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05440.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract—Halobacterium halobiumcells grown in nicotine‐containing medium synthesize bacterio‐opsin (bO) but little bacteriorhod‐opsin (bR) because nicotine inhibits retinal synthesis. In nicotine‐grown cells, bacterio‐opsin is found in a specific cell membrane fraction, the brown membrane (b.m.), which consists mainly of small round sheets. Freeze‐fracture of isolated brown membrane reveals a dense particle population on its cytoplasmic fracture face, with few particles on the external face. There is no apparent order in the particle distribution and the fracture faces are practically indistinguishable from those of red membrane (r.m.). The absorbance spectrum of b.m. shows peaks at 550 and 410nm, the circular dichroism (CD) spectrum a positive band around 540nm and positive and negative bands around 410nm with a cross‐over at 412nm. The photoreaction cycle of bR in b.m. has the same intermediates found in purple membrane (p.m.), but the apparent kinetics resemble those of bR monomers.Addition of 13‐cis‐, or all‐trans‐retinal to b.m. induces a rapid 5‐ to 10‐fold increase in the visible absorbance band, and the absorbance maximum shifts to 560nm in the dark. Kinetic analysis of the absorbance increase shows three first order kinetic constants witht½= 0.5 min, 6 min and 100 min, with most of the increase contributed by the fastest component. The CD bilobal pattern typical for purple membrane appears together with the strong negative band at 320nm. Ellipticity change at 590nm is nearly as rapid as the absorbance increase; within 5 min, 85% of the negative band is formed. Electron microscopy after addition of retinal reveals structurally distinct domains in the b.m. sheets which have the characteristic appearance of p.m. However, X‐ray reflections are more diffuse compared to p.m. Flash spectroscopy of reconstituted b.m. detects the same photocycle intermediates as in b.m. or p.m., but with apparent kinetics closer to those of purple membrane than before reconstitution. These results show that bO in the b.m. binds retinal to form bR which spontaneously aggregates into a lattice. However, the reconstituted bR lattice is not as well‐ordered as in p.m. and some bR is still present in the form of

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05441.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract—Time course of formation and crystallization of bacteriorhodopsin upon the addition of retinal to brown apo‐membrane has been studied by X‐ray diffraction, circular dichroism (CD) spectra and absorption spectra. The rate of formation of bacteriorhodopsin decreases markedly at low pH (4.8) and low temperature (5°C). Furthermore, the formation of bacteriorhodopsin does not proceed in fully dried membranes. The half‐time of the increase of exciton CD band is about 70s at 17°C and pH 7.0, and is comparable to that of the formation of bacteriorhodopsins (∼48s). The crystallization of bacteriorhodopsin proceeds to a large extent within 30 min at pH 7.0 and 5°C.The bilobed CD band of the brown holo‐membrane attributed to exciton coupling of bacteriorhodopsin molecules becomes negligibly small at pH 4.8, even though X‐ray diffraction pattern indicates the lattice structure to be similar to that of the native

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05442.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract—Suspensions of purple membranes were exposed to reversing rectangular pulses of relatively low electric field (less than 100V/cm) at various frequencies. Orientation of the membranes was estimated by measuring linear dichroism at 565nm. In the electric field of frequency lower than about 10Hz the purple membranes tended to align perpendicular to the electric field. This orientation was induced mostly by permanent dipole which was perpendicular to the membrane surface. The value of permanent dipole moment was determined to be 140D per protein at pH 7.0, 25°C, in the presence of 5mMphosphate buffer.In an electric field of frequency more than 100Hz the membrane tended to align parallel to the electric field. Electric polarizability parallel to the membrane surface was estimated to be 1 ± 10−12cm3under the same conditions.Electric dichroism of light‐adapted and dark‐adapted purple membranes was found to coincide with each other at high frequency. From this result the angle of retinal to the membrane surface was concluded to be the same between light‐adapted and dark‐adapted bacte

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05443.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract—The effect of light on purple membrane biogenesis inHalobacterium halobiumS9 strain was investigated. When bacteria were grown in the dark, the 570nm absorption due to bacteriorhodopsin increased more slowly than under illumination, but eventually after longer incubation, reached the same level as that seen in the illuminated culture.Analysis of membrane fractions by sucrose density gradient centrifugation revealed that two different membrane fractions, containing purple and brown membrane could be detected in the exponential growth phase. Another fraction whose density was higher than that of purple membrane, disappeared concomitantly with the increase in purple membrane and brown membrane, indicating that it may be related to purple membrane formation.HPLC analysis of membrane proteins showed that there was no significant difference inde novosynthesis of bacterio–opsin between dark and illuminated cultures. This led us to conclude that light stimulated retinal binding to bacterio–opsin and/or retinal biosynthesis rather than bacterio–opsin synthesis. Bacteriorhodopsin seemed to form the brown membrane fraction first, which then spontaneously reorganized into purple membrane.When incorporated in liposomes, bacteriorhodopsin in brown membrane was found to have rather higher proton pump activity than that in purple membrane. The H+pumping activity was quite heat labile. This and the CD spectra indicated that bacteriorhodopsin in brown membrane might exist without forming normal tim

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05444.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract—Two fluorescence probes, 1‐dimethylaminonaphthalene‐5‐sulfonylchloride and pyrene‐1‐sulfonylchloride, were conjugated to the protein in purple membrane and fluorescence intensity and anisotropy were measured as a function of temperature. The anisotropy of the 1‐dimethyl‐aminonaphthalene‐5‐sulfonate conjugate showed a characteristic temperature dependence, in which, the anisotropy is constant at 0.22 ± 0.01 until 25‐30°C and decreases above 30°C. The fluorescence intensity, however, varies linearly with temperature. These results imply that the transition is due to bacteri

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05445.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract—The absorption and circular dichroic spectra of the brown holo‐membrane (retinal present) and apo‐membrane (retinal absent) ofHalobacterium halobiumin solution and oriented as a film have been studied over the accessible wavelength region, 800–183nm. Since the structure of the well‐studied purple membrane can be considered to be a modification of the structure of the brown membrane and much is known about the structure of the purple membrane, interpretations of the brown membrane spectra are based on our previous interpretations of similar studies of the purple membrane. The brown membrane contains two membrane proteins, bacteriorhodopsin and cytochromeb‐561 in a 3:1 molar ratio in contrast to the purple membrane which contains only bacteriorhodopsin. Main findings are (a) degenerate oscillator coupling (exciton) among the retinyl chromophores of the bacteriorhodopsins, (b) a relatively strong in‐plane interaction between the retinal and the bacteriorhodopsin apoprotein environment, possibly due to a dissymmetric static charge distribution, (c) the planes of the aromatic rings of some of the tryptophans must be nearly parallel to the plane of the membrane, (d) the helical axes of the bacterio‐opsin polypeptide segments are significantly tilted in respect to the normal to the membrane plane in contrast to the helical axes of the bacteriorhodopsin polypeptide segments which are nearly parallel to the normal, (e) no detectable interaction between the two membrane proteins, (f) the plane of the heme of the cytochrome cannot be parallel to the membrane plane and is most likely perpendicular to it. (g) the dipole moments of the two mutually perpendicular Soret porphyrin transitions of the heme are most likely oriented at an angle to the membrane plane, (h) there seems to be a significant reduction in the symmetry of the heme group in the environment of the apoprotein, (i) the possibility of a unique geometrical arrangement and resonance interaction between the Soret porphyrin and nearby cytochrome aromatic amino acid π–π* transitions, (j) the secondary structure of the cytochrome is significantly α‐helical, and (k) the helical axes of the cytochrome polypeptide segments are randomly oriented in respect to the normal to the membrane plane. A consequence of these findings is that the fine structures of the bacteriorhodopsins of the brown and purple membranes are very similar in spite of differences in the composition and the structure of the two membranes. In addition, the orientation of the helical segments of the bacteriorhodopsin polypeptides relative to the membrane plane in the brown and purple membranes can be regulated by the retinal–apoprotein interactions. Significance of this possible regulation in respect to the proton‐pumping function of th

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05446.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract—Flash‐induced changes in surface potential and fluidity of purple membrane were studied by a spin label method. Changes in surface potential and fluidity were monitored by observing the distribution of charged and uncharged spin labels between the purple membrane and the aqueous phase.On flash illumination, a transient hyperpolarization of the surface potential and a transient fluidization of the membrane hydrophobic region are induced. The former may probably reflect the proton movement near the purple membrane surface, while the latter may result from photo‐induced conformational changes of bacteriorhodopsin.The two events are different in time course. The relationships between the two events, and the formation and decay of the intermediates of bacteriorhodopsin in the photoreaction cycle are disc

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05447.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract—The formation process of purple membrane was studied morphologically by freeze‐fracture and etching methods. The bacteria,H. halobiumR1, cultured in the medium containing 2mMnicotine, did not show the hexagonal structure of purple membrane, but a particle free area was frequently observed in the plasma membrane. However, within 24h after returning to the normal medium, hexagonal and triclinic lattices with spacings of 6 to 8nm occurred as small patches on the P face of the plasma membrane. Furthermore, it was revealed that the retinal was not required for the formation of the two‐dimensional hexagonal lattice structure of purple membrane, since the mutant bacteria,H. halobiumR1mW, lacking the ability of retinal synthesis, displayed a similar lattice. The other mutant,H. halobiumR1mR, lacking the ability of bacterio‐opsin synthesis, never showed any highly ordered arrangement. This evidence suggests that membrane proteins other than bacterio‐opsin or bacteriorhodopsin do not form highly ordered lattices in the

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05448.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY

摘要:

Abstract—The difference (in cm−1) in absorption maxima between the protonated Schiff base of retinals and the pigment derived therefrom has been defined as the opsin shift. It represents the influence of the opsin binding site on the chromophore. The analysis of the opsin shifts of a series of dihydrobacteriorhodopsins has led to the external point‐charge model, which in addition to a counter anion near the Schiff base ammonium, carries another negative charge in the vicinity of the β‐ionone ring. This is in striking contrast to the external point‐charge model proposed earlier for the bovine visual pigment. The absorption maxima of rhodopsins formed from bromo‐ and phenyl retinals support the two models. A retinal carrying a photoaffinity label has yielded a nonbleachable bacte

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1981.tb05449.x

出版商:Blackwell Publishing Ltd    

年代:1981

数据来源: WILEY