Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy

Polarized, low-temperature Fourier transform infrared (FTIR) difference spectroscopy has been used to investigate the structure of bacteriorhodopsin (bR) as it undergoes phototransitions from the light-adapted state, bR570, to the K630 and M412 intermediates. The orientations of specific retinal chromophore and protein groups relative to the membrane plane were calculated from the linear dichroism of the infrared bands, which correspond to the vibrational modes of those groups. The linear dichroism of the chromophore C=C and C-C stretching modes indicates that the long axis of the polyene chain is oriented at 20-25 degrees from the membrane plane at 250 K and that it orients more in-plane when the temperature is reduced to 81 K. The polyene plane is found to be approximately perpendicular to the membrane plane from the linear dichroism calculations of the HOOP (hydrogen out-of-plane) wags. The orientation of the transition dipole moments of chromophore vibrations in the K630 and M412 intermediates has been probed, and the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the bR570----M412 transition has been measured.     

Anthracycline-DNA interactions studied with linear dichroism and fluorescence spectroscopy

DNA-binding geometry and dynamics of a number of anthracyclines, including adriamycin and 4-demethoxydaunorubicin, interacting with DNA have been studied by means of linear dichroism and fluorescence techniques. The anthracycline chromophore is found to be approximately parallel to the plane of the DNA bases and to have a restricted mobility, as would be expected for an intercalative binding mode, but there are variations between different directions in the chromophore as well as between the drugs. From dichroic spectra of adriamycin in an anisotropic host of poly(vinyl alcohol), absorption components corresponding to transitions with mutually orthogonal polarizations have been resolved. These can be exploited to determine the orientations of the two chromophore axes in the DNA complex relative to the DNA helix axis. In a certain binding regime the long axis of the bound anthracycline chromophores (with the exception of 4-demethoxydaunorubicin) is found to be approximately 10 degrees closer to perpendicular to the helix axis than are the DNA bases. This demonstrates that the average base tilt is at least 10 degrees. By contrast, the short axis of the aglycon moiety is found to be tilted some 20-30 degrees from perpendicular. This may be because it is probing a base direction with a more pronounced, static or dynamic, inclination than the average in DNA. The drug orientation and the DNA orientation (reflecting flexibility) are observed to vary differently and nonmonotonically with binding ratio, suggesting specific binding and varying site geometries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linear-dichroism spectroscopy for the study of structural properties of proteins

This review gives an experiment directed survey of the application of linear-dichroism (LD) spectroscopy to the study of proteins. LD spectroscopy is a relatively simple technique that provides information on the orientation of chromophores in molecules, on molecular characteristies such as shape, size and electronic properties, and on binding parameters in molecular complexes. Since LD is only observed when the molecules are non-randomly oriented in the sample, particular attention is paid to various orientation techniques, viz. in electric and flow fields, in polymer films and gels, and by light induction (photoselection). Examples are given on bacteriorhodopsin and retinals, chlorosomes, lens crystallins, aspartate aminotransferase, and the interaction of gene32-and recA-protein with DNA.Abbreviations AAT aspartate aminotransferase - BR bacteriorhodopsin - CD circular dichroism - LD linear dichroism - PLP pyridoxal-5-phosphate - ss single stranded - TDM transition dipole moment     

Chromophore reorientations in the early photolysis intermediates of bacteriorhodopsin.

The photoselection-induced time-resolved linear dichroism of a bacteriorhodopsin suspension of purple membrane from 350 to 750 nm is measured by a new pseudo-null measurement technique. In combination with time-resolved absorption measurements, these linear dichroism measurements are used to determine the reorientation of the retinal chromophore of bacteriorhodopsin from 50 ns to 50 microseconds after photolysis. This time range covers the times when the K photointermediate decays to form L, as well as the early times during the formation of the M intermediate in the photocycle. An analysis of the photoselection-induced linear dichroism measured directly, along with the absorbance changes polarized parallel to the linearly polarized excitation, shows that the anisotropy is invariant over this time period, implying that the photolyzed chromophore rotates less than 8 degrees C with respect to unphotolyzed chromophores during this part of the photocycle.     

Linear dichroism of rhodopsin in air-water interface films

Air-water interface films of purified cattle rhodopsin and defined phospholipids are formed by the osmotic lysis of reconstituted membrane vesicles. The interface films thus formed consist of a phospholipid monolayer containing vesicle membrane fragments. Rhodopsin molecules at the interface are restricted within the membrane fragments where they are spectrophotometrically intact and capable of undergoing photoregeneration and chemical regeneration. Multilayers of up to 8 layers can be built from these interface films. The visible absorption band of rhodopsin in these multilayers is linearly dichroic. Quantitative analysis of the linear dichroism reveals that the dipole moment of transition of the retinal chromophore in rhodopsin forms an angle of 15 degrees +/- 4 degrees with the plane of the membrane fragments in the interface film. This orientation of the chromophore relative to the plane of the membrane is essentially the same as that observed in the intact retina. Thus, the orientation of rhodopsin in the interface films is similar to that in the intact disc membranes.     

Release of the neocarzinostatin chromophore from the holoprotein does not require major conformational change of the tertiary and secondary structures induced by trifluoroethanol

Neocarzinostatin is a potent enediyne antitumor antibiotic complex in which a chromophore is noncovalently bound to a carrier protein. The protein regulates availability of the drug by proper release of the biologically active chromophore. To understand the physiological mechanism of the drug delivery system, we have examined the trifluoroethanol (TFE)-induced conformational changes of the protein with special emphasis on their relation to the release of the chromophore from holoneocarzinostatin. The effect of the alpha helix-inducing agent, TFE, on all the beta-sheet neocarzinostatin proteins was studied by circular dichroism, fluorescence, and (1)H NMR studies. By using binding of anilinonaphthalene sulfonic acid as a probe, we observed that the protein exists in a stable, partially structured intermediate state around 45-50% TFE, which is consistent with the results from tryptophan fluorescence and circular dichroism studies. The native state is stable until 20% TFE and is half-converted into the intermediate state at 30% TFE, which starts to collapse beyond 50%. High pressure liquid chromatographic analysis of the release of the chromophore caused by TFE treatment at 0 degrees C suggests that the release process, which occurs below 20% TFE, does not result from an observable conformational change in the protein. Kinetic measurements of the release of chromophore at 25 degrees C reveal that TFE does stimulate the rate of release, which increases sharply at 15% and reaches a maximum at 20% TFE, although no major secondary or tertiary structural change of the carrier protein is observed under these same conditions. Our data suggest that chromophore release results from a fluctuation of the protein structure that is stimulated by TFE. Complete release of the chromophore occurs at TFE concentrations where no overall observable unfolding of the apoprotein is seen. Thus, the results suggest that denaturation of the protein by TFE is not a necessary step for release of the tightly bound chromophore.     

Incorporation of photoreceptor membrane into a multilamellar film.

Multilamellar arrays of photoreceptor membrane up to 50 micrometer thick have been produced using a new method. Rhodopsin chromophore orientation in the films was studied using optical linear dichroism. The rhodopsin appears to be structurally intact and capable of photobleaching and regeneration. The production of biologically active liquid-crystal films offers a promising new approach to the study of biomembranes.     

Binding geometries of benzo[a]pyrene diol epoxide isomers covalently bound to DNA. Orientational distribution

Flow linear dichroism (LD) of different benzo[a]pyrene diol epoxide (BPDE) isomers covalently bound to calf thymus DNA or poly(dG-dC) provides information about binding geometry and DNA perturbation. With anti-BPDE the apparent angle between the long axis (z) of the pyrene chromophore and the DNA helix axis is approximately 30 degrees as evidenced from the LD of z-polarized absorption bands in the pyrenyl chromophore at 252 and 346 nm. The corresponding angle for the in-plane short axis (y) is determined to be approximately 70 degrees from a y-polarized band at 275 nm. The binding of (+)-anti-BPDE to DNA is found to cause a considerable reduction of the DNA orientation. This is ascribed to a decreased persistence length of DNA, owing either to increased flexibility ("flexible joints") or to permanent kinks at the points of binding. The reduced linear dichroism (LDr), i.e., the ratio between LD and isotropic absorbance, of the long-wavelength absorption band system of BPDE bound to DNA exhibits a wavelength dependence that indicates a relatively wide orientational distribution of the z axis of pyrene. Fluorescence data support the conclusion of a heterogeneous distribution, and a very low polarization anisotropy indicates a mobility between the different orientational states, which is rapid compared to the fluorescence lifetime (nanosecond time scale). Attempts are made to simulate the observed LDr features of the (+)-anti-BPDE-poly(dG-dC) complex using different distribution models on the assumption that the angular dependence of the spectral perturbation is due to dispersive interactions with DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation of emitting dipoles of chlorophyll A in thylakoids: considerations on the orientation factor in vivo.

Orientation angles of five emitting dipoles of chlorophyll a in thylakoids were estimated from low temperature fluorescence polarization ratio spectra of magnetically oriented chloroplasts. A simple expression is given also for the evaluation of data from linear dichroism measurements. It is shown that the Qy dipoles of chlorophylls lie more in the plane of the membranes and span a larger angular interval than was previously thought. Values for the orientation factor are calculated using various models corresponding to different degrees of local order of the Qy dipoles of chlorophylls in the thylakoid. We show that the characteristic orientation pattern of the Qy dipoles of chlorophylls in the membrane, i.e., increasing dichroism toward longer wavelengths, may favour energy transfer between the antenna chlorophylls as well as funnel the excitation energy into the reaction centers.     

Probing intramolecular orientations in rhodopsin and metarhodopsin II by polarized infrared difference spectroscopy.

The light-induced conformational changes of rhodopsin, which lead to the formation of the G-protein activating metarhodopsin II intermediate, are studied by polarized attenuated total reflectance infrared difference spectroscopy. Orientations of protein groups as well as the retinylidene chromophore were calculated from the linear dichroism of infrared difference bands. These bands correspond to changes in the vibrational modes of individual molecular groups that are structurally active during receptor activation, i.e., during the rhodopsin to metarhodopsin II transition. The orientation of the transition dipole moments of bands previously assigned to the carboxyl (C=O) groups of Asp83 and Glu113 has been determined. The orientation of specific groups in the retinylidene chromophore has been inferred from the dichroism of the bands associated with the polyene C-C, C=C, and hydrogen-out-of-plane vibrations. Interestingly, the use of polarized infrared light reveals several difference bands in the rhodopsin to metarhodopsin II difference spectrum which were previously undetected, e.g., at 1736 and 939 cm(-1). The latter is tentatively assigned to the hydrogen-out-of-plane mode of the HC(11)=C(12)H segment of the chromophore. Our data suggest a significant change in orientation of this group in the late phase of rhodopsin activation. On the basis of available site-directed mutagenesis data, bands at 1406, 1583, and 1736 cm(-1) are tentatively assigned to Glu134. The main features in the amide regions in the dichroic difference spectrum are discussed in terms of a slight reorientation of helical segments upon receptor activation.     

Reinterpretation of linear dichroism of chromatin supports a perpendicular linker orientation in the folded state

We present a reinterpretation of linear dichroism data for the salt induced condensation of chromatin. A conflict between electric and flow linear dichroism data for identical chromatin samples, studied at varying degrees of Mg2+ induced folding, can be solved if the orientation in electric fields is mainly determined through the polarization of counter ions along the linker parts, whereas the orientation in flow is governed by the hydrodynamical response of the entire chromatin fiber. The orientation of a chromatin fiber in an electric field would then depend on the linker tilt angle so that at an angle larger than 55 degrees the fiber would tend to orient perpendicular to the applied field. The different orientation distributions obtained with the two methods of alignment may in this way provide extra information about the structure and folding of chromatin.     

X-ray diffraction and infrared circular dichroism of DNA-violamycin B1 complexes

X-ray diffraction and infrared linear dichroism of oriented samples of DNA-violamycin B1 complexes have been studied at different antibiotic/DNA phosphate ratios (r) as a function of relative humidity. Violamycin B1 binds to DNA according to the intercalation as well as to the outside binding model. At low r values, where the intercalation predominates the unwinding angle of DNA helix is between 6 degrees and 12 degrees per intercalation site as followed from the dependence of the pitch of helix versus r. At r greater than or equal to 0.17 the intercalation sites are saturated and the outside binding becomes prevalent; however the violamycin B1 chromophore is still oriented in the plane of DNA bases. Conformational mobility of DNA in the violamycin B1 complexes is largely inhibited compared with pure DNA, but it is higher than that of the daunomycin complexes. At least 30% of DNA in violamycin complexes has A conformation at the medium humidities as followed by IR linear dichroism. In the case of x-ray diffraction the A conformation was not detected. The distance between DNA molecules in the complex is found to be 23.2 A, that is 2 A less than in pure DNA at the same conditions and it does not depend upon r.     

Linear dichroism of acrylodan-labeled tropomyosin and myosin subfragment 1 bound to actin in myofibrils.

Muscle contraction can be activated by the binding of myosin heads to the thin filament, which appears to result in thin filament structural changes. In vitro studies of reconstituted muscle thin filaments have shown changes in tropomyosin-actin geometry associated with the binding of myosin subfragment 1 to actin. Further information about these structural changes was obtained with fluorescence-detected linear dichroism of tropomyosin, which was labeled at Cys 190 with acrylodan and incorporated into oriented ghost myofibrils. The fluorescence from three sarcomeres of the fibril was collected with the high numerical aperture objective of a microscope and the dichroic ratio, R (0/90 degrees), for excitation parallel/perpendicular to the fibril, was obtained, which gave the average probe dipole polar angle, Theta. For both acrylodan-labeled tropomyosin bound to actin in fibrils and in Mg2+ paracrystals, Theta congruent to 52 degrees +/- 1.0 degrees, allowing for a small degree of orientational disorder. Binding of myosin subfragment 1 to actin in fibrils did not change Theta; i.e., the orientation of the rigidly bound probe on tropomyosin did not change relative to the actin axis. These data indicate that myosin subfragment 1 binding to actin does not appreciably perturb the structure of tropomyosin near the probe and suggest that the geometry changes are such as to maintain the parallel orientation of the tropomyosin and actin axes, a finding consistent with models of muscle regulation. Data are also presented for effects of MgADP on the orientation of labeled myosin subfragment 1 bound to actin in myofibrils.     

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

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.     

PM-SP-LB多层膜中BR分子的有序定向排列

用垂直转移法在石英片上制成的PM—SP—LB多层膜的可见区吸收谱表明吸收峰峰位与成膜液一致,均在574nm左右,比PM水悬浮液的吸收峰位略有红移;稳态线二色性表明,除PM碎片平躺在多层膜平面内外,在提拉时的竖直方向存在BR的取向优势,优势率约为0.51左右;同时还表明,25mN/m条件下制备的PM—SP—LB多层膜中BR分子的视黄醛生色团的跃迁矩与膜平面法向所成的角接近于天然紫膜中的值。     

Orientation of the protonated retinal Schiff base group in bacteriorhodopsin from absorption linear dichroism.

Linear dichroism experiments are performed on light-adapted bacteriorhodopsin (BR568) films containing native retinal (A1) and its 3,4-dehydroretinal (A2) analogue to measure the angle between the chromophore transition dipole moment and the membrane normal. QCFF/pi calculations show that the angle between the transition moment and the long axis of the polyene is changed by 3.4 degrees when the C3-C4 bond is unsaturated. The difference vector between the two transition moments points in the same direction as the Schiff base (N----H) bond for the all-trans BR568 chromophore. Because the plane of the chromophore is perpendicular to the membrane plane, a comparison of the transition moment orientations in the A1- and A2-pigments enables us to determine the orientation of the N----H bond with respect to the absolute chromophore (N----C5 vector) orientation. The angles of the transition moments are 70.3 degrees +/- 0.4 degrees and 67.8 degrees +/- 0.4 degrees for the A1- and A2-pigments, respectively. The fact that the change in the transition moment angle (2.5 degrees) is close to the predicted 3.4 degrees supports the idea that the chromophore plane is nearly perpendicular to the membrane plane. The decreased transition moment angle in the A2-analogue requires that the N----H bond and the N----C5 vector point toward the same membrane surface. Available results indicate that the N----C5 vector points toward the exterior in BR568. With this assignment, we conclude that the N----H bond points toward the exterior surface and its most likely counterion Asp-212. This information makes possible the construction of a computer graphics model for the active site in BR568.     

Light activates rotations of bacteriorhodopsin in the purple membrane

To investigate how a photoactivated chromophore drives the proton pump mechanism of bacteriorhodopsin, we have observed how the chromophore rotates during the photocyle. To do this, we examined the dichroism induced in aqueous suspensions of purple membrane fragments by flashes of linearly polarized light. We find that the flash stimulates both the photocycling chromophores and their noncycling neighbors to undergo large (greater than 10 degrees - 20 degrees) rotations within the membrane during the photocycle, and that these two chromophore populations undergo distinctly different sequences of rotations. All these rotations could be eliminated by glutaraldehyde fixation as well as by embedding unfixed fragments in polyacrylamide or agarose gels. Thus, in these immbolizing preparations the chromophore can photocycle without rotating inside a bacteriorhodopsin monomer by more than our detection limit of 2 degrees - 5 degrees. The large rotations we observed in aqueous suspensions of purple membranes were probably due to rotations of entire protein monomers. The process by which a photocycling monomer causes its noncycling neighbors to rotate may help explain the highly cooperative behavior bacteriorhodopsin exhibits when it is aggregated into crystalline arrays of trimers.     

Orientational changes of the absorbing dipole or retinal upon the conversion of rhodopsin to bathorhodopsin, lumirhodopsin, and isorhodopsin.

The orientational change of the absorbing dipole of the retinal chromophore in vertebrate rhodopsin (rhodo) upon photo-excitation to bathorhodopsin (batho), lumirhodopsin (lumi) and isorhodopsin (iso), has been studied by polarized absorption and linear dichroism measurements on magnetically oriented frog rod suspensions that were blocked at liquid nitrogen temperature. Both the azimuthal component delta theta and the polar component delta theta of the total angular change were studied in separate experiments. Delta theta was estimated from polarized absorption measurements on rods oriented transversally with respect to the analyzing beam. The data show unequivocally that upon the rhodo leads to batho transition, the dipole shifts out of the membrane plane by only few degrees; delta theta congruent to -3 degree. This azimuthal shift was nearly exactly reversed upon the batho leads to lumi decay. A very small shift (delta theta less than or equal to 1 degree) toward the membrane plane was observed upon a rhodo leads to iso conversion. The polar component delta theta of the angular shift was estimated by studying the photoreversion of linear dichroism induced by photo-excitation with polarized light in rods oriented parallel to the analyzing beam. Upon the rhodo leads to batho transition, ther was a shift delta theta = 11 +/- 3 degrees. The overall angular shift upon this first photo-exciting step, which corresponded to the isomerisation of retinal, was only delta omega = 11 +/- 3 degrees. This is smaller than what may be expected for a cis-trans isomerization of a retinal molecule with one end fixed, and different from what has been previously estimated by another group. These discrepancies are discussed.     

Linear dichroism of the complex between the gene 32 protein of bacteriophage T4 and poly(1,N6-ethenoadenylic acid)

We performed linear dichroism measurements in compressed polyacrylamide gels on the complex between the helix-destabilizing protein of bacteriophage T4, GP32 and poly(1,N6-ethenoadenylic acid), which is used as a model system for single-stranded DNA. A strong hyperchromism for poly(1,N6-ethenoadenylic acid) in the complex indicates a strongly altered conformation. The positive linear dichroism in the wavelength region where the bases absorb must be explained by a strong tilting of the bases in the complex. This finding is in accordance with results from earlier studies, using electric birefringence and circular dichroism measurements. Our measurements show that the angle between the bases and the local helix axis is 42(+/- 6)degrees. In addition, a pronounced contribution from the tryptophan residues of GP32 can be recognized, indicating that several of these residues have a specific orientation in the complex. The sign of the dichroism due to the tryptophan residues is the same as that due to the DNA bases. However, it is not sufficient to assume that all the observed dichroism is due to one or more intercalated tryptophan residues and there must be one or more additional tryptophan residues that make an angle of less than 40 degrees with the local helix axis. Some possible structures of the DNA-protein complex are discussed.     

Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres.

In muscle fibres labelled with iodoacetamidotetramethylrhodamine at Cys707 of the myosin heavy chain, the probes have been reported to change orientation when the fibre is activated, relaxed or put into rigor. In order to test whether these motions are indications of the cross-bridge power stroke, we monitored tension and linear dichroism of the probes in single glycerol-extracted fibres of rabbit psoas muscle during mechanical transients initiated by laser pulse photolysis of caged ATP and caged ADP. In rigor dichroism is negative, indicating average probe absorption dipole moments oriented more than 54.7 degrees away from the fibre axis. During activation from rigor induced by photoliberation of ATP from caged ATP in the presence of calcium, the dichroism reversed sign promptly (half-time 12.5 ms for 500 microM-ATP) upon release of ATP, but then changed only slightly during tension development 20 to 100 milliseconds later. During the onset of rigor following transfer of the fibre from an ATP-containing relaxing solution to a rigor medium lacking ATP, force generation preceded the change in dichroism. The dichroism change occurred slowly (half-time 47 s), because binding of ADP to sites within the muscle fibre limited its rate of diffusion out of the fibre. When ADP was introduced or removed, the dichroism transient was similar in time course and magnitude to that obtained after the introduction or removal of ATP. Neither adding nor removing ADP produced substantial changes in force. These results demonstrate that orientation of the rhodamine probes on the myosin head reflects mainly structural changes linked to nucleotide binding and release, rather than rotation of the cross-bridge during force generation.