Orientation of the pigments in Photosystem II: low-temperature linear-dichroism study of a core particle and of its chlorophyll-protein subunits isolated from Synechococcus sp.

The orientation of the pigments in the Photosystem II core particle isolated from the thermophilic cyanobacterium Synechococcus sp. has been investigated by linear dichroism spectroscopy at 10 K of macroscopically oriented samples. The absorbance (A), linear dichroism (LD) and LD/A spectra are remarkably similar to those previously reported for a core complex isolated from Chlamydomonas reinhardtii (Biochim. Biophys. Acta 850 (1986) 156–161). The spectra of the Synechococcus core particle are compared to the corresponding spectra obtained on its two main constituent chlorophyll-protein complexes CP2-b (photochemically active) and CP2-c (photochemically inactive). The various features seen in the spectra of the core particle appear well segregated into the spectra of one or the other of the two subparticles without significant loss of orientation of the pigments. The orientation of the chlorophyll macrocycles, with the Y and X optical axis preferentially parallel and perpendicular to the plane of largest cross-section of the particle, respectively, is very similar in the two subparticles. CP2-b contains mainly the beta-carotene pool absorbing around 505 and 470 nm, which is oriented close to the membrane plane, while CP2-c contains the beta-carotene pool absorbing around 495 and 465 nm and oriented closer to the normal to the membrane plane. A shoulder at 682 nm in the absorbance and linear dichroism spectra of the core complex is fully segregated in the spectra of CP2-c, thus excluding the possibility that this spectral feature could be assigned to the primary donor of PS II. A negative linear dichroism component peaking around 691 nm (LD 691) in the core particle is mainly segregated in CP2-b together with the photoactive pheophytin acceptor molecule responsible for the 544 nm positive linear dichroism signal (LD 544). While the ratio of the amplitudes LD 691/LD 544 is approximately the same for the core particle and for the CP2-b complex, the amplitude of LD 691 is significantly reduced in CP2-b compared to the core particle.     

Structure of DNA within three isometric bacteriophages.

This paper describes a model for the structure of DNA contained in three morphologically similar bacteriophages--T7, P22 and phiCd-1--based on the transient electric dichroism of intact phage. The reduced dichroism of each of the phages at perfect orientation is within the range +0.12 to +0.19. Assuming that the phage orientation axis is that which passes from the apex through the tail, the measured dichroism suggests that DNA is wrapped in closely packed, co-axial solenoids with the axis of the solenoids tipped 43.5 degrees +/- 2.5 degrees from the orientation axis of the phage. All three phages show a large permanent dipole moment, with respective values of 5600, 200,000 and 500,000 Debye for T7, phiCd-1 and P22. The radius of the equivalent sphere for the three phages calculated from the rotational relaxation time for the rise of dichroism is in agreement with birefringence and electron microscope observations. The circular dichroism spectra of all three bacteriophages indicate that the local DNA helicity is similar in each case.     

Orientation of DNA in agarose gels.

An orientation of the lambda DNA during the electrophoresis in agarose gels was measured by a microscopic linear dichroism technique. The method involved staining the DNA with the dye ethidium bromide and measuring under the microscope the polarization properties of the fluorescence field around the electrophoretic band containing the nucleic acid. It was first established that the fluorescence properties of the ethidium bromide-DNA complex were the same in agarose gel and in a solution. Then the linear dichroism method was used to measure the dichroism of the absorption dipole of EB dye bound to lambda DNA. In a typical experiment the orientation of two-tenth of a picogram (2 x 10(-13)g) of DNA was measured. When the electric field was turned on, the dichroism developed rapidly and assumed a steady state value which increased with the strength of the field and with the size of DNA. A linear dichroism equation related the measured dichroism of fluorescence to the mean orientation of the absorption dipole of ethidium bromide and to an extent to which the orientation of this dipole deviated from the mean. The observed development of dichroism in the presence of an electric field was interpreted as an alignment of DNA along the direction of the field. The increase in the steady state value of dichroism with the rise in the strength of the field and with the increase of the size of DNA was interpreted as a better alignment of DNA along the direction of the field and as a smaller deviation from its mean orientation.     

Chromatin core particle unfolding induced by tryptic cleavage of histones.

Chromatin 'core particles' have been digested with trypsin to varying extents. The resulting particles are homogeneous by the criterion of ultracentrifuge boundary analysis. Sedimentation coefficients are lowered as cleavages are introduced into the histones, showing that an unfolding of the core particle occurs. This unfolding is further characterised by a lower melting temperature together with a premelting phase, higher molar ellipticity in the circular dichroism spectra at 280 nm and increased kinetics of digestion by both micrococcal nuclease and DNase I. Differences are also observed in the products of nuclease digestion. The most consistent interpretation of the data involves an unfolding process whereby free rods of DNA are released to extend from a nucleoprotein core.     

Flow orientation of brain microtubules studies by linear dichroism

Flow orientation of bovine brain microtubules has been studies using phase-modulation detected linear dichroism, LD, in a Couette cell with radial light propagation. LD could be sensitively measured in a wide flow gradient interval: 10^-3–10³ s^-1, without any apparent degradation of the microtubule structure. An extremely small flow gradient, 10^-3 s^-1 is sufficient to give significant orientation, and 10 s^-1 rapidly produced a very high degree of orientation. It is also shown that thermal convection effectively orients microtubules in vitro.The apparent linear dichroism is dominated by an anisotropic scattering from the aligned microtubules, superimposed on a weaker absorption dichroism due to intrinsic chromophores. The linear dichroism due to anisotropic turbidity, LD, is found to be an excellent tool for monitoring the formation of microtubules and in contrast to ordinary turbidity measurements, non-specific aggregates contribute to a far less extent.Time resolved LD was used to study the orientational relaxation of microtubules upon stopped shear. The relaxation towards random orientation can be described by a slow, multi-exponential decay. With increasing protein concentration the relaxation becomes slower and above approximately 1 mg/ml a fraction with a semipermanent orientation is formed. Finally, the development of orientation with time upon applying a small, constant gradient has been measured and the results are considered in terms of a model for flow orientation of rigid rods.     

Molecular Orientation in Quantasomes: III. A Flow Dichroism Apparatus and Its Application to the Study of the Structure of Spinach Quantasomes

A new apparatus is described for measuring dichroism spectra with very high sensitivity for macromolecular structures oriented in a hydrodynamic gradient. The method has been used to explore the dichroism spectrum of quantasome aggregates isolated from spinach chloroplasts. The quantasome flow dichroism resembles qualitatively that observed previously using electric field orientation, in that a pigment absorbing at wavelengths longer than 680 mμ exhibits appreciably greater dichroism than those absorbing at shorter wavelengths. It is shown that the absorption oscillator for this long wavelength absorption lies parallel to the streamlines of the sheer gradient, which is assumed to be the direction in which the planes of the chloroplast lamellae are oriented.     

Thermal transition of core particle is not a two-state process

Thermal transition of core particle which occurs before melting of DNA and can be followed by circular dichroism is not a two-state process; it is the result of two processes which cannot be dissociated in static experiments: unfolding of core particles is immediately followed by their aggregation. It is thus impossible to get thermodynamic parameters of core particle unfolding from its thermal transition monitored by circular dichroism. Thermal denaturation kinetics of core particles gives some information about their stability. Finally core particle structure is more stable in chromatin than in its isolated state.     

Orientation of gramicidin A transmembrane channel. Infrared dichroism study of gramicidin in vesicles.

Polarized infrared spectroscopy has been used to investigate the orientation of gramicidin A incorporated in dimyristoylphosphatidylcholine liposomes. Dichroism measurements of the major lipid (C = O ester, PO2-, CH2) and peptide (amide A, I, II) bands were performed on liposomes (with or without gramicidin) oriented by air-drying. The mean orientation of the lipid groups and of the pi LD helix chain in the gramicidin has been determined. It can be inferred from infrared frequencies of gramicidin that the dominant conformation of the peptide in liposomes cannot be identified to the antiparallel double-helical dimer found in organic solution. No shift in lipid frequencies was observed upon incorporation of gramicidin in the liposomes. However, a slight reorganization of the lipid hydrocarbon chains which become oriented more closely to the normal to the bilayer is evidenced by a change in the dichroism of the CH2 vibrations. The infrared dichroism results of gramicidin imply a perpendicular orientation of the gramicidin transmembrane channel with the pi LD helix axis at less than 15 degrees with respect to the normal to the bilayer.     

Cyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6301.

A procedure is described for the preparation of stable phycobilisomes from the unicellular cyanobacterium Synechococcus sp. 6301 (also known as Anacystis nidulans). Excitation of the phycocyanin in these particles at 580 nm leads to maximum fluorescence emission, from allophycocyanin and allophycocyanin B, at 673 nm. Electron microscopy shows that the phycobilisomes are clusters of rods. The rods are made up of stacks of discs which exhibit the dimensions of short stacks made up primarily of phycocyanin (Eiserling, F. A., and Glazer, A. N. (1974) J. Ultrastruct. Res. 47, 16-25). Loss of the clusters, by dissociation into rods under suitable conditions, is associated with loss of energy transfer as shown by a shift in fluorescence emission maximum to 652 nm. Synechococcus sp. 6301 phycobilisomes were shown to contain five nonpigmented polypeptides in addition to the colored subunits (which carry the covalently bound tetrapyrrole prosthetic groups) of the phycobiliproteins. Evidence is presented to demonstrate that these colorless polypeptides are genuine components of the phycobilisome. The nonpigmented polypeptides represent approximately 12% of the protein of the phycobilisomes; phycocyanin, approximately 75%, and allophycocyanin, approximately 12%. Spectroscopic studies that phycocyanin is in the hexamer form, (alpha beta)6, in intact phycobilisomes, and that the circular dichroism and absorbance of this aggregate are little affected by incorporation into the phycobilisome structure.     

Optical studies on complexes between DNA and pseudoisocyanine.

Linear dichroism (LD) results when pseudoisocyanine=PIC (1,1-diethy1-2,2-cyannine iodide) binds to flow-oriented DNA. LD may be used to follow the complexation both stoichmetrically and structurally, since when specified to unti complex concentration LD provides a measure of the average orientation of the absorbing transition dipole. Two different types of complexes can be distinguished: I. One strong, ionic-strength insensitive complex with monomeric PIC with an orientation indication intercalation. II. Several weaker complexes of electrostatic nature (only observed at I less than 0.2M Na Cl) among which those with dimeric (IIa) and with polymeric (IIb) PIC are concluded both from LD and from circular dichroism (CD). The dimer is probably formed by employing one intercalated PIC as a second site. The polymer complex is characterized by a very sharp absorption band at 553 nm polarized parallel to the DNA-axis (with positive LD and positive CL). The structure, a right-handed helical array of PIC molecules, is discussed in terms of exciton theory in relation to that of polymeric free PIC ("Scheibe polymer") which is also shown to interact with DNA (IIc) yielding a large aggregate which is degraded at a distinct flow force field...     

Orientation of intrinsic proteins in photosynthetic membranes. Polarized infrared spectroscopy of chloroplasts and chromatophores

In order to estimate the degree of orientation of the α-helices of intrinsic proteins in photosynthetic membranes, polarized infrared spectroscopy has been used to measure the dichroism of the amide I and amide II absorption bands of air-dried oriented samples of purple membranes, chloroplasts and chromatophores from Rhodopseudomonas sphaeroides. Using purple membrane, in which the orientation of the α-helices is precisely known (Henderson, R. (1977) Annu. Rev. Biophys. Bioeng. 6, 87–109), as a standard to calibrate our measurements and estimating the mosaic spread (extent of orientation) of the membranes from linear dichroism measurements performed in the visible spectral range, it is concluded that in photosynthetic membranes, the α-helices of intrinsic proteins are tilted at less than 40° with respect to the normal to the plane of the membrane.     

Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase.

Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.     

Electro-optical properties of nucleic acids and nucleoproteins. 3. Kramers-Kronig relationships in linear birefringence and dichroism. Application to a DNA-proflavine complex

The relation between the birefringence dispersion curve in a dichroic absorption band and the corresponding dichroism spectrum is given, using the Cauchy's theorem. The theory was checked on a DNA–proflavine complex with the aid of electro-optical measurements. An excellent agreement between the experimental results and the theoretical calculations was found. The birefringence and dichroism of the complex appear to be determined by the orientation of the macromolecule while no evidence for a contribution of the hyperpolarizabilities to the electro-optical effect is found. The importance of the dichroism correction in the calculation of the birefringence dispersion curve is emphasized.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls in nematic liquid crystals. I. Electric and weak magnetic field effects on the dichroism spectra

Dichroism spectra of chlorophyll a, chlorophyll b and bacteriochlorophyll a in various nematic liquid crystals are reported. The initial orientation of chlorophylls in such a sample is determined by the interaction of the aggregate formed from the pigment and the liquid crystal molecules with the electrode surface on the cell windows. Reorientation is carried out by either an electric or magnetic field. The analysis of the circular dichroism spectra obtained from these samples on the basis of the Mueller matrix shows that the intensity is predominantly related to the texture of the sample. Chlorophyll molecules can be aggregated with liquid crystals in two ways: (1) through the chlorin magnesium atom, which results in the liquid crystal chain being almost perpendicular to the porphyrin ring, or (2) attached parallel to the line connecting the first and third pyrrole rings of the chlorin, the chlorin now lying in the plane of the liquid crystal chains. By comparing the dichroism spectra of various chlorophylls in the same liquid crystal we can draw conclusions concerning the preferred type of aggregation, not only with liquid crystals, but also with biological molecules. These liquid crystal systems are models of the orientation effects found for chlorophyll in lamellae. The model studied in this work is much simpler than the lamellar system but it does exhibit several common properties with the latter. Both systems are anisotropic and show much more intense dichroism signals, often of opposite sign, compared with those observed for photosynthetic pigments in isotropic solutions. Dichroism signals of organism fragments are much more complex than those of our model, which can either be related to the occurrence in the organism of several types of pigments or, for a given type of pigment, could be the result of exciton splitting. On the basis of our model it is shown that small changes in the anisotropy of the pigment in the surroundings have a strong influence on the sign and amplitude of the observed circular dichroism signal. Such effects may be responsible for the structure of the dichroism spectra observed for biological samples. Such structures can be partially related to the superposition of the dichroism signal from various ‘domains’ of chromophore which are different in both pigment arrangement and in the anisotropy of the surroundings of the pigment molecules themselves.     

Electric dichroism and bending amplitudes of DNA fragments according to a simple orientation function for weakly bent rods

The linear dichroism is calculated for DNA fragments in their thermal bending equilibrium. These calculations are given for relatively short fragments, where bent molecules can be described by an arc model. Using the measured value of 350 A for the persistence length, the limit dichroism (corresponding to complete alignment) decreases due to thermal bending, e.g., for a fragment with 100 base pairs to 80% of the value expected for straight molecules. Thermal bending should lead to a strong continuous decrease of the dichroism with increasing chain length, which is not observed, however, in electric dichroism experiments due to electric stretching. The influence of the electric field on the bending equilibrium is described by a contribution to the bending energy, which is calculated from the movement of charge equivalents against the potential gradient upon bending. The charge equivalents, which are assigned to the helix ends, are derived from the dipole moments causing the stationary degree of orientation. By this procedure the energy term inducing DNA stretching is given for induced, permanent, and saturating induced dipole models without introduction of any additional parameter. The stationary dichroism at a given electric field strength is then calculated according to an arc model by integration over all angles of orientation of helix axes or chords with respect to the field vector, and at each of these angles the contribution to the dichroism is calculated by integration over all helices with different degrees of bending. Orientation functions obtained by this procedure are fitted to dichroism data measured for various restriction fragments. Optimal fits are found for an induced dipole model with saturation of the polarizability. The difference between orientation functions with and without electric stretching is used to evaluate dichroism bending amplitudes. Both chain length and field strength dependence of bending amplitudes are consistent with experimental amplitudes derived from the dichroism decay in low salt buffers containing multivalent ions like Mg2+, spermine, or [CoNH3)6]3+. Bending amplitudes can be used to evaluate the persistence length from electrooptical data obtained for a single DNA restriction fragment. Bending and stretching effects are considerable already at relatively low chain length, and thus should not be neglected in any quantitative evaluation of experimental data.     

Conformation and orientation of penetratin in phospholipid membranes.

The binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self-association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range approximately 50-500 microM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide-peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both alpha-helical and beta-turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the alpha-helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the beta-type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation.     

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.     

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.     

Electrooptical analysis of alpha-chymotrypsin at physiological salt concentration

The electric dichroism of alpha-chymotrypsin has been measured in a buffer containing 0.1 M Na(+), 10 mM Mg(2+) and 25 mM Tris-cacodylate pH 7.2. The reduced dichroism as a function of the electric field strength can be represented by the orientation function for permanent dipoles and is not consistent with the orientation function for induced dipoles. After correction for the internal directing field, the dipole moment is 1.1 x 10(-27) Cm (+/- 10%), corresponding to 340 D, at 20 degrees C. The assignment of the permanent dipole moment is confirmed by the shape of the dichroism rise curves, which require two exponentials with amplitudes of opposite sign for fitting. The dichroism decay time constants measured in the range of temperatures between 2 and 30 degrees C indicate a temperature induced change of the structure, which is equivalent to an increase of the hydrodynamic radius from r = 26.6 A at 2 degrees C to 28.5 A at 30 degrees C. Our results demonstrate that electrooptical investigations of proteins with a high time resolution can be extended to physiological salt concentrations without serious problems by use of appropriate instruments.     

Peptide-chain secondary structure of bacteriorhodopsin.

Ultraviolet circular dichroism spectroscopy in the interval from 190 to 240 nm and infrared spectroscopy in the region of the amide I band (1,600 cm-1 to 1,700 cm-1) has been used to estimate the alpha-helix content and the beta-sheet content of bacteriorhodopsin. Circular dichroism spectroscopy strongly suggests that the alpha-helix content is sufficient for only five helices, if each helix is composed of 20 or more residues. It also suggests that there is substantial beta-sheet conformation in bacteriorhodopsin. The presence of beta-sheet secondary structure is further suggested by the presence of a 1,639 cm-1 shoulder on the amide I band in the infrared spectrum. Although a structural model consisting of seven alpha-helical rods has been generally accepted up to this point, the spectroscopic data are more consistent with a model consisting of five alpha-helices and four strands of beta-sheet. We note that the primary amino acid sequence can be assigned to segments of alpha-helix and beta-sheet in a way that does not require burying more than two charged groups in the hydrophobic membrane interior, contrary to the situation for any seven-helix model.