The functional role of protein in the organization of bacteriochlorophyll c in chlorosomes of Chloroflexus aurantiacus.

The preparation of five different fractions containing bacteriochlorophyll (Bchl) c and their absorption and circular dichroic properties have been described. The fractions investigated were purified chlorosomes, proteolytically modified chlorosomes, chlorosomes treated with lithium dodecyl sulfate (LDS) which were subsequently subjected to size-exclusion chromatography, in vitro Bchl c aggregates and, additionally, the so-called GEF chlorosomes [prepared according to Griebenow and Holzwarth (1989) Biochim. Biophys. Acta 973, 235-240]. Proteolysis of chlorosomes caused a 35-40% decrease in absorption intensity, a 6-8 nm blue shift of the 740-nm peak and, in particular, a drastic increase of rotational strength as revealed by CD spectroscopy. Although oligomeric Bchl c aggregates and LDS-treated chlorosomes had absorption characteristics similar to Bchl c in vivo, the data clearly indicated that protein, perhaps the chlorosome-specific Mr-3700 polypeptide, was involved in the organization of Bchl c in chlorosomes from C. aurantiacus. Furthermore, the results showed that the LDS-treated chlorosome fraction was most likely comprised of a micellar complex of Bchl c with LDS which represented an entity entirely different from chlorosomes.     

Circular dichroism of green bacterial chlorosomes

Positive and negative bands in previously measured circular dichroism (CD) spectra of Chlorobium limicola chlorosomes appeared to be sign-reversed relative to those of Chloroflexus aurantiacus chlorosomes in the 740–750 nm spectral region where bacteriochlorophyll (BChl) c absorbs maximally. It was not clear, however, whether this difference was intrinsic to the chlorosomes or was due to differences in the procedures used to prepare them. We therefore repeated the CD measurements using chlorosomes isolated from both Cb. limicola f. thiosulfatophilum and Cf. aurantiacus using the method of Gerola and Olson (1986, Biochim. Biophys. Acta 848: 69–76). Contrary to the earlier results, both types of chlorosomes had very similar CD spectra, suggesting that both have similar arrangements of BChl c molecules. The previously reported difference between the CD spectra of Chlorobium and Chloroflexus chlorosomes is due to the instability of Chlorobium chlorosomes, which can undergo a hypsochromic shift in their near infrared absorption maximum accompanied by an apparent inversion in their near infrared CD spectrum during isolation. Treating isolated chlorosomes with the strong ionic detergent sodium dodecylsulfate, which removes BChl a, does not alter the arrangement of BChl c molecules in either Chloroflexus or Chlorobium chlorosomes, as indicated by the lack of an effect on their CD spectra.Abbreviations BChl bacteriochlorophyll - Cb. Chlorobium - CD circular dichroism - Cf. Chloroflexus - NIR near infrared     

Low-temperature fluorescence from single chlorosomes, photosynthetic antenna complexes of green filamentous and sulfur bacteria

Fluorescence spectra of single chlorosomes isolated from a green filamentous bacterium (Chloroflexus (Cfl.) aurantiacus) and a green sulfur bacterium (Chlorobium (Cb.) tepidum) were measured by using a confocal laser microscope at 13 K. Chlorosomes were frozen either in a liquid solution (floating chlorosome) or on a quartz plate after being adsorbed (adsorbed chlorosome). Fluorescence peak wavelengths were shorter for the adsorbed single chlorosomes than for the floating ones. Single floating Cfl. chlorosomes showed a distribution of fluorescence peak positions having a center at 759.0 nm with a full width at half maximum of 6.3 nm. Single floating Cb. chlorosomes showed a 782.7 nm center with a full width at half-maximum of 3.4 nm. The distribution shifted to the blue and became wider with increasing temperature, especially in Cb. chlorosomes, suggesting a large excitonic density of states just above the lowest level. Energy transfer from BChl-c aggregates to BChl-a molecules in the baseplate proteins was observed in the floating chlorosomes but not in the adsorbed ones. A positive correlation was found between the peak wavelength of BChl-c fluorescence and the intensity of BChl-a fluorescence in single Cfl. chlorosomes. The results suggest that the BChl-c aggregates with longer wavelengths of the fluorescence peaks have a more efficient F?rster-type energy transfer to the baseplate BChl-a.     

Bacteriochlorophyll organization and energy transfer kinetics in chlorosomes from Chloroflexus aurantiacus depend on the light regime during growth

We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 mol m^–2 s^–1) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the Q_Y band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194–202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - DAS decay associated spectrum - PMSF phenylmethylsulfonyl fluoride     

Long-range organization of bacteriochlorophyll in chlorosomes of Chlorobium tepidum investigated by cryo-electron microscopy

Intact chlorosomes of Chlorobium tepidum were embedded in amorphous ice layers and examined by cryo-electron microscopy to study the long-range organization of bacteriochlorophyll (BChl) layers. End-on views reveal that chlorosomes are composed of several multi-layer tubules of variable diameter (20-30 nm) with some locally undulating non-tubular lamellae in between. The multi-layered tubular structures are more regular and larger in a C. tepidum mutant that only synthesizes [8-ethyl, 12-methyl]-BChl d. Our data show that wild-type C. tepidum chlorosomes do not have a highly regular, long-range BChl c layer organization and that they contain several multi-layered tubules rather than single-layer tubules or exclusively undulating lamellae as previously proposed.     

Studies of the location and function of isoprenoid quinones in chlorosomes from green sulfur bacteria

The chlorosome antenna of the green sulfur bacterium Chlorobium tepidum essentially consists of aggregated bacteriochlorophyll (BChl) c enveloped in a glycolipid monolayer. Small amounts of protein and the isoprenoid quinones chlorobiumquinone (CK) and menaquinone-7 (MK-7) are also present. Treatment of isolated chlorosomes from Cb. tepidum with sodium dodecyl sulfate (SDS) did not affect the quinones, demonstrating that these are located in a site which is inaccessible to SDS, probably in the interior of the chlorosomes. About half of the quinones were removed by Triton X-100. The non-ionic character of Triton probably allowed it to extract components from within the chlorosomes. MK-10 in chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus was likewise found to be located in the chlorosome interior. The excitation transfer in isolated chlorosomes from Cb. tepidum is redox-regulated. We found a ratio of BChl c fluorescenceintensity under reducing conditions (Fred) to that under oxidizing conditions (Fox) of approximately 40. The chlorosomal BChl a fluorescence was also redox-regulated. When the chlorosomal BChl c–BChl c interactions were disrupted by 1-hexanol, the BChl c Fred/Fox ratiodecreased to approximately 3. When CK and MK-7 were extracted from isolated chlorosomes with hexane, the BChl c Fred/Fox ratio also decreased to approximately 3. A BChl c Fred/Fox ratio of 3–5 was furthermore observed in aggregates of pure BChl c and in chlorosomes from Cfx. aurantiacus which do not contain CK. We therefore suggest that BChl c aggregates inherently exhibit a small redox-dependent fluorescence (Fred/Fox 3) and that the large redox-dependent fluorescence observed in chlorobial chlorosomes (Fred/Fox 40) is CK-dependent.     

Orientation and excitonic interactions of the Fenna—Matthews—Olson bacteriochlorophyll a protein in membranes of the green sulfur bacterium Chlorobium tepidum

Linear and circular dichroism spectra of isolated bacteriochlorophyll a proteins (FMO proteins) and membrane vesicles containing FMO protein from the green sulfur bacterium Chlorobium tepidum were measured at room temperature and 77 K. The orientation of membranes and isolated FMO protein was obtained by gel squeezing. Linear dichroism (LD) data indicate that isolated FMO protein and membrane vesicles associated with the FMO protein are oriented in a similar way in a squeezed polyacrylamide gel. Both samples show a characteristic negative LD band around 814 nm with flanking positive bands at 802 and 824 nm ascribed to the Q_y excitonic transitions of BChl a of the FMO protein. This confirms that the C3 symmetry axis of the trimer is perpendicular to the membrane plane, which is supported by the model of the disc-like structure of FMO protein trimers of Cb. tepidum [Li Yi-Fen, Zhou W, Blankenship RE, and Allen JP (1997) J Mol Biol 272: 456–471]. The LD data are consistent with either BChl 3 or 6, but not 7 as the principal contributor to the low temperature band at 825 nm. The low temperature linear and circular dichroism spectra of FMO protein trimers from Chlorobium tepidum show significant differences from the low temperature LD and CD spectra of FMO protein trimers from Prosthecochloris aestuarii. The data are interpreted in terms of somewhat different pigment-protein and pigment-pigment interactions in the two complexes.     

Aggregation of bacteriochlorophyll c homologs to dimers,tetramers, and polymers in water-saturated carbon tetrachloride

Three homologs of BChl c, 2-(R)-(1-hydroxyethyl)-4-n-propyl-5-ethyl-farnesyl BChl c (PEF-BChl c), 2-(R)-(1-hydroxyethyl)-4-ethyl-5-ethyl-farnesyl BChl c (EEF-BChl c), and 2-(S)-(1-hydroxyethyl)-4-isobutyl-5-methyl/ethyl-farnesyl BChl c (iBM/EF-BChl c), formed aggregates in water-saturated carbon tetrachloride (H₂O-satd CCl₄). The water content was about 100 times higher than that of the dried CCl₄ previously used. Absorption spectra were recorded for 8 concentrations for the three homologs of BChl c and were deconvoluted in terms of standard spectra of monomer, dimer, tetramer and polymer (747-nm aggregate, Olson and Pedersen (1990) Photosynthe Res 25: 25). PEF- and EEF-BChl c formed dimers (680 nm maximum) and tetramers (705–710 nm maximum), but iBM/EF-BChl c formed polymers. Inhibition of dimer formation by water faciliated the study of the initial stages of the polymerization of BChl c. When the logarithm of polymer concentration was plotted versus the logarithm of the monomer concentration for iBM/EF-BChl c, the initial slope was 30±10 and indicated the cooperation of 20–40 BChl c molecules to form a polymer from a seed. Circular dichroism spectra of the polymers with positive and negative bands at 743 and 760 nm, respectively, were similar to those for chlorosomes (Brune et al. (1990) Photosynth Res 24: 253).Abbreviations BChl bacteriochlorophyll - CD circular dichroism - EEF 4-ethyl-5-ethyl farnesyl - iBM/EF 4-isobutyl-5-methyl/ethyl farnesyl - H₂O-satd CCl₄ water saturated carbon tetrachloride - PEF 4-n-propyl-5-ethyl farnesyl     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

Aggregation of 8,12-diethyl farnesyl bacteriochlorophyll c at low temperature

The effect of temperature on the aggregation of 3^lR-8,12-diethyl farnesyl bacteriochlorophyll c in a mixture of n-pentane and methylcyclohexane (1/1, v/v) was studied by means of absorption, circular dichroism and fluorescence spectroscopy. At room temperature essentially only two aggregate species, absorbing at 702 nm (A-702) and 719 nm (A-719), were present. Upon cooling to 219 K, A-702 was quantitatively converted to A-719. Further lowering of the temperature led to the stepwise formation of larger aggregates by the conversion of A-719 to aggregate species absorbing at 743 nm (A-743) and 755 nm (A-755). All absorption changes were reversible. A-719 was highly fluorescent (maximum at 192 K: 744 nm), while A-743 and especially A-755 were weakly fluorescent. Below 130 K the mixture solidified, and no major changes in the absorption spectrum were observed upon further cooling. At 45 K, however, a relatively strong emission at 775 nm was observed. Below 200 K, the absorption, fluorescence and circular dichroism spectra resembled that of the chlorosome. These results open up the possibility to study higher aggregates of BChl c as models for the chlorosome by various methods at low temperature, thus avoiding interference by thermal processes.Abbreviations A-680, A-702, A-719, A-743 and A-755- BChl c aggregates absorbing at the wavelengths indicated - BChl- bacteriochlorophyll - R[E,E] BChl c _F- the 3¹ R isomer of 8,12-diethyl BChl c esterified with farnesol (F), analogously - M- methyl - Pr- propyl - S- stearol (see Smith 1994) - CD- circular dichroism     

4-Isobutyl homologs of farnesyl bacteriochlorophyll c in carbon tetrachloride

Absorption (ABS) and circular dichroism (CD) spectra were recorded for 3 concentrations (2.3, 19 and ca. 75 M) of 4-isobutyl homologs. Monomer spectra were the same as those for 4-n-propyl-5-ethyl farnesyl bacteriochlorophyll c. Pure polymer spectra were obtained by subtracting the 2.3-M spectra appropriately scaled from the ca. 75-M spectra. The polymer showed an ABS peak at 742 nm and a CD trough at ca. 742 nm. These properties are in harmony with the aggregate model proposed by Smith KM, Kehrs LA and Fajer J (1983, J Am Chem Soc 105: 1387–1389). A log-logplot of absorbance at 742 nm vs. monomer concentration could be fitted by a straight line of slope 1.6.Abbreviations ABS absorbance - BChl bacteriochlorophyll - CD circular dichroism - iBM/EF 4-isobutyl-5-methyl/ethyl farnesyl - PEF 4-n-propyl-5-ethyl farnesyl     

The organization of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus and the structural role of carotenoids and protein

The organization of bacteriochlorophyll c (BChl c) molecules was studied in normal and carotenoid-deficient chlorosomes isolated from the green phototrophic bacterium Chloroflexus aurantiacus. Carotenoid-deficient chlorosomes were obtained from cells grown in the presence of 60 µg of 2-hydroxybiphenyl per ml. At this concentration, BChl c synthesis was not affected while the formation of the 5.7 kDa chlorosome polypeptide was inhibited by about 50% (M. Foidl et al., submitted). Absorption, linear dichroism and circular dichroism spectroscopy showed that the organization of BChl c molecules with respect to each other as well as to the long axis of the chlorosomes was similar for both types of chlorosomes. Therefore, it is concluded that the organization of BChl c molecules is largely independent on the presence of the bulk of carotenoids as well as of at least half of the normal amount of the 5.7 kDa polypeptide. The Stark spectra of the chlorosomes, as characterized by a large difference polarizability for the ground- and excited states of the interacting BChl c molecules, were much more intense than those of individual pigments. It is proposed that this is caused by the strong overlap of BChl c molecules in the chlorosomes. In contrast to individual chlorophylls, BChl c in chlorosomes did not give rise to a significant difference permanent dipole moment for the ground- and excited states. This observation favors models for the BChl c organization which invoke the anti-parallel stacking of linear BChl c aggregates above those models in which linear BChl c aggregates are stacked in a parallel fashion. The difference between the Stark spectrum of carotenoid-deficient and WT chlorosomes indicates that the carotenoids are in the vicinity of the BChls.     

Monomers,dimers, and tetramers of 4-n-propyl-5-ethyl farnesyl bacteriochlorophyll c in dichloromethane and carbon tetrachloride

Absorption (ABS) and circular dichroism (CD) spectra were recorded for 6 concentrations (2.0–290 M) of bacteriochlorophyll (BChl) c in each solvent. Monomer spectra were obtained by adding methanol (1:200) to each sample. The monomer showed an ABS peak and a CD trough at 664 nm in CH₂Cl₂ (ABS peak at 665 nm in CCl₄). Dimer-plus-monomer spectra were obtained by subtracting high concentration (e.g., 290 M) spectra appropriately scaled from lower concentration (e.g., 26 M) spectra. Pure dimer spectra were then obtained by subtracting monomer spectra appropriately scaled from dimer-plus-monomer spectra. The dimer showed an ABS peak at 679 nm in both CH₂Cl₂ and CCl₄ and a CD trough at ca. 670 nm in CH₂Cl₂. The optical properties of the dimer do not agree with the model for bacteriochlorophyllide d [Smith KM, Bobe FW, Goff DA and Abraham RJ (1986) J Am Chem Soc 108: 1111–1120]. Higher aggregate spectra were obtained by subtracting appropriately scaled monomer and dimer spectra from high concentration (e.g., 290 M) spectra. The aggregate showed ABS shoulders at ca. 636 and 678 nm with a peak at 702 nm in CH₂Cl₂ and at 708 nm in CCl₄; the CD spectrum in either solvent showed peaks at 638 and 679 nm with troughs at 658 and ca. 710 nm. These spectra are consistent with an excitonic interaction between 4 chromophores in the aggregate. Each of the 12 original ABS spectra was deconvoluted in terms of the appropriate monomer, dimer and aggregate spectra, and the concentrations of each component were determined. Plots of log aggregate concentration vs. log dimer concentration lay on or near a line of slope 1.9 for CH₂Cl₂ and on or near a line of slope 2.1 for CCl₄. The aggregate was thus shown to be a tetramer. The theoretical relationship between dimers and monomers (slope 2.0) was not observed in all cases.Abbreviations ABS absorbance - BChl bacteriochlorophyll - CD circular dichroism - Chl chlorophyll - DNS data not shown - PEF 4-n-propyl-5-ethyl farnesyl     

Deracemization of bilirubin as the marker of the chirality of micellar aggregates

The deracemization of bilirubin in micellar aggregates of structurally correlated chiral surfactants was studied by circular dichroism experiments and exploited as the marker of the expression of chirality of the aggregates. The obtained results suggest that the hydrophobic interactions control the transfer of chirality from the monomers to the aggregates, and that different regions of the same aggregate might feature opposite enantiorecognition capabilities.     

Excitation energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus: A specific effect of 1-hexanol on the optical properties of baseplate and energy transfer processes

The effect of 1-hexanol on spectral properties and the processes of energy transfer of the green gliding photosynthetic bacterium Chloroflexus aurantiacus was investigated with reference to the baseplate region. On addition of 1-hexanol to a cell suspension in a concentration of one-fourth saturation, a specific change in the baseplate region was induced: that is, a bleach of the 793-nm component, and an increase in absorption of the 813-nm component. This result was also confirmed by fluorescence spectra of whole cells and isolated chlorosomes. The processes of energy transfer were affected in the overall transfer efficiency but not kinetically, indicating that 1-hexanol suppressed the flux of energy flow from the baseplate to the B806-866 complexes in the cytoplasmic membranes. The fluorescence excitation spectrum suggests a specific site of interaction between bacteriochlorophyll (BChl) c with a maximum at 771 nm in the rod elements and BChl a with a maximum at 793 nm in the baseplate, which is a funnel for a fast transfer of energy to the B806-866 complexes in the membranes. The absorption spectrum of chlorosomes was resolved to components consistently on the basis, including circular dichroism and magnetic circular dichroism spectra; besides two major BChl c forms, bands corresponding to tetramer, dimer, and monomer were also discernible, which are supposed to be intermediary components for a higher order structure. A tentative model for the antenna system of C. aurantiacus is proposed.Abbreviations A670 a component whose absorption maximum is located at 670 nm - (B)Chl (bacterio)chlorophyll - CD circular dichroism - F675 a component whose emission maximum is located at 675 nm - FMO protein Fenna-Mathews-Olson protein - LD linear dichroism - LH light-harvesting - McD magnetic circular dichroism - PS photosystem - RC reaction center     

Circular Dichroism Studies on Intermolecular Interactions of Amphotericin B in Ionic Liquid‐Rich Environments

Aggregation of amphotericin B (AmB) in an ionic liquid‐rich environment was investigated using circular dichroism (CD) spectroscopy. It was found that nature of the ionic liquids’ anion had a strong impact not only on the aggregation of AmB, but more importantly on the nature of AmB aggregates, as observed in the asymmetry of the exciton couplet of the aggregate in CD spectra. Unique CD signals for AmB aggregates were observed in three different 1‐butyl‐3‐methylimidazolium ionic liquid solutions: [C₄‐mim]Br favored the formation of AmB aggregates that were similar to those found in water, whereas [C₄‐mim]BF₄ and [C₄‐mim]NO₃ produced AmB aggregates that were different from each other and those found in water. The obtained results suggest that the designer solvent ability of ionic liquids could be expanded to address numerous intermolecular processes. Chirality 25:487‐492, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

Absorption flattening in the circular dichroism spectra of small membrane fragments

The inhomogeneous distribution of chromophore occurring in a particulate suspension can result in a reduction in the apparent molar ellipticity recorded in circular dichroism (CD) spectra. The possibility of such a systematic error has often been a matter of concern when CD spectra of cell membrane proteins are recorded. The recent publication of CD spectra for bacteriorhodopsin in native and sonicated membranes, in detergent-solubilized form, and reconstituted into small unilamellar vesicles [Mao, D., & Wallace, B. A. (1984) Biochemistry 23, 2667-2673] gives a unique opportunity to apply the theoretical analysis of Gordon and Holzwarth [Gordon, D. J., & Holzwarth, G. (1971) Arch. Biochem. Biophys. 142, 481-488] so as to provide a definitive answer to the question of whether absorption flattening is significant for membrane particles. We show here that the data of Mao and Wallace can be combined with the theoretical analysis of Gordon and Holzwarth to rule out significant absorption flattening effects over the range 200-240 nm for submicrometer-sized membranes. In addition, the results show that absorption flattening can be disregarded even at 190 nm for membranous material in the size range below 100 nm. The demonstration that there are no major flattening effects in the CD spectra of bacteriorhodopsin, particularly in the region of 200-240 nm, means that the experimental spectra are incompatible with the proposal that this transmembrane protein contains seven transmembrane helices.     

An ochre suppressor of bacteriophage T4 that is associated with a transfer RNA

Ultraviolet circular dichroism spectra have been obtained for native and heat-denatured Drosophila virilis satellite DNAs I, II and III. Gall &; Atherton (1974) have found that these DNAs have simple, unique sequences. We compare here the circular dichroism spectra of these satellite sequences with the circular dichroism spectra of synthetic DNAs of simple sequences which are combined in first-neighbor calculations. We also apply an analytical procedure for determining nearest-neighbor frequencies from the DNA spectra (Allen et al., 1972). The results are an indication of the potential usefulness and present limitations of circular dichroism measurements in confirming or determining the nearestneighbor frequencies of satellite DNAs of simple sequences.     

Chirality of camphor derivatives by density functional theory

Infrared (IR) and vibrational circular dichroism (VCD) spectra of chiral camphor, camphorquinone and camphor-10-sulfonic acid (CSA), known as standard compounds for electronic circular dichroism (ECD) spectroscopy, are measured and their vibrational frequencies, infrared intensities, and rotational strengths are calculated using density functional theory (DFT). The observed IR and VCD spectra of chiral camphor and camphorquinone in carbon tetrachloride solution are reproduced by the DFT calculations, but those of CSA are not. DFT calculations of hydration models, where an anionic CSA specifically binds a few water molecules, are carried out. The average of the simulated VCD spectra in the hydration models is more consistent with the observed spectra. In addition, the wavelengths and dipole and rotational strengths for chiral camphor, camphorquinone, anionic CSA, and the hydration models were calculated by time-dependent DFT. In the region of 280-300 nm, the calculated wavelengths of the ECD bands for chiral camphor and camphorquinone coincide with the observed wavelengths that have been reported, and the calculated wavelengths for the hydration models are closer to the observed wavelengths reported than are those calculated for chiral anionic CSA. Consequently, the analysis combined with VCD and ECD spectroscopy using DFT calculations can elucidate the chirality of optically active molecules, even in an aqueous solution.