Biphasic Circular Dichroism Research Articles

Excitonic coupling effect on the circular dichroism spectrum of sodium-pumping rhodopsin KR2.

We investigate the role of excitonic coupling between retinal chromophores of Krokinobacter eikastus rhodopsin 2 (KR2) in the circular dichroism (CD) spectrum using an exciton model combined with the transition density fragment interaction (TDFI) method. Although the multimer formation of retinal protein commonly induces biphasic negative and positive CD bands, the KR2 pentamer shows only a single positive CD band. The TDFI calculation reveals the dominant contribution of the Coulomb interaction and negligible contributions of exchange and charge-transfer interactions to the excitonic coupling energy. The exciton model with TDFI successfully reproduces the main features of the experimental absorption and CD spectra of KR2, which allow us to investigate the mechanism of the CD spectral shape observed in the KR2 pentamer. The results clearly show that the red shift of the CD band is attributed to the excitonic coupling between retinal chromophores. Further analysis reveals that the weak excitonic coupling plays a crucial role in the shape of the CD spectrum. The present approach provides a basis for understanding the origin of the KR2 CD spectrum and is useful for analyzing the mechanism of chromophore-chromophore interactions in biological systems.

Glycosaminoglycan and DNA Binding Induced Intra- and Intermolecular Exciton Coupling of the bis-4-Aminoquinoline Surfen.

Despite the diverse biological activities of the glycosaminoglycan (GAG) antagonist surfen, the molecular details of its interaction with biomacromolecules remain poorly understood. Therefore, heparin and DNA binding properties of surfen were studied by circular dichroism (CD) and UV absorption spectroscopy methods. High-affinity (Ka ~ 10(7) M(-1)) association of surfen to the chiral heparin chain gives rise to a characteristic biphasic CD pattern due to the conformational twist of the aminoquinoline moieties around the central urea bridge. At higher drug loading, intermolecular stacking of surfen molecules alters the induced CD profile and also provokes strong UV hypochromism. In contrast to the right-handed heparin template, binding of surfen to the left-helicity chondroitin sulfate chains produces inverted CD pattern. Large UV hypochromism as well as polyphasic induced ellipticity bands indicate that surfen intercalates between the base pairs of calf-thymus DNA. Extensive CD spectroscopic changes observed at higher drug binding ratios refer to cooperative binding interactions between the intercalated drug molecules. The inherent conformational flexibility of surfen demonstrated here for the first time is important in its binding to distinct macromolecular targets and should be considered for rational drug design of novel GAG antagonists.

Circular dichroism and cross-linking studies of bacteriorhodopsin mutants

Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.

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 (Φ f ; for oxonols this growth is very small). The binding constants ( K) obtained from changing the absorption spectra and Φ f vary from 10 4 to (5–6)×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.

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn(2+) ions.

We present evidence of formation of an intramolecular parallel triple helix with T*A.T and G*G.C base triplets (where * represents the hydrogen bonding interaction between the third strand and the duplex while. represents the Watson-Crick interactions which stabilize the duplex). The third GT strand, containing seven GpT/TpG steps, targets the polypurine sequence 5'-AGG-AGG-GAG-GAG-3'. The triple helix is obtained by the folding back twice of a 36mer, formed by three dodecamers tethered by hydroxyalkyl linkers (-L-). Due to the design of the oligonucleotide, the third strand orientation is parallel with respect to the polypurine strand. Triple helical formation has been studied in concentration conditions in which native gel electrophoresis experiments showed the absence of intermolecular structures. Circular dichroism (CD) and UV spectroscopy have been used to evidence the triplex structure. A CD spectrum characteristic of triple helical formation as well as biphasic UV and CD melting curves have been obtained in high ionic strength NaCl solutions in the presence of Zn(2+) ions. Specific interactions with Zn(2+) ions in low water activity conditions are necessary to stabilize the parallel triplex.

Induced circular dichroism active complexes of synthetic gadolinium(III) porphyrinates with chiral amino acids

Synthetic gadolinium(III) meso-tetrakis(3,5-di-tert-butylphenyl)porphyrinates extracted zwitterionic l-phenylglycine from an aqueous solution to give a 1:1 complex in benzene through synergistic binding of both amino and carboxylic groups of the amino acids. The resulting highly coordinated complex afforded intense circular dichroism (CD) bands of reversed S-shape type at the Soret region (400–450 nm). In the case of d-phenylglycine, a complete mirror image of the CD spectrum was recorded after extraction. While the intensity of the induced CD peak was affected by the nature of the organic solvent, meso-aryl substituent and Cα-residue, its sign was well related to the stereochemistry of the bound amino acid. A combination of biphasic extraction and CD spectrometry provides a promising method for determination of the absolute configuration of unprotected α-amino acids.

Spectroscopic probes of the interactions of the dye Stains-all with deoxycholate and cholate

The biological surfactants sodium cholate (NaC) and sodium deoxycholate (NaDC) differ from the normal surfactants such as sodium dodecyl sulphate (SDS) by having hydrophilic OH groups in their hydrophobic moieties, and they failed to induce sharp blue shifted metachromasia in the common cationic dyes such as acridine orange, methylene blue, pinacyanol etc. However, both the cholates induce extremely sharp and stable blue-shifted metachromasia in the cyanine dye Stains-all (Stal), at concentrate much below the critical micellar concentrations and not disrupted by the excess of surfactants unless at above the respective critical micellar concentrations. Both chiral cholates induce very strong biphasic negative circular dichroism (CD) in Stal. At high surfactant dye both NaDC-Stal and NaC-Stal exhibit a second positive biphasic CD spectrum, indicating the formation of second species of the complexes, not immediately discernible from the respective absorption spectrum. Though the reported structures of micelles and crystals of NaDC are distinctly different from that of NaC, the induced metachromasia and circular dichroism in Stal by the two surfactants are remarkably similar. It is reasonably thought that Stal cations bound at NaC and NaDC are arranged with systematic twists in one sense, responsible for metachromasia and dichroism of the dye aggregates, the formation of NaDC-Stal and NaC-Stal are probably followed by some self organization of the complexes formed in the premicellar range of concentrations, excess surfactants added to these complexes form just part of the solvent. Only above cmc the micelles start disruption of the dye aggregates. Our results fit well with the helical model of NaDC and the Small's model of NaDC micellar aggregate with the hydrophobic surfaces oriented inside.

SPECTRAL MODIFICATIONS OF BACTERIAL ANTENNA COMPLEXES BY LIMITED PROTEOLYSIS

AbstractThe spectral effects of a number of endo‐ and exoproteases to the detergent‐solubilized antenna complexes B802–858(sphaeroides like) of strain Rhodopseudomonas acidophila 10050 and B802–824of strain 7050 were analyzed. Upon protease digestion the time course of spectral modification between 250 and 900 nm was recorded. In addition, circular dichroism (CD) signals were measured between 450 and 950 nm. The endoprotease elastase as well as the exoprotease carboxypeptidase A (CP A) altered the B802–858antenna complex, by changing the 858 nm band hypochromically (30‐50%) with an additional small hypsochromic shift (3–6nm). A combined carboxypeptidase digestion, e.g. CP A and CP B, yielded a further modification to a complex with absorption bands at 800 nm and ˜840 nm (ε˜ 50% of the 800 nm band). By exposing the isolated B803–824antenna complex to the different proteases no significant spectral change was observed. The B802–858antenna complex, modified by CP A, exhibits a60–70% decrease of the biphasic CD signal in the near infrared. The limited proteolysis experiments provide conclusive experimental evidence that the C‐terminal domains of the antenna polypeptides α and β contribute to the formation of (dimeric) bacteriochlorophyll (BChl) a molecules absorbing at around 850 nm. This agrees with the hypothesis, based on comparative amino acid sequence analysis, that in purple bacterial antenna complexes the structural requirements (specific amino acids in the vicinity of the BChl molecules, e.g. aromatic amino acids) for batho‐ and hyperchromicity of BChl molecules apparently reside to a considerable extent in the C‐terminal portions of their antenna apoproteins.

Conformational study of sodium pectate from circular dichroism induced in dyes

AbstractSodium pectate (1c) induces strong circular dichroism (CD) in 3,7‐bis(dimethylamino)‐1,9‐dimethylphenothiazinium chloride (2b) and weak CD in 1‐ethyl‐2‐[3‐(1‐ethyl‐2‐(1H)‐quinolylidene)propen‐2‐yl]quinolinium chloride (pinacyanol chloride) (4) and 2,8‐bis(dimethylamino)‐acridinium chloride (Acridine Orange) (3), and no CD in 3,7‐bis(dimethylamino)phenothiazinium chloride (Methylene Blue) (2a), besides inducing strong metachromasia in all these dyes. The signs of the CD spectra induced in the dyes 2b, 3 and 4 by pectate are opposite to those induced by hyaluronate (HYAT). Pectate is, thus, inferred to have a 31 helical conformation (like HYAT) with the sense of helicity opposite to that of HYAT. Such an inference is also in harmony with the reported 3‐fold screw axis of pectate fibre from X‐ray crystallography. Since left‐handed extended helical polyglutamate induces negative biphasic CD, 1c, depicting positive biphasic CD with the same dye, may be assigned by analogy to have a right‐handed helicity.

Cadmium-thiolate clusters in metallothionein: spectrophotometric and spectropolarimetric features.

Cd-thiolate cluster formation in rabbit liver metallothionein 1 (MT) has been followed at pH 8.4 by monitoring spectroscopic features below 300 nm as a function of increasing Cd-to-apometallothionein (apoMT) ratio. The emerging absorption profiles form a family of closely similar spectra attributable to tetrahedral Cd-tetrathiolate coordination previously established for Cd7-MT [Vasák, M., Kägi, J.H.R., & Hill, H.A.O. (1981) Biochemistry, 20, 2852-2856]. However, there is a 6-nm red shift of the unresolved lowest energy absorption band when greater than 3 equiv of Cd(II) is incorporated. This shift is paralleled by a changeover in the circular dichroism (CD) features of MT from a broad monophasic positive CD profile with ellipticity bands near 240 and 220 nm to a biphasic CD spectrum characterized by positive ellipticity bands at 260 and 224 nm and an interposed negative band at 240 nm. Both features can be attributed to a changeover from separate Cd-tetrathiolate units formed at low metal-to-apoMT ratio to Cd-thiolate clusters when the supply of cysteine ligands becomes limiting. A comparable red shift signaling the transition from the mononuclear to a trinuclear tetrahedral Cd-tetrathiolate complex is also observed upon titration of the synthetic tetrathiol dodecapeptide N-Ac-Pro-Cys-Orn-Cys-Pro-Glu-Cys-Glu-Cys-Arg-Arg-Val with Cd(II). The latter studies also provide evidence for the predominantly ligand (sulfur) character of the lowest energy Cd-tetrathiolate ligand-metal charge-transfer transition. As a corollary it is inferred that the biphasic CD profile arises from excitonic coupling of these sulfur-centered transition dipole moments dissymmetrically oriented within the Cd(II)-thiolate clusters.

Magnetic circular dichroism

Circular dichroism (CD) induced in an electronic transition by an external magnetic field has recently been employed to investigate the nature of chromophores found in biological systems, most notably the tryptophanyl chromophores of proteins and the Co(II), Fe and Fe-porphyrin chromophores of several metalloproteins. Magnetic circular dichroism (MCD) might better be referred to as the molecular Zeeman effect or Faraday effect, since MCD invites direct comparison to natural CD, a comparison which in most respects is misleading. MCD was first observed by Michael Faraday in 1845 and arises from the splitting by the field of magnetically degenerate ground or excited states. Much of the modern theory has been presented by Buckingham and Stephens [ 1,2]. MCD is generated by several mechanisms and one transition can potentially give rise to a complex, often biphasic CD spectrum. This is in marked contrast to natural CD where one gaussian CD band is associated with each optically active transition. When the Zeeman splitting is small compared to the linewidth and at temperatures where the Zeeman energies are < kT, MCD bands can be shown to arise from contributions to net ellipticity by three separate terms, the so-called A, B and C terms. A brief qualitative description of these is appropriate before discussing specific applications of MCD to biologically important chromophores. Splitting of a magnetically degenerate ground state by the magnetic field results in a population asymmetry between the new ground states, since the lower energy state will be preferentially occupied. Hence the transition from the lower state will be more intense and absorption of circularly polarized light of one sense by the lower energy transition will not be completely balanced by absorption of light of the opposite polarization by the adjacent transition of slightly higher energy. Neglecting for the moment the frequency shift, a net gaussian CD band will result, the C term, positive or negative depending on the selection rules. The population asymmetry is temperature dependent, hence the magnitude of bands due to C terms will increase at lower temperatures. Even in the absence of the-population asymmetry (e.g. when only the excited state is split by the field) cancellation of equal and opposite ellipticity from two magnetically degenerate transitions cannot occur, since there is a frequency shift between them. A net biphasic CD will remain, the A term. The frequency shift is proportional to the field strength but independent of temperature; hence, A terms are not temperature dependent. A third mechanism by which the magnetic field can induce ellipticity is by field-induced mixing of the zero-Iield states of the chromophore, the B term. This depends on the details of the particular energy levels characterizing the chromophore and delineation of this term requires a complete spectral analysis. B terms are gaussian and temperature independent, but the magnitude may vary inversely with field strength. Significant contributions from all three terms need not be present in a particular MCD spectrum and before detailed analysis can be made, it is important to sort out the contributions of the various terms, a potentially complex problem. Determination of the temperature dependence is essential and will reveal the contribution of C terms, Applications of MCD to biological systems from which useful information can be derived will depend in great measure on the specific nature of the chromophore and on what type of information is sought. The molecular Zeeman effect is insensitive to conformation. In contrast natural CD is highly sensitive to conformation and derives its great usefulness as well as its complexity of interpretation from its dependence on the dissymmetric potential N 159