Visible Circular Dichroism Spectra Research Articles

Allophycocyanin: trimers, monomers, subunits, and homodimers.

Allophycocyanin is a photosynthetic light-harvesting pigment-protein complex located in the phycobilisomes of cyanobacteria and red algae. Using dynamic light scattering and circular dichroism, solutions of purified allophycocyanin were shown to consist of homogeneous trimers (alpha3beta3) with a nonspherical shape over a very wide range of protein concentrations at pH 6.0 and 20 degrees C. Deconvolutions of the visible circular dichroism spectrum of the trimer were carried out for the first determination of the individual spectra of all six-component chromophores. The chromophores were shown to be in different microenvironments that helped determine the spectrum of the trimer. Monomers (alpha beta) that were formed in either the presence of 0.50M NaSCN or at 45 degrees C were shown to be completely reversible to trimers. However, subunits (alpha and beta) that were formed in either the presence of 8M urea or at 60 degrees C, using spectroscopy and gel-filtration column chromatography, were observed to only partially reconstitute trimers. Homodimers (alpha2 and/or beta2) formed during the regeneration of trimers. The homodimer, which was detected for the first time when both subunits were present, was shown to be in equilibrium with its subunits. Unlike the trimer situation, subunits were found to fully reconstitute monomers in the presence of 0.50M NaSCN. These results suggest a route to trimer assembly from subunits with monomers serving as intermediaries and the homodimers forming in a nonproductive step that did not interfere with the overall assembly scheme.

Viviparus ater hemocyanin: investigation of the dioxygen-binding site and stability of the oxy- and apo-forms.

The active site of Viviparus ater (mollusc) hemocyanin was investigated using the fact that the binding of dioxygen to the binuclear copper-containing sites of hemocyanins is connected with the appearance of specific dichroic bands which are very sensitive to changes in the structrure and polarity of the environment. Oxy-Viviparus ater hemocyanin exhibits near UV and visible circular dichroism spectra different from those of other molluscan and arthropodan hemocyanins. These differences are due probably to variations in the geometry or charge distribution in the dioxygen binding sites of the compared proteins. The thermostability of Viviparus ater hemocyanin and the significance of the copper-dioxygen system for the stability were also investigated. "Melting" temperatures, Tm, of 77 degrees C for the oxy-hemocyanin and 57 degrees C for the apo-protein were calculated from the denaturation curves which demonstrates the considerable role of the binuclear active site for the thermostability. Viviparus ater hemocyanin is more thermostable than other hemocyanins for which data are published.

Self-assembly of synthetic 8 1-hydroxy-chlorophyll analogues

The aggregation behavior of synthetic zinc-8 1-hydroxy-chlorins in nonpolar organic media and in solid thin film has been compared with that of zinc-3 1-hydroxy-chlorin, a model of bacteriochlorophyll- d in the chlorosomes of green photosynthetic bacteria. Zinc-8 1-hydroxy-13 1-oxo- and 3 1,13 1-dioxo-chlorins 1a and 3a self-aggregate in nonpolar organic solvents and in solid thin films. They show less red-shifted and wider visible absorption bands at the Soret and Q y regions and also more intense circular dichroism (CD) signals at the Soret region than self-aggregates of zinc-3 1-hydroxy-13 1-oxo-chlorin 5a. The 1H-NMR spectroscopic data indicate that the hydroxyl group at the 8 1-position of 1a coordinates with the central zinc of an adjacent molecule in the supramolecule. From the infrared spectra, we can deduce that the interactions of the functional groups of 1a in the aggregates are unspecific or weak. For this reason, the supramolecular structures are partially amorphous. Visible absorption and CD spectra establish that the lack of one of the three interactive moieties (8 1-hydroxy, 13-keto groups and central zinc) suppresses the aggregation ability of chlorins. Furthermore, zinc-3-acetyl-8 1-hydroxy-chlorin 2a does not self-aggregate in nonpolar organic solvents because both the hydroxyl and keto groups take various configurations. However, additional substitution of keto carbonyl group at the 3-position ( 1a→3a) affects the aggregation ability. It is concluded that the fixation of either the OH or CO group to the chlorin moiety possessing an exo-five-membered ring, as well as both the presence of OH, CO and central metal in the molecule and the linear location of the three interactive moieties are necessary for the formation of well-ordered self-aggregates of chlorophyll analogues, which are seen in native chlorosomes.

An Additional Methyl Group at the 10-Position of Retinal Dramatically Slows down the Kinetics of the Rhodopsin Photocascade

The present study focuses on ligand-protein interactions in a rhodopsin analogue generated from bovine opsin and the 10-methyl homologue of 11-cis-retinal. The analogue pigment displays a reduced alpha-band at 506 +/- 2 and a stronger beta-band at 325 nm. Remarkably, the rotational strength of these bands observed in visible circular dichroism spectra was found to be similar for both native and 10-methyl rhodopsin. The quantum yield of the analogue pigment was determined to be 0.55. All photointermediates were analyzed by Fourier transform infrared difference spectroscopy. At the batho stage, strong hydrogen-out-of-plane vibrations were observed, indicating that the 10-methyl chromophore also adopts a distorted all-trans conformation at this stage. In contrast to native rhodopsin, the batho intermediate of the 10-methyl pigment is stable up to 180 K and only slowly decays to the next intermediate between 180 and 210 K. As in native rhodopsin, the 10-methyl metarhodopsin I intermediate is generated at about 220 K, but its transition to the metarhodopsin II state is again shifted to a much higher temperature (> 293 K) than for the native pigment (> 260 K). Infrared analysis, nevertheless, shows that the conformational changes in the photointermediates of the 10-methyl pigment are basically identical with those observed in the native pigment. This is supported by a signal function assay, showing that the analogue pigment is able to activate transducin. The dual effect of the 10-methyl group on the photocascade is attributed to steric interactions which, initially, hamper the relaxation of strain in the polyene chain of the chromophore and, eventually, interfere with the conformational rearrangements of the protein moiety required to adopt the active conformation of the receptor. Our data provide direct support for the concept that the relaxation of strain in the retinal polyene chain acts as the major driving force of the photocascade dark reaction.

R-phycoerythrins having two conformations for the same aggregate

The visible circular dichroism (CD) spectrum of an R-phycoerythrin (Porphyra tenera) is composed of several positive bands. The protein in aqueous buffer very slowly exhibits changes in the CD spectrum of its chromophores, a band at 489 nm undergoes an increase in intensity and a red shift. When the band reached a 493 nm maximum, the spectrum became very stable. The aggregation state of the protein did not change during this spectral conversion. The chromophore CD spectrum was also obtained in the presence of a low concentration of urea or sodium thiocyanate, and the identical change in the CD was noted, but the change was much faster. The visible absorption and CD in the far UV spectra were unaffected by urea. Unchanged visible absorption and protein secondary structure (61% α helix) contradicted by comparatively salient alterations in the visible CD spectra suggested very subtle structural changes are influencing some of the chromophores. For a second R-phycoerythrin (Gastroclonium coulteri), the CD of the chromophores had a negative band on the blue edge of the spectrum. This is the first negative CD band observed for any R-phycoerythrin. Treatment of this protein with low concentrations of urea produced a change in the visible CD with the negative band being completely converted to a positive band. Fluorescence studies showed that the treatment by urea did not affect energy migration. Deconvolution of the CD spectra were used to monitor the chromophores. The results demonstrated that the same aggregate of each R-phycoerythrin could exist in two conformations, and this is a novel finding for any red algal or cyanobacterial biliprotein. The two forms of each protein would differ in tertiary structure, but retain the same secondary structures.

Bilin chromophores as reporters of unique protein conformations of phycocyanin 645.

At 45 degrees C, phycocyanin 645 maximally undergoes a reversible and stable conformational change. The change is observed in the visible (chromophore) region of the absorption and circular dichroism (CD) spectra. In the absorption spectrum, the absorbance is lower at 45 degrees C but remains much closer to the normal spectrum than to a strongly denatured spectrum. In the CD, a similar situation exists except that a negative band on the blue edge of the spectrum is much more strongly affected at 45 degrees C than the other bands. On returning to 20 degrees C, all these changes are restored to the original states. The protein is an alpha 2 beta 2 dimer at both 20 and 45 degrees C, and CD in the far-UV shows the identical protein secondary structures at both 20 and 45 degrees C. Fluorescence studies show that energy transfer occurs at both temperatures. At 50 degrees C the results are saliently different as the secondary structure changes and the spectral changes are mostly irreversible. At 50 degrees C, some monomers (alpha beta) are produced, and these monomers are very unstable at that temperature, resulting in the formation of some fully denatured polypeptides. Stable monomers can be produced at 20 degrees C and have visible absorption and CD spectra identical to the dimer at 45 degrees C. Therefore, the chromophores are reporting a tertiary conformational change at 45 degrees C, in which the two halves of the dimer each assume a monomer-like conformation prior to dissociating. These results are compared with a hypothesis for the chromophore topography, and the CD change at the blue edge of the spectra may result from the separation at 45 degrees C of a chromophore pair.

Chromophore topography and exciton splitting in phycocyanin 645.

The biliprotein phycocyanin 645 has been purified from a photosynthetic cryptomonad Chroomonas species. It is composed of two copies each of two polypeptides (alpha and beta); each alpha polypeptide has one chromophore, and each beta polypeptide has three. There are one cryptoviolin and two phycocyanobilins on each beta polypeptide and one 697-nm bilin on each alpha polypeptide for a total of eight chromophores on the protein. Circular dichroism (CD) spectroscopy has been used to investigate the arrangement of these chromophores. Comparisons among the intact protein (alpha 2 beta 2) and various urea-treated products have yielded a tentative model for chromophore topography. The six chromophores on the two beta polypeptides are segregated into three pairs. The chromophores of each pair are close enough to experience electronic interactions. One pair, consisting of the two cryptoviolins, produces exciton splitting on the blue edge of the visible CD spectrum, and the two pairs of phycocyanobilins cause exciton splitting on the red edge of this spectrum. Deconvolution shows that the CD spectrum of each pair has a positive and a negative band, which are nearly conservative as expected for exciton coupling. The two chromophores on the alpha polypeptides are more isolated. The pairing of cryptoviolin chromophores occurs across two beta polypeptides, but the more likely position of each of the two pairs of phycocyanobilins is probably not across a beta-beta interface but within a single beta polypeptide. The exciton splitting events both increase the range of visible light absorption for the protein and establish the routes of exciton migration through the protein.

Interaction between chromium GTP and tubulin. Stereochemistry of GTP binding, GTP hydrolysis, and microtubule stabilization

Chromium GTP (CrGTP) has been used to probe the stereochemistry of metal-GTP binding to exchangeable site of tubulin and to examine the fate and role of nucleotide-bound metal ion in GTP hydrolysis associated with microtubule assembly. The absolute stereoconfiguration of the two pairs of diastereomers of beta,gamma-bidentate CrGTP has been determined by comparison of their visible circular dichroism spectra with those of the beta,gamma-CrATP isomers whose configurations have been established (Lin, I., and Dunaway-Mariano, D. (1988) J. Am. Chem. Soc. 110, 950-956). Tubulin binds metal-GTP preferentially in the delta pseudoaxial configuration. CrGTP-tubulin shows a high propensity to undergo tubulin-tubulin interactions with associated hydrolysis of CrGTP. Hydrolysis of CrGTP in microtubule assembly develops in two consecutive steps: cleavage of the gamma-phosphate followed by release of Pi and chromium. In contrast to other NTPases (actin, hexokinase) tubulin appears able to catalyze the dissociation of the stable chromium-phosphate bonds, which implies a highly nucleophilic environment of the binding site of the metal-triphosphate moiety of GTP. Microtubules assembled from CrGTP-tubulin are made of 90% GDP subunits, and their stability is linked to a 10% proportion of CrGDP-Pi subunits, scattered along the microtubule, from which Pi does not dissociate. The possibility is evoked that some tubulin variants do not catalyze release of Pi and metal ion efficiently, and their presence could affect microtubule dynamics.

Circular dichroism and 1H NMR studies of Co2+- and Ni2+-substituted concanavalin A and the lentil and pea lectins.

Visible absorption, circular dichroism (CD) and magnetic circular dichroism spectra have been recorded for the Ca2+-Co2+ derivatives of the lentil (CCoLcH) and pea (CCoPSA) lectins (Co2+ at the S1 sites and Ca2+ at the S2 sites) and shown to be very similar for both proteins. The visible absorption and magnetic circular dichroism spectra indicate similar octahedral geometries for high spin Co2+ at S1 in both proteins, as found in the Ca2+-Co2+ complex of concanavalin A (CCoPL) (Richardson, C. E., and Behnke, W. D. (1976) J. Mol. Biol. 102, 441-451). The visible CD data, however, indicate differences in the environment around S1 of CCoLcH and CCoPSA compared to CCoPL. 1H NMR spectra at 90 MHz of the Co2+ and Ni2+ derivatives of the lectins show a number of isotropically shifted signals which arise from protons in the immediate vicinity of the S1 sites. Analysis of the spectra of the Co2+ derivatives in H2O and D2O has permitted resonance assignments of the side chain ring protons of the coordinated histidine at S1 in the lectins. Differences are observed in the H-D exchange rate of the histidine NH proton at S1 in concanavalin A compared to the lentil and pea lectins. NMR data of the Ni2+-substituted proteins, together with spectra of the Co2+ derivatives, also indicate that the side chains of a carboxylate ligand and of the histidine residue at S1 are positioned differently in concanavalin A than in the other two lectins. These results appear to account, in part, for the differences observed in the visible CD spectra of the Co2+-substituted proteins. In addition, binding of monosaccharides does not significantly perturb the spectra of the lectins. An unusual feature in the 1H NMR spectra of all three Co2+-substituted lectins is the presence of two exchangeable downfield shifted resonances which appear to be associated with the two protons of a slowly exchanging water molecule coordinated to the Ca2+ ion at S2. T1 measurements of CCoLcH have provided an estimation of the distances from the Co2+ ion to these two protons of 3.7 and 4.0 A.

Spectroscopic characterization of plastocyanin from hornwort

Plastocyanin isolated from an aquatic higher plant, Ceratophyllum demersum L. (hornwort), has been characterized by electronic absorption, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopies. The visible absorption, CD, and EPR spectra of hornwort plastocyanin indicated a complete similarity of blue copper center to those of terrestrial higher plants and algae. However, the ultraviolet absorption spectrum of hornwort plastocyanin exhibited a lower tyrosine (Tyr) and a higher phenylalanine (Phe) content of the protein comparing with other plastocyanins. The ratio of Phe Tyr residues was estimated to be 9 by amino acid analysis. The hornwort plastocyanin resembles in amino acid composition terrestrial higher plant plastocyanins rather than alga plastocyanins but is peculiar in the content of Phe and Tyr residues.

Optical effects of sodium dodecyl sulfate treatment of the isolated light harvesting complex of higher plants

The light-harvesting complex (LHC) of higher plants isolated using Triton X-100 has been studied during its transformation into a monomeric form known as CPII. The change was accomplished by gradually increasing the concentration of the detergent, sodium dodecyl sulfate (SDS). Changes in the red spectral region of the absorption, circular dichroism (CD), and linear dichroism spectra occurring during this treatment have been observed at room temperature. According to a current hypothesis the main features of the visible region absorption and CD spectra of CPII can be explained reasonably successfully in terms of an exciton coupling among its chlorophyll (Chl) b molecules. We suggest that the spectral differences between the isolated LHC and the CPII may be understood basically in terms of an exciton coupling between the Chl b core of a given CPII unit and at least one of the Chla's of either the same or the adjacent CPII. We propose that this Chl a-Chl b coupling existing in LHC disappears upon segregation into CPII, probably as a result of a detergent-related overall rotation of the strongly coupled Chl b core which changes the relative orientations of the two types of pigments and thus the nature of their coupling.

SPECTRAL PERTURBATIONS OF THE AEQUOREA GREEN‐FLUORESCENT PROTEIN

Abstract— In the jellyfish Aequorea, the green‐fluorescent protein (GFP) functions as the in vivo bio‐luminescence emitter via energy transfer from the photoprotein aequorin. Accumulated evidence has indicated that the Aequorea GFP is a relatively inflexible protein. Present evidence, however, indicates that the chromophore environment is readily accessible to a variety of external perturbants. Native Aequorea GFP has an absorbance maximum at 395 nm and a shoulder at 470 nm. In low ionic strength buffer at neutral pH and room temperature the 395/470 nm absorbance ratio is about 2.0. We show that this ratio is highly variable depending upon temperature, ionic strength, protein concentration, and pH. A maximum ratio of 6.5 (at a protein concentration of 18.6 mg/m/) and minimum of 0.42 (at a pH of 12.2) have been measured. In the latter case, the resulting absorption and excitation spectra resemble those of Renilla GFP in spectral shape (but not wavelength maximum). In all cases as the perturbant is varied the resulting spectra pass through a sharp isosbestic point, suggesting a relatively simple two‐state mechanism. These spectral perturbations are fully reversible. On the basis of these results, we suggest that the chromophore binding site is conformationally flexible. pH‐Dependent changes in the near‐UV and visible circular dichroism spectra plus spectrophotometric titration of tyrosine residues lend additional support to this hypothesis.

Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin.

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.

Circular dichroism and absorption spectra of chiral ketone. I2 complexes in solution

Difference absorption spectroscopy in the visible spectral region is used to investigate the formation of electron donor–acceptor (EDA) complexes between I2 and a series of chiral ketone compounds dissolved in chloroform. Evidence for 1 : 1 complex formation is found for the camphor + I2 and camphorquinone + I2 systems. The camphor + I2 complex exhibits a relatively strong circular dichroism (CD) band in the region of a “perturbed” molecular iodine transition (∼498 nm), and the visible CD spectrum of camphorquinone + I2 also shows evidence of induced optical activity in the bound I2 transitions.

A study of the interaction between D-amino acid oxidase and quasi-substrates.

To study the interaction between D-amino acid oxidase [EC 1.4.3.3] and quasi-substrates such as benzoate and o-, m-, and p-aminobenzoate, visible circular dichroism spectra (CD spectra) were measured and the binding rate and affinity of o-aminobenzoate to the enzyme were observed by following the absorption changes at various wavelengths. We found a new CD band around 560 nm, corresponding to the charge-transfer complexes which result from the formation of aminobenzoate complexes with the enzyme. The ellipticity of this band was positive for the p-aminobenzoate complex, but negative for the o- and m-aminobenzoate complexes. Crossover points in CD spectra were observed at 470 nm for the m-aminobenzoate complex and at 475 nm for the o-aminobenzoate complex. They probably resulted from overlapping of the positive CD band of FAD bound with the enzyme and the negative CD band of the charge-transfer complex. We propose that the amino group in aminobenzoate, not the pi-electrons of the benzene ring, is the electron donor in the charge-transfer complex and that the position of the amino group is very important for the charge-transfer interaction. The binding rate and affinity of o-aminobenzoate to the enzyme were determined using the absorption changes at 370 nm (380 nm), caused by the modification of electronic states of FAD bound with the enzyme, and at 550 nm (565 nm), caused by the formation of the charge-transfer complex of o-aminobenzoate with the enzyme. No differences between these parameters with wavelength were observed. This independence of wavelength simplifies discussion of the experimental data obtained from absorption changes.

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.

N-terminal amino acid sequences of Azotobacter vinelandii and Rhodospirillum rubrum flavodoxins

Summary The amino-terminal sequences of A. vinelandii and R. rubrum flavodoxins have been determined. These proteins have larger molecular weights (mw≅23,000) than do other flavodoxins for which sequences have previously been reported (mw≅14,000). A comparison of these results with those obtained in other laboratories for P. elsdenii, C. pasteurianum, C. MP and D. vulgaris flavodoxins shows that significant homology exists in the FMN phosphate binding site region in all of these flavoproteins. Furthermore, the sequences reveal relationships among these various proteins which correlate with properties such as riboflavin binding ability and visible circular dichroism spectra.

ChemInform Abstract: CIRCULAR DICHROISM OF COPPER(II) COMPLEXES WITH OPTICALLY ACTIVE TETRADENTATE LIGANDS IN AQUEOUS SOLUTIONS

The circular dichroism (CD) spectra of copper(II) complexes containing the asymmetric tetradentate ligands, such as ethylenediamine-N,N′-di-L-α-propionate, ethylenediamine-N,N′-di-L-α-isovalerate, and ethylenediamine-N,N′-di-L-α-hydrocinnamate, were measured in an aqueous solution. Each of the CD curves can be resolved into four components in the d–d region by Gaussian analysis; these components are related to the four possible d–d transitions of the copper(II) ion. The visible CD spectrum profile of the complexes varies with the variation in ligands; this implies that the conformation of the complexes is affected appreciably by the bulkiness of the side chain in the ligand.

Circular Dichroism of Copper(II) Complexes with Optically Active Tetradentate Ligands in Aqueous Solutions

Abstract The circular dichroism (CD) spectra of copper(II) complexes containing the asymmetric tetradentate ligands, such as ethylenediamine-N,N′-di-l-α-propionate, ethylenediamine-N,N′-di-l-α-isovalerate, and ethylenediamine-N,N′-di-l-α-hydrocinnamate, were measured in an aqueous solution. Each of the CD curves can be resolved into four components in the d–d region by Gaussian analysis; these components are related to the four possible d–d transitions of the copper(II) ion. The visible CD spectrum profile of the complexes varies with the variation in ligands; this implies that the conformation of the complexes is affected appreciably by the bulkiness of the side chain in the ligand.