Linear dichroism and the orientation of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films

Aqueous mixtures of reaction centers of Rhodopseudomonas sphaeroides and gelatin were dried to form thin films. Following hydration, these films were stretched as much as two to three times their original length. Polarized absorption spectra showing linear dichroism were obtained for both unstretched and stretched films, with the planes and stretching axes of the films mounted in various geometries relative to the electric vector of the measuring beam. These data were analyzed in terms of the following model: Reaction centers possess an axis of symmetry that is fixed in relation to the reaction center structure. In unstretched films this axis is confined to the film plane and oriented at random within the plane. In stretched films the symmetry axis is aligned with the direction of stretching. In both preparations reaction centers are distributed randomly with respect to rotation about the axis of symmetry. The data are consistent with this model when the analysis acknowledges less than perfect orientation. For perfect orientation in a stretched film the model predicts uniaxial symmetry about the axis of stretching. The approach to this condition was examined with films stretched to different extents. Extrapolation yielded dichroic ratios for the ideal case of perfect orientation, and allowed calculation of the angles between the axis of symmetry and the various optical transition dipoles in the reaction center. This treatment included the two absorption bands of the bacteriochlorophyll ‘special pair’ (photochemical electron donor) in the Q_x region, at 600 and 630 nm, which we were able to resolve in light minus dark difference spectra.     

Atomic force microscopy and force spectroscopy study of Langmuir-Blodgett films formed by heteroacid phospholipids of biological interest

Langmuir-Blodgett (LB) films of two heteroacid phospholipids of biological interest 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as a mixed monolayer with χ_POPC = 0.4, were transferred onto mica in order to investigate by a combination of atomic force microscopy (AFM) and force spectroscopy (FS) their height, and particularly, their nanomechanical properties. AFM images of such monolayers extracted at 30 mN m^− 1 revealed a smooth and defect-free topography except for the POPE monolayer. Since scratching such soft monolayers in contact mode was proved unsuccessful, their molecular height was measured by means of the width of the jump present in the respective force-extension curves. While for pure POPC a small jump occurs near zero force, for the mixed monolayer with χ_POPC = 0.4 the jump occurs at ∼ 800 pN. Widths of ∼ 2 nm could be established for POPC and χ_POPC = 0.4, but not for POPE monolayer at this extracting pressure. Such different mechanical stability allowed us to directly measure the threshold area/lipid range value needed to induce mechanical stability to the monolayers. AFM imaging and FS were next applied to get further structural and mechanical insight into the POPE phase transition (LC-LC′) occurring at pressures > 36.5 mN m^− 1. This phase transition was intimately related to a sudden decrease in the area/molecule value, resulting in a jump in the force curve occurring at high force (∼ 1.72 nN). FS reveals to be the unique experimental technique able to unveil structural and nanomechanical properties for such soft phospholipid monolayers. The biological implications of the nanomechanical properties of the systems under investigation are discussed considering that the annular phospholipids region of some transmembrane proteins is enriched in POPE.     

钝顶螺旋藻（Spirulina platensis）藻胆体Langmuir-Blodgett膜

从钝顶螺旋藻中分离制备完整藻胆体 ,然后滴加于空气 水界面上 ,应用LB膜技术制备藻胆体LB膜。结果表明 ,藻胆体在空气 水界面上具有很好的成膜性能。将藻胆体LB单层膜转移到刚揭开的云母表面 ,喷一层金 ,然后用扫描隧道显微镜观察。结果表明 ,藻胆体在Langmuir Blodgett膜中的排列方式与其在体内类囊体膜表面的排列方式类似 ,一排排聚集在一起 ,然后排列成膜。藻胆体的“核”吸附在云母表面 ,而藻胆体的“杆”伸向外面。由于钝顶螺旋藻易于规模化培养 ,藻胆体容易批量制备 ,加之藻胆体具有的独特的光物理、光化学特性和良好的成膜性能 ,以及本身就是纳米量级的颗粒 (5 0 70nm) ,预示着藻胆体在纳米光电子器件中具有很好的应用前景。     

Qualitative and quantitative analysis of the secondary structure of cytochrome C Langmuir-Blodgett films

A qualitative and quantitative analysis of the conformation of Langmuir-Blodgett (LB) dried films of cytochrome C on silicon wafers was performed by Fourier transform ir (FTIR) spectroscopy. A deconvolution procedure was applied to the amide I band analysis, in order to determine the percentage of the different secondary structures. Qualitative analysis was performed by examining difference spectra. Films obtained by spreading protein solutions at pH 7.4 and 1, dried at 25 and 100°C, on silicon wafers were also examined in order to detect spectral components associated with denatured protein domains, and to compare them with cytochrome C LB films. FTIR spectroscopy showed that the following important changes characterise LB film spectra: (a) the α-helix component is higher (its percentage is 57 and 54%) than the one estimated in dried film obtained by spreading the solutions at pH 7.4 on a silicon substrate (43%), (b) there is an increase in the intensity of bands attributed to protonated carboxy group bands, involved and not involved in the formation of hydrogen bonds, and a decrease in those attributed to deprotonated carboxy groups, (c) the intensity of several bands attributed to aromatic amino acids and aliphatic chains increases, and (d) bands due to O(SINGLEBOND)H stretching vibrations of crystallization water are present. These conformational changes could be induced by protein-protein interaction caused by the close packing of molecules that occurs during LB film formation; it cannot be excluded that they may be accompanied by partial changes in the tertiary structure of the protein. A preferential orientation of protein molecules in LB films is also a possibility. © 1997 John Wiley & Sons, Inc. Biopoly 42: 227–237, 1997     

Deposition behavior and photoelectrochemical characteristics of chlorophylla Langmuir-Blodgett films

The deposition behavior and photoelectric response characteristics of chlorophylla (Chla) monolayers and multilayers were investigated under various film fabrication conditions. Chla LB films were deposited onto quartz and pretreated ITO glass substrates under several fabrication conditions, including surface pressure and number of layers. The absorption spectra of Chla in a solution state and solid-like state (LB films) were fairly consistent with each other, and two absorption peaks were found at 678 and 438 nm, respectively. The prepared Chla LB films were set into an electrochemistry cell equipped with a Pt plate as the counter electrode, and the photoelectric response characteristics were obtained and analyzed relative to the light illumination. By considering the resulting photocurrents, the optimal fabrication conditions for Chla LB films were determined as 20 mN/m of surface pressure and 20 layers. The action spectrum of the Chla LB films was obtained in the visible region, and was found to be in good agreement with the absorption spectrum. The possible application of the proposed system as a constituent of an artificial color recognition device was suggested based on combining with the photoelectric conversion property of another lightsensitive biological pigment.     

Linear dichroism of bimolecular chlorophyll-lipid membranes. The role of anisotropy and dispersion

Polarised absorption and reflection spectra of chlorophyll-containing bimolecular lipid membranes were obtained in the spectral range of 590–710 nm. The spectra were analysed using the formalism of the complex dielectric tensor which characterizes the optical anisotropy of the membrane and the light absorption therein.The maxima of the absorption spectra recorded at a 45° angle of incidence are located at 665 and 670 nm for light in which the electric vector is oriented parallel and perpendicular, respectively, to the plane of incidence. The analysis of these spectra shows that the spectral shift is wholly due to the dispersion of the real part of the dielectric tensor.The angle between the dipole transition moment in the red and the normal to the membrane was estimated to be 42.3–45.3°.On the basis of these results, a model absorption spectrum, simulating the dichroic properties of oriented chloroplasts, was calculated for a system of parallel membranes.Some of the possible artifacts introduced into the dichroic spectra of chloroplasts due to anisotropy and dispersion are discussed.     

Linear and circular dichroism of membranes from Rhodopseudomonas capsulata

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala⁺) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala⁺ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.     

A Gaussian disorientation model for interpreting linear dichroism measurements of DNA-drug fibres and films

The optical linear dichroism of DNA-drug fibres and films can provide valuable information on the geometry of the binding and its extent, especially when used in conjunction with X-ray diffraction data from the same specimens. We have considered the macroscopic orientation of the helices within a fibre or film to be characterized by a Gaussian distribution of the helix axes about the fibre (or film) axis. Using this model we have obtained analytical expressions for the dichroic ratio without resorting to computer simulation techniques or numerical integration methods, and used them to interpret the results of experiments using DNA-phenanthridine fibres. As the humidity is increased, ethidium and dimidium bromide show an increased fraction of binding perpendicular to the helix axis, consistent with intercalation. Prothidium shows little preferred orientation in its binding, and the occurrence of a significant proportion of intercalation can be excluded.     

Fabrication and structural characterization of the Langmuir–Blodgett films from a new chitosan derivative containing cinnamate chromophores

A new amphiphilic chitosan derivative, octanoylchitosan cinnamate (OCC) was synthesized through regioselective modifications of chitosan. A solution of OCC was spread to water to form a stable monolayer at the air/water interface. The surface pressure (π)–area (A) isotherm indicated that the polymer had a limiting area of about 100 Å² per repeat unit. YZ-type multilayers were deposited onto hydrophobic substrates through Langmuir–Blodgett (LB) technique. The structural features of the LB films were investigated by UV absorption, circular dichroism (CD) and linear dichroism (LD) spectroscopy. The results showed that the intrinsic chirality originating from the helical order of the OCC backbones was maintained in the LB films. Besides, the polymer backbones were uni-axially oriented in the LB film. The ordered structures of OCC assembled in a dilute solution and in a cast film were also investigated and the results were compared with that of the LB film.     

Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

Linear dichroism of light harvesting bacteriochlorophyll proteins from Rhodopseudomonas sphaeroides in stretched polyvinyl alcohol films

Light-harvesting bacteriochlorophyll-protein complexes from Rhodopseudomonas sphaeroides 2.4.1 and R-26 mutant are solubilized in sodium dodecyl sulfate and imbedded in polyvinyl alcohol. Stretching induces orientation, and the linear dichroism of visible and near infrared absorption is analyzed. Based on a simple model, angles between the particle axis and the transition dipole moments are found. In the near infrared absorption band of the R-26 light-harvesting protein the dichroic ratio varies from 1.30 to 1.57. Using the absorption curves the band is resolved into two exciton components. In the visible absorption band the dichroic ratio has a constant value of 0.43 for the R-26 protein but varies with wavelength for the wild type light-harvesting protein. This variation is attributed to an additional bacteriochlorophyll not present in the R-26 protein.     

Macromolecular organization of chlorophyll a in aggregated chlorophyll ab protein complex as shown by circular dichroism at room and cryogenic temperatures

This report concerns the large circular dichroic (CD) signal of intact chloroplasts of higher plants. The CD spectra of chloroplasts are compared with the aggregated form of the light-harvesting chlorophyll $\text{a}\text{b}$ complex at 25°C and ?250°C. The light-harvesting chlorophyll aggregate has a CD of magnitude equal to or greater than chloroplasts, but of opposite sign, and it is not related to the CD of the unaggregated form, and hence its arrangement is an artefact compared to the arrangement in the chloroplast. We suggest that this preparation, which has pseudo-lamellar structure, is a clear example of a large CD signal being generated by macromolecular association. The asymmetry of organization in the chloroplast has an opposite sense to that of the aggregate, but affects only chlorophyll a, not chlorophyll b.     

Linear dichroism of microalgae,developing thylakoids and isolated pigment-protein complexes in stretched poly (vinyl alcohol) films at 77 K

A variety of unicellular algae, thylakoids from higher plants in different stages of maturity and isolated pigment-protein complexes were oriented in stretched polyvinyl alcohol films. Low temperature linear dichroism (LD) spectra of Chlorella pyrenoidosa and higher plant thylakoids in the films were very similar to those obtained after orientation of similar samples using magnetic or electric fields.Positive LD bands corresponding to Chl a (670) and (682) and negative bands due to Chl a (658) and Chl b (648) were resolved in spectra of the light harvesting Chl a/b protein. Chl b (648) and Chl a (658) and (670) were not seen in the LD spectrum of thylakoids from plants grown in intermittent light, the Chl b-less mutant of barley, Euglena gracilis or the cyanobacteria, Phormidium luridum and Anacystis nidulans, but did appear upon chloroplast maturation in Romaine lettuce and during the greening of etiolated and intermittent light plants. The highly oriented long wavelength Chl a (682) in the light-harvesting complex may represent residual PS II whose peak dichroism is centered at 681 nm. The PS I preparation had a Chl $\text{a}\text{b}$ ratio of approx. 6 and the LD spectrum was positive with a maximum at 690–694 nm and a band of lower amplitude at 652 nm. The minor LD band was not observed in PS I preparations from organisms that lack Chl b such as the cyanobacteria, intermittent light plants and the Chl b-less mutant of barley. We suggest that the 652 nm band is due to Chl b molecules associated with the antenna of PS I and are distinct from those on the light harvesting complex whose orientation is different. We also conclude that all the Chl a forms are oriented and that the long geometric axes of the pigment-protein complexes, as deduced from the configuration they assume in the stretched films, are axes that normally lie parallel to the plane of the native thylakoid.     

Isotope-labeled vibrational circular dichroism studies of calmodulin and its interactions with ligands

In this work we have studied ligand-induced secondary structure changes in the small calcium regulatory protein calmodulin (CaM) using vibrational circular dichroism (VCD) spectroscopy. We find that, due to its chiral sensitivity, VCD spectroscopy has increased ability over IR spectroscopy to detect changes in the structure and flexibility of secondary structure elements upon ligand binding. Moreover, we demonstrate that the uniform isotope labeling of CaM with (13)C shifts its amide I' VCD band by about approximately 43 cm(-1) to lower wavenumbers, which opens up a spectral window to simultaneously visualize a bound target protein. Therefore this study also provides the first example of how isotope labeling enables protein-protein interactions to be studied by VCD with good separation of the signals for both isotope-labeled and unlabeled proteins.     

Orientation of plgments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. IV. The 100 K linear dichroism spectra of thylakoids from wild-type and chlorophyll b-less barley thylakoids

Using a polyacrylamide gel squeezing technique, linear dichroism spectra of thylakoids from wild-type and chlorophyll-b less barley have been obtained at 100 K. The calculated difference linear dichroism spectra, based on normalization at 690–695 nm, are identical to those of the light-harvesting complex (LHC) isolated by Triton solubilization. This observation is in agreement with previous conclusions (Tapie, P., Haworth, P., Hervo, G. and Breton, J. (1982) Biochim. Biophys. Acta 682, 339–344) regarding: (i) scattering artifacts are absent in linear dichroism spectra determined using polyacrylamide gels, (ii) the in vivo orientation of LHC pigments is maintained in the isolated complex and (iii) the largest dimension(s) of the isolated LHC is (are), in vivo, parallel to the plane of the photosynthetic membrane.     

Mechanism of the light-state transition in photosynthesis: V. 77 K linear dichroism of Anacystis nidulans in State 1 and State 2

Linear-dichroism spectra of Anacystis nidulans at 77 K were determined for whole cells chemically fixed in light State 1 and light State 2. Whole cells were oriented by the squeezed gel technique using 5% gelatin 2.2 M sucrose gels. Peaks with positive dichroism were observed at 638 nm and 688 nm with shoulders at approx. 650 nm and 700 nm. The amplitude of the 650 nm shoulder was greater for cells in State 2 than those in State 1, and the State-2-minus-State 1 difference spectrum had a single peak at 656 nm. The linear dichroism spectrum of phycobilisomes isolated from A. nidulans showed peaks at 635 nm (phycocyanin) and 656 nm (allophycocyanin). The spectrum for thylakoid membranes free of phycobilisomes had one peak at 685 nm with a shoulder at 698 nm. We suggest that the change in dichroism at 656 nm between cells in State 1 and State 2 results from a change in orientation of the allophycocyanin core of the phycobilisome. This result is discussed in the context of our model for the light-state transition in phycobilisome-containing organisms.     

Orientation of the pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. III. A quantitative comparison of the low-temperature linear dichroism spectra of thylakoids and isolated pigment-protein complexes

The low-temperature linear dichroism spectrum of thylakoids oriented in polyacrylamide gel can be adequately described by a linear combination of the corresponding spectra of particles of light-harvesting complex, Photosystem I and Photosystem II, isolated by Triton X-100 extraction. The main conclusions which can be derived from this observation are: (1) The in vivo orientation of the pigments within each of the three complexes is not significantly affected by the extraction and purification procedures. (2) The various photosynthetic pigments are oriented roughly to the same extent in each of the three main biochemical constituents of the thylakoid. (3) All the complexes investigated behave like ellipsoids, the largest dimensions of which are lying in the plane of the photosynthetic membrane.     

Conformational stabilities, infrared, and vibrational dichroism spectroscopy studies of tris(ethylenediamine) zinc(II) chloride

The conformational stabilities of the transition metal complex of Zn (en)₃Cl₂ were studied using density functional theory (DFT). Deformational potential energy profiles (PEPs), and pathways between the different isomeric conformational energies were calculated using DFT/B3LYP/6–31G. The relative conformational energies of Δ(λλλ), Δ(λλδ), Δ(λδδ) and Δ(δδδ) are 10.48, 7.08, 3.56, and 0.0 kcal/mol, respectively, which are small compared to the barrier heights for reversible phase transitions (49.56, 49.55, 49.52 kcal/mol, respectively). Frequency assignment was carried out by decomposing Fourier transform infrared (FTIR) spectra using Gaussian and Gaussview. The theoretical IR and vibrational dichroism spectroscopy (VCD) absorption spectra are presented for all conformations within the range of 400–3,500 cm^-1.     

Orientation of pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. I. Determination of optimal conditions for linear dichroism measurement

The nature and possible causes of polarized light-scattering artefacts in linear dichroism measurements are investigated. Using criteria described in this article, the available orientation techniques have been critically assessed in order to obtain the linear dichroism spectra of thylakoids and of pigment-protein complexes isolated from pea. It is demonstrated here that the polyacrylamide gel squeezing technique of Abdourakhmanov et al. (Abdourakhmanov, I.A., Ganago, A.O., Erokhim, Yu.E., Solov'ev, A.A. and Chugunov, V.A. (1979) Biochim. Biophys. Acta 546, 183–186) does not lead to pigment degradation and that the linear dichroism spectra obtained in these conditions are essentially free of scattering artefacts. The linear dichroism spectra of light-harvesting complex isolated in different states of aggregation or incorporated into phospholipid vesicles are compared to the spectra of thylakoids. This comparison indicates: (1) that the isolation procedure of Burke et al. (Burke, J.J., Ditto, C.L. and Arntzen, C.J. (1978) Arch. Biochem. Biophys. 187, 252–263) leads to light-harvesting complex in which the in vivo orientation of pigments is preserved; (2) that the antenna chlorophyll a molecules of this complex have a significant degree of orientation with respect to the plane of the thylakoid.     

The orientations of reaction center transition moments in the chromatophore membrane of Rhodopseudomonas sphaeroides, based on new linear dichroism and photoselection measurements

Chromatophore membranes from Rhodopseudomonas sphaeroides were oriented by drying suspensions on the surfaces of glass slides. Polarized spectra of light-induced absorption changes were obtained between 500 and 1000 nm. As observed earlier, these spectra showed negative bands, reflecting photooxidation of the bacteriochlorophyll ‘special pair’ in the reaction centers, centered near 870, 810, 630 and 600 nm. These bands have been designated BY₁, BY₂, BX₁ and BX₂, respectively, corresponding to two Q_y transitions and two Q_x transitions of the dimeric special pair. We found the BY₁ and BX₁ transition moments to be parallel (within 20°) to the plane of the membrane, whereas the BX₂ moment makes an angle of 55–63° with the plane.Using the photoselection technique we found that the angle between the BY₁ and BX₁ transition moments is 30°, while that between BY₁ and BX₂ is 75°. The BX₁ and BX₂ moments were found to be orthogonal, consistent with the prediction of molecular exciton theory for a dimer.By combining these data, we have calculated the orientations of the transition moments of the bacteriochlorophyll dimer in spherical polar coordinates, with the pole of the coordinate system normal to the plane of the membrane. The orientations of the Q_y and Q_x transition moments of the two bacteriopheophytin molecules in the reaction center were also computed in this coordinate system by transforming the data reported by Clayton, C.N., Rafferty, R.K. and Vermeglio, A. ((1979) Biochim. Biophys. Acta 545, 58–68). We have derived the transformation equations for two polar coordinate systems: in one, the pole is an axis of symmetry as defined by the orientations of purified reaction centers in stretched gelatin films (Rafferty, C.N. and Clayton, R.K. (1979) Biochim. Biophys. Acta 545, 106–121). In the other, the pole is normal to the plane of the chromatophore membrane. These two polar axes are approximately orthogonal.