The differential scattering of circularly polarized light by chloroplasts and evaluation of their true circular dichroism

Chloroplasts isolated from pea leaves display an intense circular dichroism in the range 600 to 720nm. Circularly polarized light is also differentially scattered by chloroplasts, and this effect can be confused with circular dichroism. By using an instrumental modification it was possible to distinguish, and record separately, the ellipticities of the transmitted light (circular dichroism) and of the scattered light in the same c.d. instrument. By means of a light-scattering apparatus, the intensity of unpolarized light scattered by chloroplasts was measured as a function of wavelength and of angle. This measurement allowed the aforementioned ellipticities to be corrected for mutual interference. At a concentration of 4mug of chlorophyll/ml (the optimum practical concentration of chloroplasts at which there was no significant interaction of scattering and absorption effects) spectra of true circular dichroism (circular differential absorption) and circular differential scattering were obtained. The former showed maxima, positive at 688nm and negative at 676nm, with an intensity Deltatheta=8.3m degrees .litre.(mg of chlorophyll)(-1).cm(-1). The latter had a maximum at 683nm with an intensity of +47m degrees with respect to the solvent baseline; this value is independent of the concentration of chloroplasts in dilute suspensions. It is suggested that the intense circular dichroism of chloroplasts reflects specific chlorophyll-chlorophyll interactions in the light-harvesting pigment. The advantages of this method for determining the c.d. of scattering suspensions over those of other investigators are discussed.     

Heat aggregation studies of phycobilisomes, ferritin, insulin, and immunoglobulin by dynamic light scattering.

Dynamic laser light scattering studies on the heat aggregation behavior of phycobilisomes (PBS), ferritin, insulin, and immunoglobulin (IgG) in dilute aqueous solutions has been reported. Except for PBS, results are reported for heat aggregation trends in these proteins for three different pH environments (4.0, 7.5, 9.1). For PBS, studies were performed only in the neutral buffer medium (pH 7.5). The experiments were performed in the very dilute concentration regime (between 0.23 and 1.8 gL-1). For all these samples heat aggregation and dissociation trends were found to be linear with temperature. Upon temperature reversal (self-cooling), hysteresis-like behavior observed in insulin was found to be predominantly large at pH 7.5. PBS, ferritin, and IgG showed no such behavior at any of three pH values, and retraced their path of aggregation while dissociating on temperature reversal. Heat aggregation and dissociation processes in ferritin were found to be independent of pH. The IgG samples showed smooth aggregation tendency only up to 35 degrees C in the buffer media pH 4.0 and 9.1, whereas for pH 7.0 the same could be observed until 60 degrees C. Low polydispersity in the correlation spectra was observed in case of all these samples.     

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans. I. Soluble complexes.

The interaction of acridine orange with dermatan and chondrotin sulfates results in the formation of complexes containing bound dye molecules ordered into dissymmetric arrays. Complexes containing an excess of available disaccharide residues compared to dye are completely soluble, and exhibit biphasic circular dichroism bands. Analysis of the dependence of the extrinsic circular dichrosim on dye aggregation indicates the presence of extended dye stacks bound to the glycosaminoglycan. Complexes formed in solutions containing an excess of dye are only partially soluble, and exhibit circular dichroism spectra having band shifts and intensity changes relative to the soluble complexes. The latter complexes show a sharp drop in induced circular dichroism with temperature, due to a cooperative change in the structure of the complex. The structural order of the dye–glycosaminoglycan complex may differ from the intrinsic structure of the glycosaminoglycan itself in dilute solution.     

Studies of the optical properties of the proflavine–DNA complex

We report studies of the optical properties of the proflavine–DNA complex, using absorbance and circular dichroism spectroscopy. From comparison of the absorption spectra of proflavine complexed with calf thymus and T2 DNA, we conclude that stacking of the dyes external to the double helix is comparatively much weaker with T2 DXA, probably because of its glucosylation. Several sources are found for the circular dichroism induced in proflavine when it is complexed with DNA. There is a relatively weak circular dichroism induced when the dye is infinitely dilute on the DNA lattice; this presumably arises from the environmental asymmetry of the binding site. Stronger circular dichroism effects are induced by interaction of intercalated and stacked dyes; studies with T2 DNA, for which stacking seems to be blocked, permit a tentative resolution of effects due to the two modes of binding. One recurring theme of these studies is the observation that the optical properties are quite dependent on environment. The most dramatic example is a strong variation with salt concentration of the amplitude of the circular dichroism induced in the isolated (intercalated) monomer by the surrounding DNA. This suggests that the structure of the intercalated complex is quite sensitive to external conditions.     

Photoselection studies on the orientation of chlorophyll aI in the functional membrane of photosynthesis

The orientation of chlorophyll a_I in the functional membrane of photosynthesis in green plants is studied by a photoselection technique. On excitation of an isotropic suspension of isolated spinach chloroplasts with a linearly polarized flash of light linear dichroism of the absorption changes of chlorophyll a_I (wavelengths 705 and 430 nm) is observed. The dichroism is maximum for excitation at wavelengths greater than 690 nm, medium at excitation into the blue band of the chloroplast absorption spectrum, and it is small if excitation goes into all red transition moments above 600 nm. This reflects the degree of order between the transition moments of the antennae system around Photosystem I. We conclude as to a higher order between the transition moments at the long-wavelength end of the spectrum in comparison with a lower degree of order between the transition moments belonging to the intervall from 600 to 680 nm. This confirms the results of other authors which were obtained with oriented chloroplasts. However, the photoselection approach avoids characteristic artifacts which may affect linear-dichroism studies with oriented membranes.A quantitative interpretation of the observed photoinduced dichroism of chlorophyll a_I to yield the orientation of the respective porphyrin rings in the membrane is not feasible yet due to the absence of specific information on the symmetry properties of the antennae system and on the geometry of the chlorophyll a_I aggregate. Under the assumption of a circular degenerate antennae system a rather flat inclination of chlorophyll a_I has to be expected.     

Condensation of DNA by the C-terminal domain of histone H1. A circular dichroism study

The condensation of DNA by the C-terminal domain of histone H1 has been studied by circular dichroism in physiological salt concentration (0.14 M NaF). As the intact H1 molecule, its C-terminal domain induces the so-called psi state of DNA that is characterized by a nonconservative circular dichroism spectrum which is currently attributed to ordered aggregation of the DNA molecules. On a molar basis, intact H1 and its C-terminal domain give spectra of similar intensity. Neither the globular domain of H1 nor an N-terminal fragment, that includes both the globular and N-terminal domains, has any effect on the conservative circular dichroism of DNA. From these results it is concluded that the condensation of DNA mediated by histone H1 is mainly due to its C-terminal domain. The effect of the salt concentration and the size of DNA molecules on the circular dichroism of the complexes are also examined.     

Differential light-scattering of granal and agranal chloroplasts and their fragments.

Intact (class-A) granal and agranal maize chloroplasts and chloroplast fragments were examined for differential scattering of circularly polarized light (measured at 90 degrees) and c.d. (circular dichroism) (measured at 0 degrees) by using a modified spectropolarimeter with a large acceptance angle. Useful c.d. information was obtained by making corrections for scattered light. Chloroplast fragments exhibited a large and characteristic differential scattering of circularly polarized light recognized in the presence of granal chloroplasts. It is confirmed that agranal chloroplasts do not have the intense 682 nm c.d. peak that is assigned to the presence of grana.     

Conformational change and aggregation of concanavalin A at high temperatures

Increase of the beta sheet content and aggregation of concanavalin A (con A) induced at about 60 C were followed with circular dichroism (c.d.) and scattered light intensity (I90) on both metal-complexed and demetallized species. The conversion occurred at a higher temperature for metal-complexed species than for demetallized one. A concentration-independent conversion curve of metal-complexed species, obtained for a concentration range below around 6 microgram/ml (6 x 10(-3) kg m-3) with a midpoint at 57 degrees C, was well described in terms of a conformational equilibrium between two conformers. However, aggregation did exist even at a low concentration of 1 microgram/ml. Aggregation also occurred without the conformational change as found at the initial stage or in Tris buffer, which suggested the absence of direct coupling between the conformational change and the aggregation. Changes of c.d. at 222 nm, expected to represent the main chain conformation, differed from those at 290 nm reflecting the environment of side chain chromophores. Time courses of three properties examined, c.d. at 222 nm, at 290 nm, and I90, always exhibited a lag in the case of metal-complexed species while the lag was not observed in the case of demetallized species, however. Lag became longer in c.d. but it became shorter in I90 as the protein concentration increased.     

Inhibitory effect of post-micellar SDS concentration on thermal aggregation and activity of papain

Papain, a cysteine protease isolated from the latex of Carica papaya, is known to undergo irreversible thermal unfolding. In this study, we found that thermal unfolding of papain is accompanied by a simultaneous self-assembly process where this protein is observed to aggregate above 50°C. The extent of aggregation increased with increasing protein concentration from 3–40 μM. The aggregation was confirmed by enhanced turbidity, light scattering intensity, 1-anilino-8-naphthalene sulfonate (ANS) fluorescence intensity and by transmission electron microscopy. Furthermore, we noted that post-micellar concentration of sodium dodecyl sulfate (SDS) remarkably suppresses the thermal aggregation of papain. Far-UV circular dichroism studies revealed that SDS significantly enhances α-helical content of the protein and also tends to prevent its unfolding, and thus inhibits aggregation. Additionally, papain showed maximal activity at 65°C in neutral buffer. However, in the presence of 6 mM SDS (above its critical micellar concentration), the enzyme lost activity by about 10-fold. Thus, promoting the helical propensity of the protein does not appear to be a suitable strategy to overcome the aggregation related problems of industrially important proteins such as papain, which are not only required to be protected against aggregation but also need to remain functionally active in the presence of aggregation inhibitors.     

Characterization of protein aggregation: the case of a therapeutic immunoglobulin

In this paper, a therapeutic immunoglobulin (Antibody A) has been characterized in two solutions: (1) 0.1% acetic acid containing 50 mM magnesium chloride, a solution in which the immunoglobulin is stable, and (2) 10 mM sodium phosphate buffer pH approximately 7. The protein solutions were characterized by microscopy, asymmetrical flow field-flow fractionation (FFF), light scattering, circular dichroism, fluorescence and fluorescence lifetime spectroscopy. The results show that Antibody A dissolved in 0.1% acetic acid containing 50 mM magnesium chloride exists as 88% monomer, 2% low molecular weight aggregates and 10% high molecular weight aggregates (>1 million Dalton). In phosphate buffer, Antibody A formed micrometre-sized aggregates that were best characterized by fluorescence microscopy. The aggregation of Antibody A in phosphate buffer was shown to be concomitant with conformational changes in amino acid residue side chains. The aggregates formed in phosphate buffer were easily disrupted during FFF analysis, indicating that they are formed by weak interactions. The combination of microscopy, asymmetrical flow field-flow fractionation (FFF) and spectroscopy allowed a reliable assessment of protein self association and aggregation.     

Conformational structure of bombesin as studied by vibrational and circular dichroism spectroscopy

Raman and Fourier transform infrared (FTIR) spectroscopies and circular dichroism (CD) have been applied to investigate the secondary structure of bombesin in the solid state and in phosphate buffer solution (pH 3.8). At concentrations around 10⁻⁵ M, circular dichroism reveals that bombesin exists as an irregular or disordered conformation. However, the secondary structure of the peptide appears to be a mixture of disordered structure and intermolecular β-sheets in 0.01 M sodium phosphate buffer when the peptide concentrations are higher than around 6.5 mM. The tendency of bombesin to form aggregated β-sheet species seems to be originated mainly in the sequence of the residues 7–14, as supported by the Raman spectra and β-sheet propensities (P_β) of the amino-acid residues. It is the hydrophobic force of this amino-acid sequence, and not a salt bridge effect, that is the factor responsible for the formation of peptide aggregates.     

Circular dichroism of cattle rhodopsin and bathorhodopsin at liquid nitrogen temperatures

The photoevent in vision has been considered to be the conversion of rhodopsin to bathorhodopsin, which is caused by photoisomerization of the chromophoric retinal. Recently some objections were raised to this hypothesis. The reliability of the hypothesis was verified by measurement of circular dichroism of bathorhodopsin.The measurement of circular dichroism of rhodopsin extract (containing 66% or 75% of glycerol) at liquid nitrogen temperatures (?195°C) by a conventional spectropolarimeter induced an extraordinary large signal, owing to linear dichroism originated from conversion of rhodopsin to bathorhodopsin by the measuring light. The similar linear dichroism can be induced by irradiation of rhodopsin extract at ?195°C with polarized light or natural light. At photosteady state the linear dichroism disappeared.Circular dichroism spectrum of cattle rhodopsin displayed two positive peaks ([θ]_max = 80 800 degrees at 335 nm, and [θ]_max = 42 600 degrees at 500 nm) at ?195°C, whereas, bathorhodopsin displayed a positive peak ([θ]_max = 43 100 degrees at 334 nm) and a negative peak ([θ]_max = 163 000 degrees at 540 nm).The change of the positive sign to negative one at α-band of circular dichroism spectrum supports the hypothesis that the conversion of rhodopsin is due to rotation of the chromophoric retinal about C-11—12 double bond (‘photoisomerization model’).     

Partial analysis of the bands seen in circular dichroism spectra of green and blue-green algal cells and thylakoids

A comparison was made of the circular dichroism (C.D.) spectra of Chlorella, Euglena, and Anacystis cells and thylakoids. Analyses of the spectra reveal that these C.D. bands are similar to those observed previously in whole spinach choloroplasts and subchloroplast particles. C.D. spectra of Euglena chloroplasts show bands at longer wavelengths than previously reported. From comparisons of circular dichroism spectra and fine structure, it was concluded that: (a) bands seen in circular dichroism spectra were not the result of light scattering from thylakoid membranes; and (b) bands seen in the C.D. spectra of nonmembranous systems (previously reported) could account for circular dichroism of algae. We also concluded that comparisons would have to be made with model systems in order to correct for effects of absorption flattening, concentration obscuring, and differential light scattering of membranous systems.     

Solute effects on the irreversible aggregation of serum albumin

Thermal stress on bovine serum albumin (BSA) promotes protein aggregation through the formation of intermolecular beta-sheets. We have used light scattering and chromatography to study effects of (<1 M) Na(2)SO(4), NaSCN, sucrose, sorbitol and urea on the rate of the thermal aggregation. Both salts were strong inhibitors of BSA aggregation and they reduced both the size and number (concentration) of aggregate particles compared to non-ionic solutes (or pure buffer). Hence, the salts appear to suppress both nucleation- and growth rate. The non-electrolyte additives reduced the initial aggregation rate (compared to pure buffer), but did not significantly limit the extent of aggregation in samples quenched after 27 min. heat exposure (40-50% aggregation in all samples). The non-electrolytes did, however, modify the aggregation process as they consistently brought about smaller but more concentrated aggregates than pure buffer. The results are discussed along the lines of linkage- and transition state theories. In this framework, the rate of the aggregation process is governed by the equilibrium between a thermally denatured state (D) and the transition state D( not equal). Thus, the effect of a solute relies on its preferential interactions with respectively D and D( not equal). The current results do not show any correlation between the solutes' preferential interactions with native BSA and their effect on the rate of aggregation. This suggests that non-specific, "Hofmeister-type" interactions, which scale with the solvent accessible surface area, are of minor importance. Rather, salt induced suppression of aggregation is suggested to depend on the modulation of specific electrostatic forces in the D( not equal) state.     

Amyloid nucleation triggered by agitation of beta2-microglobulin under acidic and neutral pH conditions

Amyloid nucleation through agitation was studied with beta2-microglobulin, which is responsible for dialysis-related amyloidosis, in the presence of salt under acid and neutral pH conditions. First, the aggregation of beta2-microglobulin in NaCl solutions was achieved by mildly agitating for 24 h at 37 degrees C protein solutions in three different states: acid-unfolded, salt-induced protofibrillar, and native. The formation of aggregates was confirmed by an increase in light scattering intensity of the solutions. Then, the aggregated samples were incubated without agitation at 37 degrees C for up to 25-45 days. The structural changes in the aggregated state during the incubation period were examined by means of fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy. The results revealed that all the samples in the different states produced a mature amyloid nucleus upon agitation, after which the fibrils elongated without any detectable lag phase during the incubation, with the acid-unfolded protein better suited to undergoing the structural rearrangements necessary to form amyloid fibrils than the more structured forms. The amount of aggregate including the amyloid nucleus produced by agitation from the native conformation at neutral pH was estimated to be about 9% of all the protein by an analysis using ultracentrifugation. Additionally, amyloid nucleation by agitation was similarly achieved for a different protein, hen egg-white lysozyme, in 0.5 M NaCl solution at neutral pH. Taken together, the agitation-treated aggregates of both proteins have a high propensity to produce an amyloid nucleus even at neutral pH, providing evidence that the aggregation pathway involves amyloid nucleation under entirely native conditions.     

A circular dichroism study of the structure of Apis mellifera melittin

The secondary structure of the polypeptide melittin has been examined employing circular dichroism. In water or dilute buffer at pH 7, this protein is approximately 25–30% helical. Helicity is increased to near 50% by increasing the pH to 9 or above. High concentrations of phosphate buffer dramatically increase mean residue ellipticity at 222 nm to a value near the maximum expected for a totally helical molecule. Dilute solutions of sodium dodecyl sulfate have the same effect. In contrast, the secondary structure of melittin is little affected by bromide ion, calcium ions, or dihexanoyl-phosphatidylcholine.     

Honey bee odorant-binding protein 14: effects on thermal stability upon odorant binding revealed by FT-IR spectroscopy and CD measurements

In the present work, we study the effect of odorant binding on the thermal stability of honey bee (Apis mellifera L.) odorant-binding protein 14. Thermal denaturation of the protein in the absence and presence of different odorant molecules was monitored by Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). FT-IR spectra show characteristic bands for intermolecular aggregation through the formation of intermolecular β-sheets during the heating process. Transition temperatures in the FT-IR spectra were evaluated using moving-window 2D correlation maps and confirmed by CD measurements. The obtained results reveal an increase of the denaturation temperature of the protein when bound to an odorant molecule. We could also discriminate between high- and low-affinity odorants by determining transition temperatures, as demonstrated independently by the two applied methodologies. The increased thermal stability in the presence of ligands is attributed to a stabilizing effect of non-covalent interactions between odorant-binding protein 14 and the odorant molecule.     

Membrane-induced folding and structure of membrane-bound annexin A1 N-terminal peptides: implications for annexin-induced membrane aggregation

Annexins constitute a family of calcium-dependent membrane-binding proteins and can be classified into two groups, depending on the length of the N-terminal domain unique for each individual annexin. The N-terminal domain of annexin A1 can adopt an α-helical conformation and has been implicated in mediating the membrane aggregation behavior of this protein. Although the calcium-independent interaction of the annexin A1 N-terminal domain has been known for some time, there was no structural information about the membrane interaction of this secondary membrane-binding site of annexin A1. This study used circular dichroism spectroscopy to show that a rat annexin A1 N-terminal peptide possesses random coil structure in aqueous buffer but an α-helical structure in the presence of small unilamellar vesicles. The binding of peptides to membranes was confirmed by surface pressure (Langmuir film balance) measurements using phosphatidylcholine/phosphatidylserine monolayers, which show a significant increase after injection of rat annexin A1 N-terminal peptides. Lamellar neutron diffraction with human and rat annexin A1 N-terminal peptides reveals an intercalation of the helical peptides with the phospholipid bilayer, with the helix axis lying parallel to the surface of membrane. Our findings confirm that phospholipid membranes assist the folding of the N-terminal peptides into α-helical structures and that this conformation enables favorable direct interactions with the membrane. The results are consistent with the hypothesis that the N-terminal domain of annexin A1 can serve as a secondary membrane binding site in the process of membrane aggregation by providing a peripheral membrane anchor.     

Conformational changes of lysozyme refolding intermediates and implications for aggregation and renaturation

It is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding.     

Low cetyltrimethylammonium bromide concentrations induce reversible amorphous aggregation of tobacco mosaic virus and its coat protein at room temperature

Ordered and amorphous protein aggregation causes numerous diseases. Tobacco mosaic virus coat protein for many decades serves as the classical model of ordered protein aggregation ("polymerization"). It was also found to be highly prone to heat-induced amorphous aggregation and the rate of this aggregation could be easily manipulated by changes in solution ionic strength and temperature. Here, we report that rapid amorphous aggregation of this protein can be induced at 25 degrees C in phosphate buffer by low micromolar (start at about 15 microM) concentrations of cationic surfactant cetyltrimethylammonium bromide. At equilibrium four surfactant molecules bound to the protein subunit. As judged by circular dichroism and fluorescence spectroscopy data, the coat protein molecules retained their native structure upon the cetyltrimethylammonium bromide induced aggregation. No aggregation was observed at the higher surfactant concentrations (above 300 microM). Micromolar concentrations of anionic surfactant sodium dodecylsulfate rapidly reversed the cetyltrimethylammonium bromide induced aggregation of the coat protein due to formation of mixed surfactant-surfactant micelles. Cetyltrimethylammonium bromide (100-300 microM) also induced the reversible intact tobacco mosaic virus virion aggregation. The possible liability to the cetyltrimethylammonium bromide induced amorphous aggregation of other ordered aggregate-producing proteins has been discussed.