Changes In Circular Dichroism Research Articles

Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability. If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy and entropy of unfolding, the midpoint of the unfolding transition and the free energy of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength requires approximately 8 h.

Structural Transitions in Polyribocytidylic Acid Induced by Changes in pH and Temperature: Vibrational Circular Dichroism Study in Solution and Film States

Changes in vibrational absorption and vibrational circular dichroism (VCD) spectra of polyribocytidylic acid (polyC) in buffered D(2)O solution as a function of pH and temperature are reported. Analysis of these spectral data led us to establish the absorption band at approximately 1693 cm(-1) and associated negative VCD couplet as diagnostic markers of the double-helical form of polyC. An alternate interpretation suggesting quadruplex formation for polyC is also discussed. In addition to the solution state spectral data, pH-dependent absorption and VCD spectra for polyC films derived from dilute H(2)O solutions are also presented. The pH-dependent changes in the absorption and VCD spectra of polyC films are found to be similar to those observed for polyC in solution.

Encapsulation of Ferricytochrome c into the Nanoparticle Made from a Natural Polysaccharide: Schizophyllan

Abstract A novel protein/polysaccharide complexation was found when unfolded ferricytochrome c (Cyt-c) was mixed with denatured schizophyllan (SPG) and the renaturation of both protein and polysaccharide was carried out simultaneously. Upon the complexation, the Q band was split, indicating that ferric Cyt-c was converted into the ferrous state. The changes in circular dichroism and fluorescence from tryptophan showed that the entire protein conformation was drastically deformed.

Structural characterization of globulin from common buckwheat ( Fagopyrum esculentum Moench) using circular dichroism and Raman spectroscopy

Raman and far-UV circular dichroism (CD) spectroscopy was used to study the conformation of globulin from common buckwheat ( Fagopyrum esculentum Moench) (BWG) under the influence of various buffer environments and heat treatments. Secondary structural analysis of BWG by CD spectroscopy yielded 15.0% α-helical, 25.8% β-sheet, 28.9% β-turn and 30.3% random coil contents. Raman spectrum also showed β-sheets as the major secondary structure in native BWG. Chaotropic salts caused band shifts and intensity changes in Raman amide III vibration, indicating transitions from β-sheet to disordered structure following the lyotropic series of anions. Extreme pHs and several protein structure perturbants led to changes in CD and Raman spectral characteristics, demonstrating protein unfolding and denaturation. Increasing heating time at 100 °C induced the appearance of anti-parallel β-sheet (1235–1237 cm −1) and caused a progressive increase in random coil content, suggesting protein denaturation and aggregation. Both non-covalent and covalent interactions play important roles in stabilizing the conformation of BWG.

Design of Functional Ferritin-Like Proteins with Hydrophobic Cavities

Ferritin four-helix bundle subunits self-assemble to create a stable multimer with a large central hydrophilic cavity where metal ions bind. To explore the versatility of this reaction vessel, computational design was used to generate cavities with increasingly apolar surface areas inside a dodecameric ferritin-like protein, Dps. Cavity mutants, in which as many as 120 surface accessible hydrophilic residues were replaced with hydrophobic amino acids, were shown to still assemble properly using size-exclusion chromatography and dynamic light scattering measurements. Wild-type Dps exhibited highly cooperative subunit folding and assembly, which was monitored by changes in Trp fluorescence and UV circular dichroism. The hydrophobic cavity mutants showed distinctly less cooperative unfolding behavior, with one mutant forming a partially assembled intermediate upon guanidine denaturation. Although the stability of Dps to such denaturation decreased with increasing apolar surface area, all proteins exhibited high melting temperatures, T(m) = 74-90 degrees C. Despite the large number of mutations, near-native ability to mineralize iron was maintained. This work illustrates the versatility of the ferritin scaffold for engineering large protein cavities with novel properties.

Allosterism in the binding of AGT to short single‐ and double‐stranded DNAs

O6-alkylguanine-DNA alkyltransferase (AGT) is an important cellular defense against the mutagenic effects of DNA alkylating agents. Here, we report circular dichroism (CD), fluorescence and analytical ultracentrifugation studies that characterize the interaction of AGT with 16 nt single-stranded (ss) and 16 bp double-stranded (ds) DNAs. Sedimentation equilibrium (SE) studies indicate that AGT binds cooperatively to these DNAs and reaches similar limiting stoichiometries (n = 4 ± 0.5) with both ss- and ds-forms. Changes in circular dichroism at long wavelengths (~280 nm) suggest that at least partial unstacking and untwisting accompanies binding to both forms. Enhanced fluorescence of 2-aminopurine-substituted DNAs with binding is also consistent with base-unstacking. This DNA allosterism may reflect the formation of extrahelical bases as part of the DNA-surveillance mechanism. CD analyses suggest that the alpha-helix content of AGT is increased slightly upon ssDNA binding. This conformational change is accompanied by a small but reproducible decrease in tryptophan fluorescence intensity but no detectable change in the wavelength of maximum emission. Since the crystal structure of a single AGT molecule bound to dsDNA is closely similar to that of the free protein, the observed changes may reflect a conformational adjustment that is necessary for cooperative binding. Supported by NIH grant GM070662.

Change in conformation upon complexation of double-armed terephthalamide hosts: dynamic molecular recognition of ditopic guests with strong CD signaling

Conformation of novel terephthalamide hosts 1 changes from anti to syn upon complexation with a bidentate guest ( 2 or 3 ). Chiral sense in the helical syn-form of the double-armed host molecules 1 is biased by the asymmetric centers on the chiral guest [ ( R, R)/( S, S)- 2a ], which can be detected by the drastic change in circular dichroism (CD) spectrum thanks to the exciton coupling of two chromophores (‘arms’) linked to the amide nitrogens. Asymmetric centers on the host molecule also exhibit preference for the twisting direction upon change in geometry from the anti- to syn-form. Thus, the achiral guests ( 2b or 3 ) can also be detected by modulation of CD spectrum upon complexation with the chiral host [ ( R, R)- 1a ].

Initial repertoire of anti-(4-hydroxy-3-nitrophenylacetyl) antibodies as potential donors for effective affinity maturation

We previously found that there are two distinct antibody (Ab) maturation pathways for the immune response of C57BL/6 mice to 4-hydroxy-3-nitrophenylacetyl (NP), one involving Abs with high evolvability (group-H) and the other involving Abs with low evolvability (group-L). Commitment to whichever pathway is followed pre-determined in B cells at an early developmental stage. Candidates for the group-L or -H pathway are thus expected to pre-exist in the initial repertoire of the immune response. In the present study, we examined the initial Ab repertoire from the viewpoint of the latent potential of these Abs for effective affinity maturation. At first, we prepared anti-NP B cell hybridomas at 1 week postimmunization. Although the diversity of the obtained repertoire was maintained mainly by the third complementarity determining region of the heavy chain (CDR-H3), their changes in the near UV circular dichroism resulting from NP-binding allowed for classification into three groups according to the same rules applied in the pathway classification of the maturated Abs. This suggested that the innate structural properties of CDR-H3 were conserved throughout maturation. In other words, in exploring the structure of CDR-H3, it is possible to distinguish the latent potentials of Abs in effective affinity maturation even those making up the initial Ab repertoire. We then examined an artificially designed group-H Ab prototype and found its NP-binding ability sufficient for engagement in the initial repertoire. The question arose here as to why the majority of the actual initial repertoire consisted of the group-L ancestors regardless of their middling NP-binding affinity, which called for further discussion from the viewpoint of the dynamics possibly shaping the repertoire.

Optically Active Electrochromism of Poly(3,4-ethylenedioxythiophene) Synthesized by Electrochemical Polymerization in Lyotropic Liquid Crystal of Hydroxypropyl Cellulose/Water: Active Control of Optical Activity

The electrochemical polymerization of 3,4-ethylenedioxythiophene (EDOT) in a water solution of hydroxypropyl cellulose (HPC) as a polymer lyotropic liquid crystal (LC) gives polyEDOT*/HPC hybrid (PEDOT*/HPC) as an optically active polymer. Circular differential interference contrast (C-DIC) microscopy of the polymer reveals a well-resolved ordered structure, and circular dichroism (CD) measurements indicate a Cotton effect for the polymer film. PEDOT prepared using this chiral nematic LC field exhibits good electrochemical stability and switching between a dark blue reduced state and sky blue oxidized state with corresponding changes in Cotton effect. The polymer thus shows “electrochiroptical effect”, such as optically active electrochromism, and repeating change in CD through electrochemical redox process. The proposed method represents a new technique for the preparation of chiral conducting polymer films.

Structural Transitions in Polyriboadenylic Acid Induced by the Changes in pH and Temperature: Vibrational Circular Dichroism Study in Solution and Film States

A correlation of the changes in vibrational absorption and vibrational circular dichroism (VCD) spectral features with the structural changes of polyriboadenylic acid (polyA) as a function of pH is reported. Analysis of the solution spectral data as a function of pH led us to establishing the importance of a previously unrecognized absorption band at approximately 1665 cm(-1). The present studies indicate that this absorption band and associated VCD originate from the double-helical structure of polyA. The observed changes in solution-state VCD features are indicative of the pH-dependent transitions among the three acidic forms of polyA (A, B, and "frozen" form). In addition to the solution-state spectral data, pH-dependent absorption and VCD spectra for films of polyA, derived from dilute H2O solutions, are also presented. The pH-dependent changes in the absorption and VCD spectra of films are also correlated to the polyA structural changes.

Optically detected magnetic resonance of Cu, Fe and Mn defects inLiNbO3

The optical absorptions of Cu2+, Fe3+ and Mn2+ in LiNbO3 are investigated using optically detected magnetic resonance (ODMR). Themethod is based on the changes of the magnetic circular dichroism (MCD)bands of the ions, caused by the magnetic resonances of the paramagneticground states of these ions. All transitions reached from the ground state ofCu2+ lie between 1180 and 860 nm. Three bands are observed, resulting from combined spin–orbitand trigonal field splitting of the first excited crystal field states. All features resulting fromFe3+ have to be ascribed to the forbidden transitions from the6A tothe 4G cubic precursor states. Their weak and narrow optical transitionsare superimposed on the wide and strong intervalence absorption ofFe2+. This ion leads to a diamagnetic contribution to the MCD, resultingfrom the Zeeman admixture of the first excited spin state ofFe2+ to its diamagnetic ground state. The MCD ofMn2+ is caused by an intervalence transition to the conduction band. ODMR allows us to detectthese bands, lying near 400 nm, which in plain optical absorption are coveredby strong precursor absorptions before the onset of the fundamental transition.

Photoinduced changes of absorption and circular dichroism in a chiral nematic phase containing a photochromic fulgide

Mesophases composed of a nematic mixture (ZLI1132) alone or with a chiral inductor (twisted nematic phase) were doped with a photochromic furylfulgide (FFU) and irradiated with visible and UV light. FFU shows great fatigue resistance and high repeatability of photochromic cycles in these media. Quantum yields for the photoconversions are very similar to values previously reported in solution. Beside from the photochromic changes in absorption spectra, a great and reversible circular dichroism change in the chiral nematic phase was observed in the visible absorption band of the cyclic isomeric form of FFU upon UV irradiation. This change is due to the mesophase-induced chiral absorption. The potential of this signal for information storage is discussed.

Crucial role of the lipid part of lipopolysaccharide for conformational change of minor spike H protein of bacteriophage ϕX174

The contribution of the lipid part of lipopolysaccharide (LPS) to recognition by minor spike H protein of bacteriophage ϕX174 was investigated by comparing the interactions of H protein with LPS and its deacylated derivatives. The fluorescence and circular dichroism (CD) spectra of H protein increased upon binding to intact LPS and a partially deacylated derivative. In contrast, completely deacylated derivatives showed lower affinities and almost no fluorescence or CD changes of H protein. These results demonstrate that the lipid part of LPS is responsible for the conformational change of minor spike H protein, which would function as a trigger for phage DNA ejection for infection of the host cell.

Protonated structures of naturally occurring deoxyribonucleic acids and their interaction with berberine

Protonation-induced conformational changes in natural DNAs of diverse base composition under the influence of low pH, low temperature, and low ionic strength have been studied using various spectroscopic techniques. At pH 3.40, 10 mM [Na +], and at 5 °C, all natural DNAs irrespective of base composition adopted an unusual and stable conformation remarkably different from the canonical B-form conformation. This protonated conformation has been characterized to have unique absorption and circular dichroic spectral characteristics and exhibited cooperative thermal melting profiles with decreased thermal melting temperatures compared to their respective B-form counterparts. The nature of this protonated structure was further investigated by monitoring the interaction of the plant alkaloid, berberine that was previously shown from our laboratory to differentially bind to B-form and H L-form of poly[d(G–C)] [ Bioorg. Med. Chem. 2003, 11, 4861]. Binding of berberine to protonated conformation of natural DNAs resulted in intrinsic circular dichroic changes as well as generation of induced circular dichroic bands for the bound berberine molecule with opposite signs and magnitude compared with B-form structures. Nevertheless, the binding of the alkaloid to both the B and protonated forms was non-linear and non-cooperative as revealed from Scatchard plots derived from spectrophotometric titration data. Steady state fluorescence studies on the other hand showed remarkable increase of the rather weak intrinsic fluorescence of berberine on binding to the protonated structure compared to the B-form structure. Taken together, these results suggest that berberine can detect the formation of significant population of H L-form structures under the influence of protonation irrespective of heterogeneous base compositions in natural DNAs.

Circular-dichroism study of the interaction of aspartate-aminotransferase isoenzymes with a coenzyme analog.

The interaction between a coenzyme derivative, 4'-N-(2,4-dinitro-5-fluorophenyl)-pyridoxamine 5'-phosphate, and the apoenzyme of cytoplasmic and mitochondrial aspartate aminotransferase, was studied by circular dichroism. The specific complexes initially formed were characterized by their circular dichroic spectra. The spectra indicate that the complex is very probably the same for the two isoenzymes. In contrast the spectra recorded during further reaction, in agreement with previous results, monitor different reaction paths and characterize the irreversible labeling at the active site of the cytoplasmic enzyme and regeneration of pyridoxal 5'-phosphate in the mitochondrial enzyme. By following circular dichroic changes in the mitochondrial enzyme, initial kinetic characterization of the cleavage of 4'-N-(2,4-dinitro-5-fluorophenyl)-pyridoxamine 5'-phosphate to form pyridoxal 5'-phosphate at the active site, is provided.

Role of Putative Anion-Binding Sites in Cytoplasmic and Extracellular Channels ofNatronomonas pharaonisHalorhodopsin

Natronomonas (Natronobacterium) pharaonis halorhodopsin (NpHR) is an inward light-driven Cl(-) ion pump. For efficient Cl(-) transport, the existence of Cl(-)-binding or -interacting sites in both extracellular (EC) and cytoplasmic (CP) channels is postulated. Candidates include Arg123 and Thr126 in EC channels and Lys215 and Thr218 in CP channels. The roles played by these amino acid residues in anion binding and in the photocycle have been investigated by mutation of the amino acid residues at these positions. Anion binding was assayed by changes in circular dichroism and the shift in the absorption maximum upon addition of Cl(-) to anion-free NpHR. The binding affinity was affected in mutants in which certain EC residues had been replaced; this finding revealed the importance of Arg123. On the other hand, mutants in which certain residues in the CP channel were replaced (CP mutants) did not show changes in their dissociation constants. The photocycles of these mutants were also examined, and in the case of the EC mutants, the transition to the last step was greatly delayed; on the other hand, in the CP mutants, L2-photointermediate decay was significantly prolonged, except in the case of K215Q, which lacked the O-photointermediate. The importance of Thr218 for binding of Cl(-) to the CP channel was indicated by these results. On the basis of these observations, the possible anion transport mechanism of NpHR was discussed.

Effect of Mutation of the Sac7d Intercalating Residues on the Temperature Dependence of DNA Distortion and Binding Thermodynamics

Sac7d is a small chromatin protein from the hyperthermophile Sulfolobus acidocaldarius which kinks duplex DNA by approximately 66 degrees at a single base pair step with intercalation of V26 and M29 side chains. Site-directed mutagenesis coupled with calorimetric and spectroscopic data has been used to characterize the influence of the intercalating side chains on the structure and thermodynamics of the DNA complex from 5 to 85 degrees C. Two single-alanine substitutions (V26A and M29A) and five double-glycine, -alanine, -leucine, -phenylalanine, and -tryptophan substitutions of the surface residues have been created. NMR and fluorescence titrations indicated that the substitutions had little effect on the structure of the protein or DNA binding site size. Each of the mutant proteins demonstrated a temperature-dependent binding enthalpy which was correlated with a similar temperature dependence in the structure of the complex reflected by changes in fluorescence and circular dichroism. A positive heat capacity change (DeltaC(p)) for DNA binding was observed for only those mutants which also demonstrated a thermotropic structural transition in the complex, and the temperature range for the positive DeltaC(p) coincided with that observed for the structural transition. The thermodynamic data are interpreted using a model in which binding is linked to an endothermic distortion of the DNA in the complex. The results support the proposal that the unfavorable enthalpy of binding of Sac7d at 25 degrees C is due in part to the distortion of DNA.

Gold Nanoparticles as a Colorimetric Sensor for Protein Conformational Changes

Spherical gold nanoparticles and flat gold films are prepared in which yeast iso-1-cytochrome c (Cyt c) is covalently bound to the gold surface by a thiol group in the cystein 102 residue. Upon exposure to solutions of different pH, bound Cyt c unfolds at low pH and refolds at high pH. This conformational change causes measurable shifts in the color of the coated nanoparticle solutions detected by UV-VIS absorption spectroscopy and in the refractive index (RI) of the flat gold films detected by surface plasmon resonance (SPR) spectroscopy. Both experiments demonstrate the same trend with pH, suggesting the use of protein-covered gold nanoparticles as a simple colorimetric sensor for conformational change.

Chiral imides as potential chiroptical switches: synthesis and optical properties

A series of chiral aromatic imides and diimides were synthesized and their electrochemical, absorption, fluorescent, and chiroptical properties were examined for their potential application as molecular chiroptical switches. These compounds exhibit strong UV–vis absorptions, and can be electrochemically reduced to radical anions that absorb in the near infrared (NIR) region. Further reduction to the dianionic states results in new absorptions in the visible region. The changes in circular dichroism upon redox switching were apparent in the UV–vis region but were absent in the NIR region.

Solvent/Gelator Interactions and Supramolecular Structure of Gel Fibers in Cyclic Bis-Urea/Primary Alcohol Organogels

An organogel system consisting of trans-(1S,2S)-bis(ureidododecyl)cyclohexane (SS-BUC) and a series of primary alcohols was explored with optical polarizing microscopy (OPM), electron microscopy, circular dichroism (CD), wide-angle X-ray scattering (WAXS), and synchrotron small-angle X-ray scattering (SAXS). OPM, SAXS, and especially WAXS showed that the gel fiber of SS-BUC/methanol gels essentially consists of SS-BUC crystal itself. SAXS showed that the SS-BUC crystal in the gel takes a lamella with a domain spacing of 5.2 nm. When we left the gel at room temperature, the spacing decreased to 3.1 nm after several months. This distance change may correspond to the structural transition from a double-layer structure to an intercalated-layer structure, which was proposed by Feringa et al. (Chem.-Eur. J. 1999, 5, 937-950) as a possible arrangement of the molecular packing. When the gels in ethanol, propanol, butanol, or octanol were examined, they never showed crystalline peaks in WAXS and SAXS, indicating the amorphous nature of the gels. With increasing the alkyl chain length from ethanol to octanol, dramatic changes were observed in the CD spectrum in the 200-500-nm range. Because these CD changes are correlated to the absorbance of urea, those can be considered as the evidence that the solvents strongly relate to the spatial arrangement between the adjacent urea groups. For the amorphous gels, the cross-sectional correlation function [gammaCu] was directly obtained by the inverse Hankel transform of the SAXS data. The value of gammaCu for the gels is decreased with increasing u (distance between the two scattering bodies, see eq 5). Furthermore, it more rapidly decreases than that of the rigid cylinder model. This feature can be explained by the speculation that many solvent molecules permeate into the SS-BUC fiber. There was a clear difference between ethanol and the other gels, indicating that the solvents with a longer alkyl chain give the more permeated and diffused fiber. This permeated fiber (i.e., wet fiber) can rationalize the dramatic CD change, by presuming that the permeated solvent molecules alter the molecular stacking form.