Richard J. Cherry

Transient and linear dichroism studies on bacteriorhodopsin: Determination of the orientation of the 568 nm all-trans retinal chromophore☆

Abstract The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways. In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a time-independent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78 ° ± 3 ° is determined. In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of the light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient dichroism method, linear dichroism measurements on oriented samples lead to an angle of 71 ° ± 4 °. No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substantially smaller than the recently suggested value of 20 nm (Ebrey et al., 1977). The orientation of the 568 nm transition dipole moment, which coincides with the direction of the all-trans polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.

Anomalous Diffusion of Major Histocompatibility Complex Class I Molecules on HeLa Cells Determined by Single Particle Tracking

Single-particle tracking (SPT) was used to determine the mobility characteristics of MHC (major histocompatibility complex) class I molecules at the surface of HeLa cells at 22 degrees C and on different time scales. MHC class I was labeled using the Fab fragment of a monoclonal antibody (W6/32), covalently bound to either R-phycoerythrin or fluorescent microspheres, and the particles were tracked using high-sensitivity fluorescence imaging. Analysis of the data for a fixed time interval suggests a reasonable fit to a random diffusion model. The best fit values of the diffusion coefficient D decreased markedly, however, with increasing time interval, demonstrating the existence of anomalous diffusion. Further analysis of the data shows that the diffusion is anomalous over the complete time range investigated, 4-300 s. Fitting the results obtained with the R-phycoerythrin probe to D = D0talpha-1, where Do is a constant and t is the time, gave D0 = (6.7 +/- 4.5) x 10(-11) cm2 s-1 and alpha = 0.49 +/- 0.16. Experiments with fluorescent microspheres were less reproducible and gave slower anomalous diffusion. The R-phycoerythrin probe is considered more reliable for fluorescent SPT because it is small (11 x 8 nm) and monovalent. The type of motion exhibited by the class I molecules will greatly affect their ability to migrate in the plane of the membrane. Anomalous diffusion, in particular, greatly reduces the distance a class I molecule can travel on the time scale of minutes. The present data are discussed in relation to the possible role of diffusion and clustering in T-cell activation.

Temperature-dependent aggregation of bacteriorhodopsin in dipalmitoyl- and dimyristoylphosphatidylcholine vesicles*

Bacteriorhodopsin has been incorporated into large unilamellar lipid vesicles. Its aggregation behaviour was investigated using X-ray diffraction, electron microscopy, circular dichroism and rotational diffusion measurements. At temperatures below the lipid phase transition, bacteriorhodopsin crystallizes into patches with the same hexagonal lattice observed in the purple membrane. Above the phase transition, the lattice disaggregates and the protein molecules are monomeric provided the lipid to protein ratio is sufficiently high.

Anisotropic rotation of bacteriorhodopsin in lipid membranes. Comparison of theory with experiment.

Rotational diffusion of bacteriorhodopsin in dimyristoyl phosphatidylcholine vesicles has been measured at different temperatures and lipid; protein ratios by the technique of flash-induced transient linear dichroism. The data are used to evaluate critically the theory of anisotropy decay due to protein rotation in the lipid bilayer. The theoretical model assumes that rotation of the protein occurs only around the membrane normal. Under conditions favoring completely monomeric bacteriorhodopsin, namely at molar lipid; protein ratios greater than or approximately 100 and for temperatures sufficiently above the lipid phase transition, it is found that the theoretical model provides an excellent description of the experimental data. Curve-fitting analyses of the experimental decay curves show that the retinal is oriented at an angle of 78 +/- 2 degrees with respect to the membrane normal. Between 25 and 37 degrees C, the protein rotates with a relaxation time of 15 +/- 5 micros in the lipid liquid crystalline phase, corresponding to the membrane viscosity of 3.7 +/- 1.3 P. The curve analysis also provides a sensitive test for the presence of protein aggregates in the lipid bilayer.

Rotational diffusion of bacteriorhodopsin in lipid membranes.

New techniques for measuring the diffusion of proteins in cell membranes have recently been reported [l-7] . Such measurements should in principle test to what extent proteins are freely floating in the fluid lipid bilayer, as envisaged in currently popular concepts of membrane structure [8]. They may also provide a method of investigating structural features of membranes which restrict or prevent diffusion. Before these aims can be fully achieved, it is necessary to have a sound basis for interpreting diffusion measurements in membranes. The familiar Stokes-Einstein equations are not applicable in two dimensional systems and indeed it is not certain that diffusion in lipid bilayers can in any case be treated by classical hydrodynamics. Model calculations of diffusion in membranes have recently been presented [9] but the results have yet to be critically tested. Experimental data which are sufficiently unambiguous to test theoretical predictions are most likely to be obtained with simple model systems rather than with cell membranes. Here we report the incorporation of bacteriorhodopsin, one of the best characterised membrane proteins, into phospholipid bilayers. We propose that this system should prove valuable for a detailed investigation of diffusion in membranes and give results of preliminary measurements of protein rotation.

Physical properties of lecithin-cerebroside bilayers.

Abstract The incorporation of ox brain cerebroside into egg-lecithin model membranes (bilayers) typically increases the electrical resistance and breakdown voltage and decreases the a.c. capacitance. At 1:1 mole ratio the capacitance measured at 1000 Hz is 0.24 ± 0.02 μ F/cm 2 as compared with 0.38 ± 0.02 μ F/cm 2 for egg lecithin alone. By comparison with the effects of including sphingomyelin and dibehenoyl lecithin in bilayers, it is concluded that the reduced capacitance is in part due to a thicker hydrocarbon region resulting from the relatively longer hydrocarbon chains of the cerebroside. Combined measurements of a.c. and d.c. capacitance indicate the presence of a low frequency dispersion from which it is inferred that the remaining reduction in a.c. capacitance is due to a contribution from the polar groups. An analysis of the bilayer equivalent circuit shows that such a contribution can be expected only if the polar region has a low conductance and a low dielectric constant. Calorimetric studies demonstrate that hydrated ox brain cerebroside has a gel-liquid crystalline phase transition at about 55°. The transition temperature of hydrated cerebroside-egg lecithin samples falls as the proportion of lecithin is increased, reaching 20° at about 1:1 mole ratio. There is circumstantial evidence that this is close to the maximum amount of cerebroside that can be incorporated into the bilayer at this temperature.

Rotational diffusion and exciton coupling of bacteriorhodopsin in the cell membrane of Halobacterium halobium

Bacteriorhodopsin forms a two-dimensional hexagonal crystalline lattice in the purple membrane of Halobacterium halobium [l-4]. Rotational motion of bacteriorhodopsin may be investigated by measuring the decay of dichroism of flash-induced absorbance changes; in the rigid lattice of the purple membrane the protein is immobilised [S]. Rotational motion is observed when the lattice is disrupted by organic solvents [6] or when bacteriorhodopsin is incorporated into lipid vesicles [7]. Exciton coupling effects due to the interaction of the retinal chromophores of adjacent bacteriorhodopsin molecules have been observed in the CD spectra of the purple membrane in the 568 nm absorption band [g-lo]. The necessary conditions for the occurrence of such effects are satisfied in the crystalline lattice. The results of recent structural investigations suggest that the lattice is formed from trimers [4]. The exciton CD-spectra could likewise be interpreted as arising from the interactions of the three chromophores within a trimer [9], neglecting inter-trimer interactions. The exciton coupling bands disappear when bacteriorhodopsin is solubilised into micelles containing protein monomers [8,10,1 l] and when the bacteriorhodopsin molecules within the membrane acquire rotational mobility, either by addition of organic solvents [6] or by incorporation into artificial lipid membranes [7,12]. There appears to be a clear correlation between loss of exciton coupling, observation of rotational diffusion and protein disaggregation. The exciton CD effects in combination with rotational diffusion measurements thus provide useful information concerning the state of aggregation of the bacteriorhodopsin molecules within the membrane. Bacteriorhodopsin synthesis in the cells depends on the rate of aeration during growth. If bacteriorhodopsin synthesis is not optimal, besides the crystalline lattice of the purple membrane a second bacteriorhodopsin membrane fraction is found. This fraction, which has been termed ‘brown’ membrane [ 131, does not show a crystalline lattice of protein molecules [ 141. Cells grown in the presence of nicotine ((nicotine’ cells) fail to synthesise retinal but continue to produce at a reduced level the protein bacteria-opsin [ 131. Such cells contain large amounts of lycopene, since nicotine inhibits the synthesis of retinal by blocking

Phase Transitions and Heterogeneity in Lipid Bilayers

The optical reflectivity of several well-characterized lipid bilayer systems has been correlated with calorimetric studies of the membrane components. There is a large increase in mean membrane thickness when a bilayer is cooled below the transition temperature of the membrane lipid. Similar studies on membranes generated from a mixture of two lipids possessing different degrees of unsaturation suggest that between the characteristic transition temperatures of the two lipids, the bilayer contains clusters of gel and liquid crystalline lipid which coexist within the plane of the membrane.

Protein mobility in membranes

The investigation of the mobility of components of biological membranes is currently attracting considerable attention and has recently been the subject of two detailed reviews [ 1,2], The possibility of rapid diffusion arises from the concept that many membranes contain regions of lipid bilayer which are predominantly in a liquid-crystalline, that is, fluid state [ 3 -8 ] . Proteins embedded in the lipid bilayer might be expected to exhibit relatively rapid rotational and translational diffusion. Of course, diffusion might also be restricted by protein-protein interactions in the membrane or by attachment to microfilaments or other assemblies within the cell [9]. The purpose of this review letter is to summarise measurements and observations of protein mobility in membranes. In addition, progress towards developing new techniques for measuring protein diffusion will be briefly described.

Polarised absorption spectroscopy of chlorophyll-lipid membranes

Abstract A technique has been developed for measuring visible absorption spectra of chlorophyll in lipid membranes. An expression is derived which enables the directions of the transition moments of the different absorption bands to be determined from polarisation data. It is found that the transition moments of the principal blue and red absorption bands of chlorophyll a make angles of 26° and 36.5° respectively with the plane of the membrane. On the assumption that these two transitions lie in the plane of the porphyrin ring and are mutually perpendicular, it may be deduced that the plane of the porphyrin ring is tilted at approx. 48° to the membrane surface. For chlorophyll b the transition moments of the blue and red bands are found to make angles of 29.5° and 36.5° with the plane of the bilayer, giving an angle of tilt of the porphyrin ring of approx. 51°. These results are compared with measurements of dichroism in vivo .