The effect of resonance energy homotransfer on the intrinsic tryptophan fluorescence emission of the bothropstoxin-I dimer.

Bothopstoxin-I (BthTX-I) is a homodimeric Lys49-PLA2 homologue from the venom of Bothrops jararacussu in which a single Trp77 residue is located at the dimer interface. Intrinsic tryptophan fluorescence emission (ITFE) quenching by iodide and acrylamide has confirmed that a dimer to monomer transition occurs on reducing the pH from 7.0 to 5.0. Both the monomer and the dimer showed an excitation wavelength-dependent increase in the fluorescence emission maximum, however the excitation curve of the dimer was blue-shifted with respect to the monomeric form. No differences in the absorption or circular dichroism spectra between pH 5.0 and 7.0 were observed, suggesting that this curve shift is due neither to altered electronic ground states nor to exciton coupling of the Trp residues. We suggest that fluorescence resonance energy homotransfer between Trp77 residues at the BthTX-I dimer interface results in excitation of an acceptor Trp population which demonstrates a red-shifted fluorescence emission.     

Why is it important to simultaneously use more than one chiroptical spectroscopic method for determining the structures of chiral molecules?

In recent years, four different chiroptical spectroscopic methods, namely vibrational circular dichroism, vibrational Raman optical activity, electronic circular dichroism, and optical rotatory dispersion, have become popular for establishing the absolute configuration and predominant conformations of chiral molecules in solution state. Many individual laboratories normally utilize only one of these methods to derive the molecular structural information. Although that approach may be satisfactory for most of the molecules studied, it is to be noted that in some instances a single method can give ambiguous conclusions or may not give complete structural information. This article summarizes the situations where simultaneous use of more than one chiroptical spectroscopic method is required to obtain molecular structural information and recommends the routine application of more than one chiroptical spectroscopic method for any given molecule.     

Conformation change of hornet silk proteins in the solid phase in response to external stimulation

Hornet silks adopt a variety of morphology such as fibers, sponge, films, and gels depending on the preparation methods. We have studied the conformation change of hornet silk proteins (Vespa mandarina) as regenerated films, using chiroptical spectrophotometer universal chiroptical spectrophotometer 1, which can measure true circular dichroism spectra without artifact signals that are intrinsic to solid‐state samples. The spectra showed that the proteins in films alter the conformation rapidly from the α‐helix to the coiled coil and then to a β‐sheet structure in response to heat/moisture treatment, but the transformation stopped at the coiled coil state when the sample was soaked in EtOH/water solution. Water is required for the α‐helix to the coiled coil transition, and extra energy is required for the further transition to a β‐sheet structure. This is the first successful circular dichroism study of fibril silk proteins to follow the conformation changes in the solid state. This work shows that proteins can undergo conformational changes easily even in the solid phase in response to external stimuli, and this can be traced by solid‐phase‐feasible chiroptical spectrophotometers. Application of unnatural stress to proteins gives valuable insights into their structure and characteristics.     

Absorption Circular Dichroism Induced by Contorted Electrical Oscillations in Rectangular Nanoholes

The chiroptical response of plasmonic chiral nanostructures can be tuned by combining different structures. In this paper, an L-shaped metal strip is introduced into a rectangular metal nanohole to generate absorption circular dichroism (ACD). The results of a finite-element method calculation show that ACD effects result from contorted electrical oscillations in the L-shaped strip. Additional calculations show that the ACD effects of the proposed metasurface depend strongly on the structure parameters. These findings provide not only a mechanism for enhancing chiroptical responses in planar structures but also a general strategy for the chiral manipulation of light and creation of chiroptical devices.

     

Chiroptical properties of phospholipids

The chiroptical properties of phospholipids were investigated by optical rotatory dispersion and circular dichroism. The spectra of phosphatidylcholine varied with the solvent used. The sign of the circular dichroism effects differed in some cases from previously reported. The results show that it is possible to distinguish different types of phospholipids from each other. The results also show that optical rotatory dispersion and circular dichroism might be a very useful tool for the determination of the configuration of various phospholipids.     

Chiral superstructures of insulin amyloid fibrils

Hydrodynamic forces are capable of inducing structural order in dispersed solid phases, and of causing symmetry-breaking when chiral crystals precipitate from an achiral liquid phase. Until it was observed upon vortex-assisted fibrillation of insulin, such behavior had been thought to be confined to few unbiological systems. In this paper we are discussing chiroptical properties of two chiral variants of insulin amyloid, termed +ICD and -ICD, which form during the process of chiral bifurcation in vortexed solutions of aggregating insulin. As conventional measurements of circular dichroism of solid, anisotropic substances are particularly vulnerable to overlapping influences of linear birefringence and linear dichroism, we have employed complementary tools including dedicated universal chiroptical spectrophotometer to rule out such artifacts. We propose that the strong chiroptical properties of +ICD and -ICD insulin fibrils are an aspect of genuine superstructural chirality of amyloid fibrils and of powerful excitonic couplings taking place within them. A comparison of thioflavin T complexes with fibrils formed by insulin and polyglutamic acid suggests that the extrinsic Cotton effect stemming from the level of single twisted dye molecules is weaker, although diagnostically useful, and cannot account for the overall magnitude of ICD of the dye bound to ±ICD insulin amyloid.     

Enantioselective sensing of chiral amino alcohols with a stereodynamic arylacetylene-based probe

Enantioselective induced circular dichroism analysis of amino alcohols has been accomplished using a conformationally flexible arylacetylene-based probe exhibiting two terminal aldehyde groups. The chirality of the amino alcohol substrates is imprinted on the stereodynamic receptor upon [1 + 2] condensation, which ultimately generates a strong chiroptical response. The distinct induced circular dichroism effects of the diimines obtained can be used for enantioselective sensing and enantiomeric excess determination of a wide range of substrates.     

Urea-induced denaturation of human serum albumin labeled with acrylodan

We induced the denaturation of unlabeled human serum albumin (HSA) and of similar albumin labeled with acrylodan (6-acryloyl-2-dimethylamino naphthalene) with urea and studied the transition profiles using circular dichroism and fluorescence spectroscopy. The circular dichroism spectra for both albumin preparations resulted in the same curves, thus indicating that labeling with acrylodan does not perturb the conformation of HSA. Our results indicate that the denaturation of both albumin preparations takes place at a single, two-state transition with midpoint at about 6 M urea, due to the unfolding of its domain II. It is important to point out that even at 8 M urea, some residual structure remains in the HSA. Great changes in the fluorescence of the dye bound to the protein were observed by addition of solid guanidine hydrochloride to the protein labeled with acrylodan dissolved in 8 M urea, indicating that domain I of this protein was not denatured by urea.     

Ligand-induced conformational changes in cytosolic protein kinase C

The changes in intrinsic spectral properties of protein kinase C were monitored upon association with its divalent cation and lipid activators in a model membrane system. The enzyme demonstrated changes in both its intrinsic fluorescence and far ultraviolet circular dichroism spectra upon association with lipid vesicles in the absence of calcium. The acidic phospholipid, phosphatidylserine, significantly quenched the intrinsic tryptophan fluorescence and was also the most potent lipid support for the phosphorylating activity of the enzyme. The enzyme was fully activated by a number of Ca2(+)-lipid combinations which correlated with maximal fluorescence quenching (40-50%) of available tryptophan residues in hydrophobic domains. The circular dichroism structure of the associated active-protein Ca2(+)-lipid complexes suggested different active enzyme secondary structures. However, the Ca2(+)-dependent changes in fluorescence and circular dichroism spectra were observed only after the enzyme associated with the lipid vesicles. These data suggest that protein kinase C has the properties of a complex multidomain protein and provides an additional perspective into the mechanism of protein kinase C activation.     

Study on the Absolute Configurations of 3‐Alkylphthalides using TDDFT Calculations of Chiroptical Properties

Absolute configurations (ACs) of 3‐alkylphthalides including natural products (?)‐3‐n‐butylphthalide ( (S)‐1 ) and fuscinarin have been studied using chiroptical properties and quantum chemical calculation. Electronic circular dichroism and optical rotatory dispersion spectra of (S)‐1 predicted adopting time‐dependent density functional theory and hybrid functionals coincide very well with the experimental and literature data of (S)‐1 , leading unambiguously to AC assignment as S for levorotatory isomer. The relationship between structures and chiroptical properties of 3‐alkylphthalides were also studied using theoretical calculation. It is found that when the alkyl group is adjacent to the single chiral center in the molecule, both the length of the alkyl side chain and the polarity of solvent may exert significant effect on electronic circular dichroism spectra. On the basis of these observations, it is recommended that the long‐chain alkyl group may be replaced by at least propyl instead of methyl group in such compounds. The present work shows that combination of chiroptical properties and ab initio calculations can provide a feasible and reliable way to the AC establishment of novel 3‐alkylphthalide derivatives with a high degree of confidence. Chirality 24:987‐993, 2012. © 2012 Wiley Periodicals, Inc.     

Thyroglobulin structure-function: the effect of iodination on the structure of human thyroglobulin

Thyroglobulin of very low iodine content has been prepared from a single non-toxic human goitre. The initial iodine content of the protein (0.038%) has been increased to levels of 0.16% and 0.85% by in vitro treatment with thyroid peroxidase and the resulting proteins studied with respect to their intrinsic fluorescence, circular dichroism spectra and binding of the hydrophobic probe 1,8-anilinonaphthalene sulfonic acid (ANS). While significant differences were observed between levels of iodination in both the ANS binding and intrinsic fluorescence of the thyroglobulin, no significant differences in the near and far UV circular dichroism spectra of the protein as a function of iodine content were observed. These data suggest that, the iodination of thyroglobulin effects specific areas of the protein without significant disruption of its overall secondary structure.     

Exploring potentialities and limitations of stapled o‐oligo(phenyleneethynylene)s (o‐OPEs) as efficient circularly polarized luminescence emitters

In this paper, we have studied the chiroptical properties of a family of o‐oligo(phenyleneethynylene) (o‐OPE) derivatives with different steric hindrance. Experimental results show high dissymmetry factors (g_abs and g_lum up to 1.1 × 10^?2) and very similar electronic circular dichroism (ECD) and circularly polarized luminescence (CPL) for all the derivatives that make this basic o‐OPE scaffold a robust pure organic emitter. Vibrational circular dichroism spectra are used to characterize conformational properties in solution. Density functional theory and time‐dependent density functional theory calculations support experimental results also proving that ECD and CPL are almost exclusively linked to helical moiety and not to size or conformation of substituents. As chiroptical properties of these emitters are independent of substituents, this OPE scaffold can be used as basic skeleton for the design of sensing probes with high CPL efficiencies.     

Chirality responsive helical poly(phenylacetylene) bearing L-proline pendants

A novel, optically active, cis-transoidal poly(phenylacetylene) bearing an L-proline residue as the pendant group (poly-1) was prepared by the polymerization of the corresponding monomer using a rhodium catalyst in water, and its chiroptical property was investigated using circular dichroism spectroscopy. Poly-1 showed intense Cotton effects in the UV-visible region of the polymer backbone in water, resulting from the prevailing one-handed helical conformation induced by the covalent-bonded chiral L-proline pendants and exhibited a unique helix-sense inversion in response to external, achiral, and chiral stimuli, such as the solvent and interactions with chiral small molecules. We found that poly-1 could enantioselectively trap 1,1'-2-binaphthol within its hydrophobic helical cavity inside the polymer in aqueous media and underwent an inversion of its helical sense in the presence of one of the enantiomers. The effect of the optical purity of 1,1'-2-binaphthol on the chiroptical properties of poly-1 was also investigated.     

Chirality sensing with stereodynamic copper(I) complexes

Three Cu(I) complexes derived from stereodynamic diphosphine ligands were synthesized and used for chirality sensing. The coordination of diamines and amino acids to these complexes generates distinct circular dichroism signals. The chiroptical sensor response allows determination of the absolute configuration and the enantiomeric excess of the analyte at low concentrations. This method is operationally simple, fast, and attractive for high‐throughput sensing applications.     

Circular polarization of fluorescence of chlorophyll in solution and in native structures

Chlorophyll dimers in solution, subchloroplast particles and chloroplasts were investigated by their circular dichroism and circular polarization of their fluorescence, which reflect their optical rotatory power in the ground state and electronically excited state, respectively. The chlorophyll dimers in fluid solution lose their optical activity upon electronic excitation, reflecting a marked concomitant change in the structure of the dimers. This change is arrested in a solution of very high viscosity. The pronounced difference between the circular polarization of the dimers in fluid media and that of subchloroplast particles and chloroplasts indicates that the former are not suitable models for associated chlorophyll in native structures in electronically excited states. Impairment of the photochemical activity of chloroplasts by heat treatment is accompanied by a reduction of the circular polarization of the fluorescence, which probably reflects a disorganization in structure. The same extent of circular polarization was observed in the fluorescence of chloroplasts regardless whether the reaction centers are open or closed; thus either the same molecules are emitting in the two cases or, if different molecules emit, they are packed in a similar way.     

Specific optical rotation is a versatile tool for the identification of critical micelle concentration and micellar growth of tartaric acid–based diastereomeric amphiphiles

Four novel tartaric acid–based diastereomeric chiral amphiphiles, two being enantiomers of the other two, have been synthesized and investigated using chiroptical spectroscopic methods, along with tensiometry and dynamic light scattering experiments. We found that an inflection point in specific optical rotation (SOR) values at ~0.32 mM corresponds to the critical micelle concentration (CMC). The increase in magnitude of SOR values beyond CMC corresponds to the growth of aggregates. For enantiomers, oppositely signed SOR values were observed, ruling out the possibility for the presence of aggregation size mediated artefacts. SOR values did not exhibit concentration dependence for a chiral tartaric acid based non‐aggregating analogue further establishing the absence of artefacts or anomalous interaction of tartaric acid based head group with solvent. Electronic circular dichroism spectra showed no significant changes in band positions or intensities with concentration. Due to the requirement for higher concentrations (~200 mM) needed to obtain vibrational circular dichroism spectra, these measurements are not found to be useful for studying concentration dependent properties of chiral amphiphiles.     

Investigations of intermediates appearing in the reassociation of the light-harvesting 1 complex of Rhodospirillum rubrum

We investigated the temperature-mediated reassociation of the B820 subunit of Rs. rubrum to form a light-harvesting 1 complex (LH 1). By combining several spectroscopic techniques with global spectral data analysis fitting, we present evidence for the occurence of two spectral intermediates that appear during the reassociation process. At high temperatures, halfway the reassociation reaction, a prominent intermediate appears that has an absorption maximum around 850 nm, a fluorescence maximum around 860–867 nm, a high anisotropy (0.3 to 0.4) and a circular dichroism spectrum with three or four bands with alternating signs. At lower temperatures, more towards the end of the reassociation process, a second intermediate tends to appear that has an absorption maximum around 860 nm, a fluorescence maximum around 885 nm, a medium to high anisotropy (0.1 to 0.3) and a circular dichroism spectrum with two bands with alternating signs. The latter circular dichroism spectrum has a blueshifted zero-crossing compared to the spectrum of the LH 1 complex. Both intermediates have the spectroscopic features of a small oligomer. In the Q_y region, the fluorescence anisotropy of both intermediates slightly increases at longer excitation wavelengths, indicative for energy transfer among the pigments within the intermediate oligomers. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electronic circular dichroism for the detection of microalbuminuria

Over the past decades, chiroptical spectroscopy has proved its incomparable ability to elucidate the structure and spatial arrangement of chiral molecules. Systematic analysis of biomolecules in the natural environment of biofluids, however, remains challenging. In this study, we used chiroptical spectroscopy to monitor urinary levels of human serum albumin. Not only severe proteinuria but even just a slightly increased urinary excretion of albumin (microalbuminuria) may indicate serious health complications, especially for diabetic individuals. Given the chiral nature of albumin and its typical spectral pattern, it may be easily observable by chiroptical spectroscopy, particularly electronic circular dichroism. The performed chiroptical analysis of urine not only allowed the detection of clinically confirmed microalbuminuria but was also able to reveal this pathological condition in cases beyond the diagnostic capability of common clinical procedures. Thus, our approach suggests that electronic circular dichroism is a useful tool for the fast and reliable qualitative monitoring of microalbuminuria with the potential for a quantitative analysis in the future.     

Determination of the absolute configurations of natural products via density functional theory calculations of vibrational circular dichroism, electronic circular dichroism, and optical rotation: the iso-schizozygane alkaloids isoschizogaline and isoschizogamine

The development of density functional theory (DFT) methods for the calculation of vibrational circular dichroism (VCD), electronic circular dichroism (ECD), and transparent spectral region optical rotation (OR) has revolutionized the determination of the absolute configurations (ACs) of chiral molecules using these chiroptical properties. We report the concerted application of DFT calculations of VCD, ECD, and OR to the determination of the ACs of the isoschizozygane alkaloid natural products, isoschizogaline, and isochizogamine, whose ACs have not previously been determined. The ACs of naturally occurring (-)-isoschizogaline and (-)-isoschizogamine, are both determined definitively to be 2R, 7R, 20S, 21S.