Spectroscopy on individual light-harvesting 1 complexes of Rhodopseudomonas acidophila

In this paper the fluorescence-excitation spectra of individual LH1-RC complexes (Rhodopseudomonas acidophila) at 1.2 K are presented. All spectra show a limited number of broad bands with a characteristic polarization behavior, indicating that the excitations are delocalized over a large number of pigments. A significant variation in the number of bands, their bandwidths, and polarization behavior is observed. Only 30% of the spectra carry a clear signature of delocalized excited states of a circular structure of the pigments. The large spectral variety suggests that besides site heterogeneity also structural heterogeneity determines the optical spectrum of the individual LH1-RC complexes. Further research should reveal if such heterogeneity is a native property of the complex or induced during the experimental procedures.     

Spectral diffusion of the lowest exciton component in the core complex from Rhodopseudomonas palustris studied by single-molecule spectroscopy

We have recorded fluorescence-excitation spectra from individual RC–LH1 complexes from Rhodopseudomonas palustris. The spectra feature a few broad bands accompanied by a sharp line at the low-energy side of the spectrum which is ascribed to the lowest exciton state of the BChl a assembly. Recording several fluorescence-excitation spectra from the same individual complex in rapid succession reveals that the linewidth of the lowest exciton transition is determined by spectral diffusion which increases for higher excitation energies.     

Single-Molecule Spectroscopy Unmasks the Lowest Exciton State of the B850 Assembly in LH2 from Rps. acidophila

We have recorded fluorescence-excitation and emission spectra from single LH2 complexes from Rhodopseudomonas (Rps.) acidophila. Both types of spectra show strong temporal spectral fluctuations that can be visualized as spectral diffusion plots. Comparison of the excitation and emission spectra reveals that for most of the complexes the lowest exciton transition is not observable in the excitation spectra due to the cutoff of the detection filter characteristics. However, from the spectral diffusion plots we have the full spectral and temporal information at hand and can select those complexes for which the excitation spectra are complete. Correlating the red most spectral feature of the excitation spectrum with the blue most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Hence, application of fluorescence-excitation and emission spectroscopy on the same individual LH2 complex allows us to decipher spectral subtleties that are usually hidden in traditional ensemble spectroscopy.     

Single-Molecule Spectroscopy Unmasks the Lowest Exciton State of the B850 Assembly in LH2 from Rps. acidophila

We have recorded fluorescence-excitation and emission spectra from single LH2 complexes from Rhodopseudomonas (Rps.) acidophila. Both types of spectra show strong temporal spectral fluctuations that can be visualized as spectral diffusion plots. Comparison of the excitation and emission spectra reveals that for most of the complexes the lowest exciton transition is not observable in the excitation spectra due to the cutoff of the detection filter characteristics. However, from the spectral diffusion plots we have the full spectral and temporal information at hand and can select those complexes for which the excitation spectra are complete. Correlating the red most spectral feature of the excitation spectrum with the blue most spectral feature of the emission spectrum allows an unambiguous assignment of the lowest exciton state. Hence, application of fluorescence-excitation and emission spectroscopy on the same individual LH2 complex allows us to decipher spectral subtleties that are usually hidden in traditional ensemble spectroscopy.     

Fluorescence-excitation spectra of acridine orange-DNA and -RNA systems

Summary The absorption, fluorescence and fluorescence-excitation spectra were investigated in free AO, AO-DNA and AO-RNA systems. In the case of Complex I, AO seems to be metachromatically bound to the two kinds of binding sites, phosphates neighboring purine bases and those neighboring pyrimidine bases, and becomes non-fluorescent.In the case of Complex II, the AO dimer originally present in solution dissociates into monomers and the monomer is intercalated between base pairs, resulting in the enhancement of fluorescence emission. These are suggested by the difference between the absorption band and the fluorescence-excitation spectrum normalized at the maximum to the absorption. The electronic processes of optical behaviours in free and bound AO are discussed on the basis of the emission properties at 77 °K.     

Discrimination of petroleum fluorescence spectra.

This paper presents studies of the total spectra (fluorescence-excitation matrix) of petroleum with regard to the utilization of fluorescence for determining petroleum pollutants. Thorough testing of one group, comprising almost forty lubricating oils in the form of their hexane solutions, points out their discrimination.     

Isolation and purification of the major photosynthetic antenna, fucoxanthin-Chl a/c protein, from cultured discoid germilings of the brown Alga, Cladosiphon okamuranus TOKIDA (Okinawa Mozuku)

A chlorophyll c binding membrane intrinsic light-harvesting complex, the fucoxanthin-chlorophyll a/c protein (FCP), was isolated from cultured discoid germilings of an edible Japanese brown alga, Cladosiphon (C.) okamuranus TOKIDA (Okinawa Mozuku in Japanese). The discoid germiling is an ideal source of brown algal photosynthetic pigment-protein complexes in terms of its size and easiness of cultivation on a large scale. Ion-exchange chromatography was crucial for the purification of FCP from solubilized thylakoid proteins. The molecular weight of the purified FCP assembly was estimated to be ~56?kDa using blue native-PAGE. Further subunit analyses using 2D-PAGE revealed that the FCP assembled as a trimer consisting of two distinguishable subunits having molecular weights of 18.2 (H) and 17.5 (L)?kDa. Fluorescence and fluorescence-excitation spectra confirmed that the purified FCP assembly was functionally intact.     

Investigation on chlorosomal antenna geometries: tube,lamella and spiral-type self-aggregates

Molecular mechanics calculations and exciton theory have been used to study pigment organization in chlorosomes of green bacteria. Single and double rod, multiple concentric rod, lamella, and Archimedean spiral macrostructures of bacteriochlorophyll c molecules were created and their spectral properties evaluated. The effects of length, width, diameter, and curvature of the macrostructures as well as orientations of monomeric transition dipole moment vectors on the spectral properties of the aggregates were studied. Calculated absorption, linear dichroism, and polarization dependent fluorescence-excitation spectra of the studied long macrostructures were practically identical, but circular dichroism spectra turned out to be very sensitive to geometry and monomeric transition dipole moment orientations of the aggregates. The simulations for long multiple rod and spiral-type macrostructures, observed in recent high-resolution electron microscopy images (Oostergetel et al., FEBS Lett 581:5435–5439, 2007) gave shapes of circular dichroism spectra observed experimentally for chlorosomes. It was shown that the ratio of total circular dichroism intensity to integrated absorption of the Q _y transition is a good measure of degree of tubular structures in the chlorosomes. Calculations suggest that the broad Q _y line width of chlorosomes of sulfur bacteria could be due to (1) different orientations of the transition moment vectors in multi-walled rod structures or (2) a variety of Bchl-aggregate structures in the chlorosomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hysteresis and conformational drift of pressure-dissociated glyceraldehydephosphate dehydrogenase

Pressure dissociation of yeast glyceraldehydephosphate dehydrogenase (GAPDH) was studied by fluorescence spectroscopy. Observations in the range of -5 to 30 degrees C indicate that monomer association into the tetramer proceeds with an enthalpy change of -14 kcal mol-1 and a large increase in entropy which at 25 degrees C amounts to 18 kcal mol-1. The large conformational drift and the low-temperature stability of the tetramer recovered after decompression facilitated a comparison of its properties with those of the native tetramer. Significant differences in absorption and fluorescence-excitation polarization spectra, yield of tryptophan fluorescence, and binding of anilinonaphthalenesulfonate and NADH were observed. At 0 degree C the standard free energies of association of the monomers into the native and drifted tetramers were respectively -32 and -29 kcal mol-1. The volume change upon association measured from the pressure span of the compression curves was 200-230 mL mol-1 but four times as large when derived from the displacement of the compression curves with total protein concentration. This large discrepancy can be explained by the existence in the native tetramer population of a distribution of free energies of association with a dispersion from the mean of about 6 kcal mol-1. At 0 degree C and 1 bar ATP and ADP decreased the stability of the GAPDH tetramer by changes in free energy of association of +3.7 and +4.1 kcal mol-1, respectively. NAD and c-AMP stabilized it by -2.3 and -1.3 kcal mol-1. The variation in sign and magnitude of the ligand-induced changes in free energy of association observed in this case, and previously in hexokinase [Ruan, K., & Weber, G. (1988) Biochemistry 27, 3295], and the heterogeneity of the free energy of association of GAPDH, revealed as indicated above, lead to the conclusion that oligomeric aggregates exist in a variety of conformations that depend upon the protein concentration, temperature, pressure, and the presence of specific ligands. The multiplicity of species revealed by the energetics raises questions about the significance of the structures of oligomeric proteins determined by X-ray crystallography.     

Spectroscopy on the B850 band of individual light-harvesting 2 complexes of Rhodopseudomonas acidophila. I. Experiments and Monte Carlo simulations

The electronic structure of the circular aggregate of 18 bacteriochlorophyll a (BChl a) molecules responsible for the B850 absorption band of the light-harvesting 2 (LH2) complex of the photosynthetic purple bacterium Rhodopseudomonas acidophila has been studied by measuring fluorescence-excitation spectra of individual complexes at 1.2 K. The spectra reveal several well-resolved bands that are obscured in the single, broad B850 band observed in conventional absorption measurements on bulk samples. They are interpreted consistently in terms of the exciton model for the circular aggregate of BChl a molecules. From the energy separation between the different exciton transitions a reliable value of the intermolecular interaction is obtained. The spectra of the individual complexes allow for a distinction between the intra- and the intercomplex disorder. In addition to the random disorder, a regular modulation of the interaction has to be assumed to account for all the features of the observed spectra. This modulation has a C(2) symmetry, which strongly suggests a structural deformation of the ring into an ellipse.     

13C-1H magnetic double-resonance study of fetal enamel matrix proteins

Recently developed ¹³C-¹H nuclear magnetic double-resonance techniques have been used to study proteins in the intact fetal enamel matrix. Enamel protein chains undergoing rapid, almost isotropic motion were detected in scalar decoupled ¹³C-nmr spectra, while motionally restricted enamel protein chains were principally observed in proton-enhanced spectra. The latter spectra were obtained using a matched Hartmann-Hahn contact to transfer polarization from protons to carbons (cross-polarization). Both mobile and motionally restricted enamel protein chains were observed in dipolar decoupled ¹³C-nmr spectra. A comparison of integrated intensities obtained from the scalar decoupled and dipolar decoupled spectra showed that 70% of the fetal enamel protein chains exhibit rapid, nearly isotropic molecular motion (τ ? 10^?6 sec), while the remaining 30% are rigid or undergo only anisotropic molecular motion.     

A study of subtilisin types Novo and Carlsberg by circular polarization of fluorescence.

The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.     

Fluorescence polarization studies on four biliproteins and a bilin model for phycoerythrin 545.

Fluorescence (excitation) polarization spectroscopy in the wavelength region of the bilin chromophores was applied to phycoerythrocyanin (CV-phycocyanin), phycocyanins 645 and 612, and phycoerythrin 545. The cryptomonad biliproteins - phycoerythrin 545 and phycocyanins 612 and 645 - were studied as both protein dimers having an alpha(2)beta(2) polypeptide structure and as alphabeta monomers. The cyanobacterial phycoerythrocyanin (CV-phycocyanin) was a trimeric oligomer. The changes in polarization across the spectrum were attributed to transfers of energy between bilins. Cryptomonad biliproteins are isolated as dimers. The similarities between their steady-state fluorescence polarization spectra and those of the corresponding monomers suggested that the monomers' conformations were analogous to the dimers. This supports the use of monomers in the study of dimer bilin organization. The unusual polarization spectrum of phycoerythrin 545 was explained using a model for the topography of its bilins. Obtaining the emission spectra of phycoerythrin 545 at several temperatures and a deconvolution of the dimer circular dichroism spectrum also successfully tested the bilin model. Circular dichroism spectroscopy was used to determine which polarization changes are formed by F?rster resonance energy transfers and which may be produced by internal conversions between high- and low-energy states of pairs of exciton-coupled bilins. Attempts were made to assign energy transfer events to the corresponding changes in fluorescence polarization for each of the four biliproteins.     

Deconvolution of C-phycocyanin beta-84 and beta-155 chromophore absorption and fluorescence spectra of cyanobacterium Mastigocladus laminosus.

Absorption and fluorescence spectra of the C-phycocyanin beta-subunit were quantitatively deconvoluted into component spectra of the beta-84 and beta-155 chromophores. The deconvolution procedure was based on a theoretical treatment of polarization properties. Four kinds of spectra (absorption, emission, emission polarization, and excitation polarization) measured on C-phycocyanin isolated from the cyanobacterium Mastigocladus laminosus were used as the experimental data set. Without any assumption of spectral shape, the absorption and fluorescence spectra of both chromophores were unambiguously resolved and their fluorescence quantum yields were evaluated. By combining the spectra of the alpha-subunit, independently measured, with the resolved spectra of the beta-subunit, the fluorescence and fluorescence polarization spectra and the fluorescence quantum yield of the monomer were estimated; they agree with experimental values to within an acceptable error. Further, the matrix of energy transfer rates in the monomer was estimated; it gave a significantly different result (by up to 40%) from previously estimated ones.     

Fluorescein excitation and emission polarization spectra in living cells: changes during the cell cycle.

Changes in the fluorescein fluorescence emission and excitation polarization spectra in synchronized cultured S3 fibroblasts at G1, mid-S, and mitosis, as well as in human lymphocytes before and after stimulation with mitogens, were studied. In contrast to those measured in aqueous solutions the emission and excitation polarization spectra in living cells exhibit a wavelength dependence characteristic for the state of the cell cycle. Changes in the temperature and in the amount of intracellular water result in quantitative wavelength-independent changes in the polarization spectra. Possible mechanisms for the qualitative wavelength-dependent changes in the fluorescein emission and excitation polarization spectra during the cell cycle are discussed.     

Spectroscopy of individual light-harvesting 2 complexes of Rhodopseudomonas acidophila: diagonal disorder, intercomplex heterogeneity, spectral diffusion, and energy transfer in the B800 band

This paper reports a detailed spectroscopic study of the B800 absorption band of individual light-harvesting 2 (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila at 1. 2 K. By applying single-molecule detection techniques to this system, details and properties can be revealed that remain obscured in conventional ensemble experiments. For instance, from fluorescence-excitation spectra of the individual complexes a more direct measure of the diagonal disorder could be obtained. Further spectral diffusion phenomena and homogeneous linewidths of individual bacteriochlorophyll a (BChl a) molecules are observed, revealing valuable information on excited-state dynamics. This work demonstrates that it is possible to obtain detailed spectral information on individual pigment-protein complexes, providing direct insight into their electronic structure and into the mechanisms underlying the highly efficient energy transfer processes in these systems.     

6-Aminohexanoic acid-plasminogen interactions studied by fluorescence

Circular polarization of luminescence spectra of human plasminogen and of its derivatives were measured in solutions of ligand-free proteins and with saturating amounts of 6-aminohexanoic acid. Spectroscopic changes induced by the ligand reveal similar perturbations of the binding sites in all the protein derivatives. It is concluded that the gross conformational change induced by 6-aminohexanoic acid binding to the native plasminogen involves changes of sterical relations of entire protein domains.     

Fluorescence polarization and spin-label studies of the fluidity of stromal and granal chloroplast membranes

The lipid fluidity of thylakoid membrane regions separated by Yeda press and sonication methods has been investigated using diphenylhexatriene fluorescence polarization measurements and rotational correlation times derived from the ESR spectra of the spin-labels 5-doxyldecane and 12-doxylstearate. According to both techniques, stromal lamellae vesicles with essentially only Photosystem I activity were more fluid than the granal membranes. The differences in lipid fluidity between the two fractions were interpreted in terms of the ratio of the amounts of protein compared to lipid in the membranes. Stromal lamellae fractions contained lower protein/lipid ratios compared with the granal membranes.     

Temperature dependence of protein globule polarization and electron transport in preparations of photosynthetic reaction centers from Rhodopseudomonas sphaeroides

Differential "light-minus-dark" spectra were obtained for reaction center (RC) preparations cooled in the light and in the dark at 77 K. The two types of preparations were found to display different spectral features in the spectral regions 760-770, 790-810, 880-990 nm. Differences in the spectra of the two types were found to exist in preparations cooled to temperatures below 120-100 K, whereas at temperatures above 130 K such differences were not observed. The observed spectral changes may be associated with the polarization processes occurring in the RC globule. Samples cooled in the light and in the dark show different temperature dependencies of the efficiency of electron transfer to the secondary quinone acceptor. The differences are irreversible after cooling to temperatures below 170 K, and reversible after cooling to 180-200 K. It is postulated that the observed kinetic changes are reflections of changes in the configuration of the acceptor complex of the RC. The possible existence of a correlation between the polarization processes in the protein globule and the structural configurations of the RC is discussed.