Structural studies on aqueous and hydroalcoholic solutions of a polyene antibiotic: Amphotericin B

Circular dichroism and absorption and light scattering have been used to study the effect of ethyl alcohol on an aqueous solution of Amphotericin B (“Fungizone”), which is an antifungal heptene. In aqueous solution, light-scattering studies show that Amphotericin B exists in an aggregated form. The estimated mass of these aggregates is about 2 × 10⁶ daltons, representing about 2000 molecules. Since the aggregated form is high molecular weight and scattering, the CD involves probably the differential scattering of right and left polarized light. In aqueous solution, Amphotericin B exhibits a strong dissymetric couplet in CD at the wavelength of the absorption maximum (328 nm). This latter maximum presents a blue shift when compared with the normal absorption in polar organic solvents. In hydroalcoholic solutions, for alcohol concentrations below 35%, the molecular weight of the aggregates is unchanged, while the absorption and CD spectra are modified. For alcohol concentrations greater than 35%, the aggregates mass decreases quickly and becomes undetectable at 50% ethyl alcohol concentration. For these solutions, the CD and absorption spectra are practically constant and characteristic of unaggregated Amphotericin B form.     

Anisotropic circular dichroism signatures of oriented thylakoid membranes and lamellar aggregates of LHCII

In photosynthesis research, circular dichroism (CD) spectroscopy is an indispensable tool to probe molecular architecture at virtually all levels of structural complexity. At the molecular level, the chirality of the molecule results in intrinsic CD; pigment–pigment interactions in protein complexes and small aggregates can give rise to excitonic CD bands, while “psi-type” CD signals originate from large, densely packed chiral aggregates. It has been well established that anisotropic CD (ACD), measured on samples with defined non-random orientation relative to the propagation of the measuring beam, carries specific information on the architecture of molecules or molecular macroassemblies. However, ACD is usually combined with linear dichroism and can be distorted by instrumental imperfections, which given the strong anisotropic nature of photosynthetic membranes and complexes, might be the reason why ACD is rarely studied in photosynthesis research. In this study, we present ACD spectra, corrected for linear dichroism, of isolated intact thylakoid membranes of granal chloroplasts, washed unstacked thylakoid membranes, photosystem II (PSII) membranes (BBY particles), grana patches, and tightly stacked lamellar macroaggregates of the main light-harvesting complex of PSII (LHCII). We show that the ACD spectra of face- and edge-aligned stacked thylakoid membranes and LHCII lamellae exhibit profound differences in their psi-type CD bands. Marked differences are also seen in the excitonic CD of BBY and washed thylakoid membranes. Magnetic CD (MCD) spectra on random and aligned samples, and the largely invariable nature of the MCD spectra, despite dramatic variations in the measured isotropic and anisotropic CD, testify that ACD can be measured without substantial distortions and thus employed to extract detailed information on the (supra)molecular organization of photosynthetic complexes. An example is provided showing the ability of CD data to indicate such an organization, leading to the discovery of a novel crystalline structure in macroaggregates of LHCII.     

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-beta,D-maltoside, n-octyl-beta,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.     

Exciton theory for supramolecular chlorosomal aggregates: 1. Aggregate size dependence of the linear spectra

The interior of chlorosomes of green bacteria forms an unusual antenna system organized without proteins. The steady-spectra (absorption, circular dichroism, and linear dichroism) have been modeled using the Frenkel Hamiltonian for the large tubular aggregates of bacteriochlorophylls with geometries corresponding to those proposed for Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes. For the Cf. aurantiacus aggregates we apply a structure used previously (V. I. Prokhorenko., D. B. Steensgaard, and A. R. Holzwarth, Biophys: J. 2000, 79:2105-2120), whereas for the Cb. tepidum aggregates a new extended model of double-tube aggregates, based on recently published solid-state nuclear magnetic resonance studies (B.-J. van Rossum, B. Y. van Duhl, D. B. Steensgaard, T. S. Balaban, A. R. Holzwarth, K. Schaffner, and H. J. M. de Groot, Biochemistry 2001, 40:1587-1595), is developed. We find that the circular dichroism spectra depend strongly on the aggregate length for both types of chlorosomes. Their shape changes from "type-II" (negative at short wavelengths to positive at long wavelengths) to the "mixed-type" (negative-positive-negative) in the nomenclature proposed in K. Griebenow, A. R. Holzwarth, F. van Mourik, and R. van Grondelle, Biochim: Biophys. Acta 1991, 1058:194-202, for an aggregate length of 30-40 bacteriochlorophyll molecules per stack. This "size effect" on the circular dichroism spectra is caused by appearance of macroscopic chirality due to circular distribution of the transition dipole moment of the monomers. We visualize these distributions, and also the corresponding Frenkel excitons, using a novel presentation technique. The observed size effects provide a key to explain many previously puzzling and seemingly contradictory experimental data in the literature on the circular and linear dichroism spectra of seemingly identical types of chlorosomes.     

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.     

A structural study of bovine lens alpha-crystallin and its subunits by absorption and linear dichroism spectroscopy

The structure of the bovine alpha-crystallin aggregate and its reaggregated isolated subunits has been studied by measurement of their absorption and linear dichroism spectra over the range 250-350 nm. Also, changes in structure with respect to time have been monitored in this way. From the absorption spectra it appears that the aromatic residues in subunit aggregates are in the same chemical environment as those in native protein. The light scattering due to the size of the protein molecules increases when the proteins are kept in solution, this effect being much stronger for the subunits. The linear dichroism spectra point to strong structural ordering in alpha-crystallin, the absorption transition dipoles of the aromatic residues being preferentially aligned along the long axis of the molecules. Moreover, a considerable deviation from a spherical or tetrahedrally symmetric structure of alpha-crystallin is inferred. The subunit aggregates show less ordering and might be more spherical. When kept in solution, their structural order seems to be decreased. The linear dichroism spectra show absorption at 325 nm, which is not detectable in the normal absorption spectra.     

Structural transitions of calf thymus DNA in concentrated LiCl solutions

The solubility, sedimentation, circular dichroism, and absorption spectral characteristics of calf thymus DNA have been examined in concentrated solutions of LiCl (6-13 m) at 25 to 27 degree C. At all concentrations of LiCl, the DNA is base stacked and exhibits normal hypochromicty, At the upper end of this range of LiCl concentrations, DNA aggregates and ultimately precipitates completely from solution between 13 and 14 m LiCl. This aggregation process is dependent on concentration, base composition, and molecular weight of DNA. The sedimentation velocity data taken together with the absorbance spectral data suggest that the aggregation process leading to the formaiton of large structures beings at approximately equal to 9 m. Prior to the onset of aggregation, the circular dichroism (CD) spectra can be adequately fitted by a linear combination of contributions of the B, C, and A forms of DNA (Hanlon, S., Brudno, S., Wu, T. T., and Wolf, B. (1975), Biochemistry 14, 1648). Above 9 m LiCl, both factor analysis and a primitive version of matrix rank order analysis indicate that at least one additional spectral component is required to account for the observed CD spectra above 260 nm. The general shape of this additional component or distortion resembles the psi form of DNA.     

Structure of amphotericin B aggregates based on calculations of optical spectra

The Degenerate ground state approximation was used to calculate the optical absorption and CD spectra for helical polymer models of amphotericin B aggregates in aqueous solution. Comparisons with experimental spectra indicate that a two-molecule/unit cell helical polymer model is a possible structure for aggregates of amphotericin B.     

Effect of intramolecular interaction on the electron excitation state of nucleic acid components

A theoretical and experimental investigation of absorption and luminescence features of crystals and aggregates of nucleic acid bases were carried out. The long wavelength low intensity bands in UV-absorption nd excitation spectra, bathochromic shift of fluorescence spectra, the change of correlation between the intensity of fluorescence and phosphorescence spectra were obtained. The interpretation of these experimental results was proposed on the basis of pair interaction calculations (exciton-resonance and charge-resonance) in different conformations of cytosine dimers. The energy transfer after excitation at lambda 280 and 312 nm was investigated in nucleic acid base aggregates.     

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.     

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     

Covalent attachment of an alkylamine prevents the B to Z transition in poly(dG-dC)

Covalent complexes of n-butylamine and double-stranded poly(dG-dC) were prepared by coupling the amine to exocyclic amino groups of guanine bases with CH2O. Neither the absorption spectrum above 230 nm nor the s020,w of the complexes in low to moderate ionic strength solvents, freed of excess unreacted reagents, differs significantly from that of unreacted poly(dG-dC) or a control which had been exposed only to CH2O. In contrast, the CD spectra are profoundly altered. The minimum at 252 nm becomes more negative, and the rotational strength of the positive band above 260 nm is reduced as a linear function of the extent of amine attachment. At 0.22 mol of amine per mole of nucleotide, the transformation is similar to that observed by others in poly(dG-dC) when complexed to core histones in reconstituted core particles or in concentrated LiCl solvents at temperatures below the B----Z transition. Sedimentation studies reveal that these changes in the circular dichroism (CD) spectra reflect secondary structural effects rather than the formation of aggregates or psi type structures. Raman spectra reveal, however, that these secondary structural changes must occur within the B family as the amine complex retains B backbone geometry. The conformation produced by the attachment of the amine is probably a higher winding angle (overwound) B variant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protochlorophyll forms with different molecular arrangements

Spectral properties of protochlorophyll (PChl) forms were investigated in solid-film model systems by absorption. fluorescence and circular dichroism (CD) spectroscopy. The solid films were prepared from diethyl ether solution of PChl on a cover glass surface by evaporation of the solvent. After preparation the films usually showed an absorption maximum at 635 nm or in some cases at 640 nm. The PChl form with 635 nm absorption maximum had no CD signal, whilst the films with absorption maximum at 640 nm gave an intense negative CD band at about 640 nm and a positive one at 668 nm. The treatment of the films with ammonia or acetone vapour resulted in a red shift of the absorption maximum from 635 nm or 640 nm to 650 nm. The study of the CD spectra of the films with different PChl forms showed that, depending on the treatment, forms of PChl with similar absorption and fluorescence spectra, but with opposite CD signals, can exist. It is suggested that the differences of the CD spectra are mainly due to different arrangements of the aggregates.     

Optical properties of complexes of antimalarial drugs with ferriprotoporphyrin IX in aqueous medium. II. The system ferriprotoporphyrin IX-quinidine

Light absorption and circular dichroism (CD) were recorded at 26 to 27 degrees C in dilute aqueous solutions (10(-4) M) of ferriprotoporphyrin IX (FP) in the presence of (+)-quinidine. In contrast to the appearance of relatively small and positive CD bands between pH 7 and 10, two bands of opposite sign, having unusually large molar ellipticities of the order of 10(6) deg X cm2 X dmol-1 FP were observed in the Soret region near 400 nm at pH 11.0-11.5. This unique complex A was formed only slowly over periods of hours or days at 26 degrees C. By lowering the pH of Complex A below 10, under certain conditions, an "enantiometric" mirror-image CD spectrum, with all bands having opposite sign to Complex A, was obtained in the range of 650 to 300 nm (Complex B), while the light-absorption spectra of Complexes A and B were similar. The formation of Complex A was inhibited at mole fractions of FP greater than 0.5. Also, this complex was not measurably formed at low salt concentrations. Ultracentrifugation measurements of the complex solutions indicated the presence of very high aggregates. Possible interpretations of the optical properties observed are based on interactions between FP molecules, which are assumed to be arrayed chirally within aggregates of FP with quinidine. A comparison between quinine and quinidine complexes of FP is presented.     

Complexation of polymethine dyes with human serum albumin: a spectroscopic study

Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Phif; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Phif vary from 10(4) to (5-6) x 10(7) M(-1). K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis-->trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.     

Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films.

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.     

Tubular exciton models for BChl c antennae in chlorosomes from green photosynthetic bacteria

Exciton calculations on tubular pigment aggregates similar to recently proposed models for BChl c/d/e antennae in light-harvesting chlorosomes from green photosynthetic bacteria yield electronic absorption spectra that are super-impositions of linear J-aggregate spectra. While the electronic spectroscopy of such antennae differs considerably from that of linear J-aggregates, tubular exciton models (which may be viewed as cross-coupled J-aggregates) may be constructed to yield spectra that resemble that of the BChl c antenna in the green bacterium Chloroflexus aurantiacus. Highly symmetric tubular models yield absorption spectra with dipole strength distributions essentially identical to that of a J-aggregate; strong symmetry-breaking is needed to simulate the absorption spectrum of the BChl c antenna.Abbreviations BChl bacteriochlorophyll - [E,M] BChl c _S bacteriochlorophyll c with ethyl and methyl substituents in the 8- and 12-positions, and with stearol as the esterifying alcohol     

Molecular interactions between micellar polysialogangliosides and affinity-purified tetanotoxins in aqueous solution

Two-affinity purified tetanotoxin forms, TeToA and TeToB, with different affinities for gangliosides were characterized by analytical ultracentrifuge, circular dichroism (CD), and amino acid composition. Both toxin forms share a common sedimentation coefficient of about 6-7 S and similar alpha-helicity values, but they vary in amino acid composition. Incubation of TeToB with micellar polysialogangliosides results in formation of high (21-24 S) and medium (13-15 S) size toxin-micellar ganglioside aggregates as revealed by analytical ultracentrifuge technique. At TeToB/[N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosylceramide (GT1b) molar ratios of greater than 26, high molecular weight aggregates (Mr greater than or equal to 700,000) which contain between 3 and 5 toxin monomers are formed, whereas at molar ratios less than 15, about 1-2 monomers are present. TeToA does not form aggregates in the presence of gangliosides. A marked increase in the alpha-helix from about 20 to 39% is apparent in the CD spectrum of TeToB after interaction with ganglioside mixture (G1b). Cerebrosides, sulfatides, sphingomyelin, and phosphatidylserine also increase the alpha-helix, presumably because of an overall effect of lipids on the protein. TeToA and fragment B but not C also undergo similar changes in the presence of G1b, suggesting that the effect of ganglioside is not specific. The polarity of the CD spectra of a number of gangliosides is shifted from a negative to a positive value after interaction with tetanotoxin. The data are consistent with the interpretation of a discrete hydrophobic domain on the toxin heavy chain which interacts with micellar gangliosides to form macromolecular complexes.     

Steady-state and transient polarized absorption spectroscopy of photosystem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus

Core antenna and reaction centre of photosystem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669-670(+), 673(+), 680(-), 683-685(-), 696-697(-), and 711(-) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700(+)A(1)(-) or (3)P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on P(A), whereas the cation is localized most likely on P(B).