Circular Dichorism Research Articles

Characterization of the Calcium Binding Domain of NADPH Oxidase 5 (NOX5)

Superoxide generated by non-phagocyte NADPH oxidases (NOXs), such as NOX5, is of growing importance for vascular physiology and pathology. NOX5 enzyme consists of a transmembrane heme domain that is linked to a flavoprotein domain that contains FAD and binds NADPH. It appears to be regulated by self-contained Ca2+ binding domains (CaBD), which contains four EF-hands motifs. Previously we demonstrated that this calcium binding gates the heme reduction in NOX5, possibly thorough the CaBD-flavorprotein interaction. To better understand its structure and function, here we characterized the metal binding properties of the recombinant CaBD by fluorescent spectroscopy. Our data revealed that CaBD binds to Ca2+, Mg2+, terbium (Tb3+) in the range of μM to mM. The data are further supported by the studies using the site-directed labeled CaBD. The rate of calcium association was too fast to be determined by a stopped-flow device, but the dissociation rate constant was determined to be 5 s−1 at 20 °C. The ANS titration and Stern-Volmer plots suggested that there was a significant conformational change upon the metal bindings. Interestingly, the spectra of circular dichorism indicated otherwise no change on the context of its secondary structure. However, this conformational change can be observed using the Surface Plasmon Resonance with the CaBD immobilized in the sensor chip. Because our data and other studies suggest there are two different types of calcium bindings in CaBD, currently we are performing the similar studies using N- and C-terminal halves of CaBD (aa 1-78 and 79-184), and mutants. We also are investigating the metal bindings in the CaBD of Dual Oxidase (DUOX), in which its hydrogen peroxide activity is controlled by calcium binding.

Human serum albumin unfolding pathway upon drug binding: A thermodynamic and spectroscopic description

The interest on phenothiazine drugs has been increased during last years due to their proved utility in the treatment of several diseases and biomolecular processes. In the present work, the binding of the amphiphilic phenothiazines promazine and thioridazine hydrochlorides to the carrier protein human serum albumin (HSA) has been examined by ζ-potential, isothermal titration calorimetry (ITC), fluorescence and circular dichorism (CD) spectroscopies, and dynamic light scattering (DLS) at physiological pH with the aim of analyzing the role of the different interactions in the drug complexation process with this protein. The ζ-potential results were used to check the existence of complexation. This is confirmed by a progressive screening of the protein charge up to a reversal point as a consequence of drug binding. On the other hand, binding causes alterations on the tertiary and secondary structures of the protein, which were observed by fluorescence and CD spectroscopies, involving a two-step, three-state transition. The thermodynamics of the binding process was derived from ITC results. The binding enthalpies were negative, which reveal the existence of electrostatic interactions between protein and drug molecules. In addition, increases in entropy are consistent with the predominance of hydrophobic interactions. Two different classes of binding sites were detected, viz. Binding to the first class of binding sites is dominated by an enthalpic contribution due to electrostatic interactions whereas binding to a second class of binding sites is dominated by hydrophobic bonding. In the light of these results, protein conformational change resembles the acid-induced denaturation of HSA with accumulation of an intermediate state. Binding isotherms were derived from microcalorimetric results by using a theoretical model based on the Langmuir isotherm. On the other hand, the population distribution of the different species in solution and their sizes were determined through dynamic light scattering (DLS). Aggregation of drug/protein complexes was found as a result of a possible expansion of protein structure induced at high drug concentrations. In addition, the presence of free drug aggregates at concentrations below the drug critical micelle concentration was also detected.

Tetrameric β3‐Peptide Bundles

Tetrameric β³‐Peptide Bundles

Binding analysis of pazufloxacin mesilate to catalase using spectroscopic methods

The interaction of pazufloxacin mesilate (PZFX) with catalase was studied by spectroscopic methods under simulative physiological conditions. It was observed that the PZFX quenched the intrinsic fluorescence of catalase through a static quenching procedure. Gradual addition of catalase led to a marked increase in fluorescence anisotropy ( r), it was suggested that the drug was located in a restricted environment of catalase. The negative Δ H 0 and Δ S 0 values in the case of PZFX with catalase complex implied that both hydrogen bonds and electrostatic interactions might play a significant role in PZFX binding to catalase. Circular dichorism (CD) spectra detection supported a change in the secondary structure of protein caused by the interaction of PZFX with catalase.

Four New Nonaoxygenated C18 Dibenzocylcooctadiene Lignans from Kadsura philippinensis

Four new nona-oxygenated C18 dibenzocyclooctadiene lignans, kadsuphilins C-F (1-4), were isolated from the EtOAc soluble portion of the alcoholic extract of the aerial parts of Kadsura philippinensis. The structures of 1-4 were elucidated on the basis of extensive spectroscopic analyses, including 2D NMR (HMQC, HMBC, and NOESY) experiments, comparison of the spectral data with those of the related metabolites. The stereochemistries of the biphenyl and octadiene moieties were deduced from circular dichorism (CD) and the NOESY spectra, respectively. The in vitro antiplatelet aggregation activity of metabolites 1-4 also have been evaluated.

Novel extended and branched N-terminal analogs of VIP

The effects of vasoactive intestinal peptide (VIP) are primarily mediated through VPAC1 and VPAC2, receptors that are preferentially coupled to adenylate cyclase activation. As a large majority of the potent VIP antagonists have modifications in the N-terminal domain of the peptide, the effect of multiplication of this domain on VIP was examined with the aim of possibly amplifying peptide-receptor (VPAC1) activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the N-terminal domain that was obtained by either linear tandem extension or by parallel branching. Circular dichorism (CD) analysis revealed that these extended/branched peptides maintained an α helical structure in organic environment, similar to VIP. A specific branched VIP analog was found to be slightly more potent towards VPAC1-related cAMP production as compared to VIP. This analog could have potential therapeutic value in several disorders, similar to VIP. Two branched N-terminal VIP sequences demonstrated superior receptor binding and activation as compared to two N-terminals in tandem. The results suggest that correct alignment of the VIP N-terminal region is important for receptor binding and activation. However, increased receptor binding was not directly associated with increased cAMP production suggesting steric dynamic interactions.

Main chain helical transformation induced through UV light in arylidene polymer

Arylidene polymers are showing a preferred main chain conformation in the solution phase when subjected to the circular dichorism (CD) analysis. Probably the first time a non-azo, achiral and acrylate derivative of arylidene compound when subjected to polymerization is oriented itself in a definite helical form. Ultraviolet (UV) light cross-linking of these polymers induces the main chain asymmetric transformation from one preferred conformation to another conformation. Concentration independent nature of the conformational change was observed and the CD spectrum showing a blue shift at 270 nm due to loss in the conjugation of the molecule upon irradiation with UV light.

Induced Magnetic Polarization in Cu Layers of Gd/Cu Multilayers Studied by X-ray Magnetic Circular Dichroism

The induced magnetic polarization in nonmagnetic layers and its relationship with the magnetization in ferromagnetic layers are investigated for Gd/Cu multilayers with X-ray magnetic circular dichorism (XMCD). The XMCD measurements show that the peak intensities at the Cu K -edge decrease in proportion to those at the Gd L 3 -edge as the temperature increases. The overall shape of the XMCD spectra at the Cu K -edge, on the other hand, does not change. These results suggest that the size of the magnetic polarization in the Cu layers changes but the depth profile remains unchanged when the Gd magnetization varies in size. The XMCD spectra at the Cu K -edge are composed of several positive and negative peaks. A possible origin of the specific shape of the spectra is discussed.

A tradeoff between protein stability and conformational mobility in homotrimeric dUTPases

Oligomerization directs active site formation in homotrimeric 2 ′-deoxyuridine triphosphate pyrophosphatases (dUTPases). Stability of the homotrimer is a central determinant in enzyme function. The present comparative studies of bacterial and fruitfly dUTPases with homologous 3D structures by differential scanning microcalorimetry; fluorescence, circular dichorism and infrared spectroscopies, demonstrate that unfolding is a two-state highly cooperative transition in both dUTPases excluding a significantly populated intermediate state of dissociated and folded monomers. The eukaryotic protein is much less resistant against either thermal or guanidine hydrochloride-induced denaturation. Results suggest that hydrophobic packing of the inner threefold channel of the dUTPase homotrimer greatly contributes to stability.

A new family of spin crossover complexes with a tripod ligand containing three imidazoles: synthesis, characterization, and magnetic properties of [FeIIH3LMe](NO3)2.1.5H2O, [FeIIILMe].3.5H2O, [FeIIH3LMe][FeIILMe]NO3, and [FeIIH3LMe][FeIIILMe](NO3)2 (H3LMe) = Tris[2-(((2-methylimidazol-4-yl)methylidene)amino)ethyl]amine).

A new family of spin crossover complexes, [Fe(II)H(3)L(Me)](NO(3))(2).1.5H(2)O (1), [Fe(III)L(Me)].3.5H(2)O (2), [Fe(II)H(3)L(Me)][Fe(II)L(Me)]NO(3) (3), and [Fe(II)H(3)L(Me)][Fe(III)L(Me)](NO(3))(2) (4), has been synthesized and characterized, where H(3)L(Me) denotes a hexadentate N(6) tripod ligand containing three imidazole groups, tris[2-(((2-methylimidazol-4-yl)methylidene)amino)ethyl]amine. It was found that the spin and oxidation states of the iron complexes with this tripod ligand are tuned by the degree of deprotonation of the imidazole groups and by the 2-methyl imidazole substituent. Magnetic susceptibility and Mössbauer studies revealed that 1 is an HS-Fe(II) complex, 2 exhibits a spin equilibrium between HS and LS-Fe(III), 3 exhibits a two-step spin transition, where the component [Fe(II)L(Me)](-) with the deprotonated ligand participates in the spin transition process in the higher temperature range and the component [Fe(II)H(3)L(Me)](2+) with the neutral ligand participates in the spin transition process in the lower temperature range, and 4 exhibits spin transition of both the Fe(II) and Fe(III) sites. The crystal structure of 3 consists of homochiral extended 2D puckered sheets, in which the capped tripodlike components [Fe(II)H(3)L(Me)](2+) and [Fe(II)L(Me)](-) are alternately arrayed in an up-and-down mode and are linked by the imidazole-imidazolate hydrogen bonds. Furthermore, the adjacent 2D homochiral sheets are stacked in the crystal lattice yielding a conglomerate as confirmed by the enantiomeric circular dichorism spectra. Compounds 3 and 4 showed the LIESST (light induced excited spin state trapping) and reverse-LIESST effects upon irradiation with green and red light, respectively.

Conformation of the transmembrane domains in peripheral myelin protein 22. Part 1. Solution‐phase synthesis and circular dichroism study of protected 17‐residue partial peptides in the first putative transmembrane domain*

Charcot-Marie-Tooth disease (CMT) is the most commonly inherited peripheral neuropathy. DNA duplication and point mutation of the gene encoding peripheral myelin protein 22 (PMP22) have been found in CMT type 1A dominants. To investigate the influence of the point mutation of PMP22 on the secondary structure, protected partial peptides in the putative first transmembrane domain, wild type Boc-IVLH(Bom)VAVLVLLFVSTIV-OMe (1) and its Pro16 mutant Boc-IVLH(Bom)VAVPVLLFVSTIV-OMe (2) were synthesized. Circular dichorism (CD)-spectral analysis suggested that peptide 1 adopts a stable alpha-helical conformation in membrane-mimetic solvent,1-BuOH/1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) system. On the contrary, the mutant 2 favors beta-sheet conformation in the same solvent system. Interestingly, alpha-helix to beta-sheet transition of 2 was observed at higher contents of 1-BuOH than 70%.

Synthesis of metallointercalator-DNA conjugates on a solid support.

Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.

The Synthesis of N4‐(6‐Aminopyridin‐2‐yl)‐2′‐deoxycytidine for Recognizing the CG Base Pair at Neutral pH by Oligodeoxyribonucleotide‐Directed Triple Helix Formation

AbstractThe sequence‐specific recognition of double‐helical DNA by oligodeoxyribonucleotide‐directed triple helix (triplex) formation is limited mostly to purine tracts. To interrupt the purine tract in a target sequence, a non‐natural deoxyribonucleoside N4‐(6‐aminopyridin‐2‐yl)‐2′‐deoxycytidine (pC) was designed to interact with the C base in the CG base pair. The protected phosphoramidite synthon of pC was synthesized in seven steps and then was incorporated into an oligodeoxyribonucleotide by an automatic DNA synthesizer. Two 22‐mers, designated as C2 and P, with a common sequence of 5′‐d‐TmCTXTmCTTCTGTCTCCAGACAG were synthesized in this study. mC is 5‐methyl‐2′‐deoxycytidine and X is either 2′‐deoxycytidine (C) or pC for C2 and P, respectively. C2 is able to form a paper clip type triplex with one C · CG mismatched base triad in slightly acidic conditions but not at the neutral pH. On the other hand, P forms a stable triplex under both acidic and neutral conditions. This indicates that pC is able to form a pC · CG base triad in the triplex. Their physical properties were studied by UV thermal melting experiments and circular dichorism spectroscopy (CD). The thermal melting results imply that the pC · CG base triad is as stable as the C+ · GC triad at pH 6.0, and pC helps the triplex formation preferably at neutral to acidic pH. In addition to the hydrogen bonding interaction with the CG base pair, the hydrophobic interaction of pC may also play an important role in stabilizing the triplex formation of oligodeoxyribonucleotides. In the presence of spermine at either pH 5.0 or pH 6.0, the melting temperature of the third strand of P was elevated about 30 and 21 °C, respectively. Thus, spermine can enhance the stability of the triple‐helical structure.

Circular Dichroism and Magnetic Circular Dichroism Studies of the Mixed-Valence Binuclear Non-Heme Iron Active Site in Uteroferrin and Its Anion Complexes

Uteroferrin (Uf) is a purple acid phosphatase for which the reduced [FeIIFeIII] form of the enzyme is catalytically active in the hydrolysis of phosphate esters. The binuclear non-heme iron active site of the native reduced enzyme (1/2metUf) and its molybdate- and phosphate-bound forms have been studied with absorption (ABS), circular dichorism (CD), and magnetic circular dichroism (MCD) spectroscopies to probe their geometric and electronic structure. CD and MCD in the UV−Vis region give the tyrosine (phenolate) to FeIII charge transfer bands which probe the ferric center. CD and MCD in the NIR region probe the d−d transitions of both the FeII and the FeIII centers. Variable-temperature variable-field (VTVH) MCD combined with EPR data are analyzed to determine the g values and energies of the ground state and the excited sublevels. These parameters are further interpreted in terms of a spin Hamiltonian model, which includes the zero-field splitting (ZFS) of FeII and FeIII centers and the exchange couplin...

Triazinylaniline derivatives as fluorescence probes. Part 3. Effects of calcium and other metal ions on the steady-state and time-resolved fluorescence of bovine brain calmodulin labelled at lysine-75

The fluorescence characteristics of bovine brain calmodulin labelled at lysine-75 by the reactive triazinylaniline (TA) dye p-Et2NC6H4C3N3(Cl)2 reveal structural changes critically dependent on calcium ion binding. Binding of two calcium ions to the C-terminal lobe exposes a hydrophobic region to the TA fluorophore, enhancing the fluorescence yield four-fold. Addition of two further calcium ions, to the N-terminal lobe, decreases the TA probe fluorescence three-fold by a displacement of the hydrophobic probe imposed by mutual interaction of two protein lobes/regions. The protein matrix induces circular dichorism in the attached TA dye in a calcium-dependent manner. Auxiliary binding of divalent metal ions at mmol dm–3 concentrations greatly enhances probe fluorescence. Despite the above changes the probe rotational relaxation times (8.0 ns) and limiting anisotropies (0.340) are insensitive to the calcium status of the triazinylaniline-labelled calmodulin (TACaM). Close co-rotation of the probe with a single lobe of the calmodulin carrier is likely, but the possibility of the TA probe optical transition axis lying parallel to a major axis of protein deformations is not excluded. Complex formation with melittin, and the potential utility of TACaM in the study of calmodulin-peptide interaction kinetics, are described.

Tertiary interactions in the folding pathway of hen lysozyme: kinetic studies using fluorescent probes.

The refolding kinetics of hen lysozyme have been studied using a range of fluorescent probes. These experiments have provided new insight into the nature of intermediates detected in our recent hydrogen-exchange labeling studies [Radford, S.E., et al. (1992) Nature 358, 302-307], which were performed under the same conditions. Protection from exchange results primarily from the development of stabilizing side-chain interactions, and the fluorescence studies reported here have provided a new perspective on this aspect of the refolding process. The intrinsic fluorescence of the six tryptophan residues and its susceptibility to quenching by iodide have been used to monitor the development of hydrophobic structure, and these studies have been complemented by experiments involving binding to a fluorescent hydrophobic dye 1-anilino-naphthalenesulfonic acid (ANS). Formation of fixed tertiary interactions of aromatic residues has been monitored by near-UV circular dichorism, while development of a competent active site has been probed by binding to a competitive inhibitor bearing a fluorescent label, 4-methylumbelliferyl-N,N'-diacetyl-beta-chitobiose. The combination of these techniques has enabled us to monitor the development both of the hydrophobic core of the protein and of interactions between the two folding domains. If the behavior of the tryptophans is representative of the hydrophobic residues of the protein in general, it seems that collapse is already substantial in species formed within the first few milliseconds of refolding and is highly developed in later intermediates which nonetheless appear to lack many fixed tertiary interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the hormone binding domain of human beta 1 thyroid hormone nuclear receptor: is it an alpha/beta barrel?

To understand the structure of the hormone binding domain (HBD) of human beta 1 thyroid hormone nuclear receptor (h-TR beta 1), truncated h-TR beta 1 fragments, MD32 (M169-D456), KD29 (K201-D456), DD28 (D211-D456), KD25 (K235-D456), and KP28 (K201-P448), were analyzed by circular dichorism (CD). MD32 and KD29 show intense CD spectra with double minima at 222 and 208-210 nm, indicating the presence of extensive regions of alpha-helix. DD28 and KD25 have spectra which are reduced in intensity with minima around 215 nm, characteristic of a beta-sheet. The observed spectra are compatible with sequence analysis which predicts that HBD contains alternating stretches of alpha-helix and beta-strand. These extensive decreases in secondary structure in DD28 and KP28 in which the predicted first beta-strand or last alpha-helix was deleted, respectively, were accompanied by the loss of hormone binding activity. On the basis of these results, we suggest a new model for h-TR beta 1 consisting of the known DNA binding domain linked by an alpha-helical hinge to the HBD, with the tertiary structure of an alpha/beta barrel. The model is compatible with previous chemical and genetic studies on the structure of this protein.

The secondary structure of a membrane-embedded peptide from the carboxy terminus of lipophilin as revealed by circular dichorism

Several intramembranous peptides have been isolated from the major myelin proteolipid protein (lipophilin) isolated from normal human myelin membrane after labelling the protein with a membrane-permeable photolabel, 3-( trifluoromethyl)-3-(m-[ 125 I] iodophenyl)diazirine . Peptide T-3, comprising residues 205–268, represents the C-terminal portion of the protein. Reconstitution of peptide T-3 into lipid vesicles prepared from egg phosphatidylcholine (PC) or into lysoPC micelles yielded visually transparent preparations, free of scattering artifacts, which were used for circular dichroism studies to assess the extent of secondary structure in the peptide. Peptide T-3 had a high degree of α-helix in various environments. In aqueous environment, the secondary structure was 45% α-helix, 33% β-structure and 9% β-turns. Transfer of the peptide to PC vesicles or lysoPC micelles increased the proportion of α-helix and decreased that of β-structure. In PC vesicles, the α-helical content was 80% with little or no β-structure. Small amounts of other structures such as β-turns and unordered structures were also present. The partitioning of this C-terminal section of lipophilin into membranes may have an important role initiating and/or stabilizing the native conformation of lipophilin in the myelin membrane.

Relativistic scattered wave calculations of hexachloro- and hexabromoiridate (IV)

Dirac scattered-wave (DSW) calculations are reported for the ground and several excited states [IrCl6]2− and [IrBr6]2−, and comparisons are made to electron spin resonance, optical absorption, and magnetic circular dichorism spectra. Metal and ligand-centered contributions to the Zeeman and hyperfine tensors differ from those estimated from ligand field theory, although both calculations are in qualitative agreement with experiment. The predicted symmetries and spin-orbit splittings of ligand-to-metal charge transfer transitions are approximately correct, but the estimated transitions energies are too low by 0.2–0.7 eV. The implications of this behavior for future relativistic Xα calculations are discussed, with an emphasis on various approximate methods of including spin-polarization effects in relativistic calculations.

Conformational studies on [formula omitted

Poly(N-δ- trimethyl- l-ornithine ), (Me 3Orn) n , is usually not able to attain the α-helical conformation in aqueous solution independent of its pH value; however, it becomes α-helical at low concentrations of sodium perchlorate over a wide pH range according to the circular dichorism (c.d.) spectra. Cl −, SO 4 2− and H 2PO 4 − do not induce α-helix formation. One can conclude that a distinct topology of the anions bound by the side chains is responsible for the α-helix-inducing effect of some water-structure-breaking anions such as perchlorate. This means that the anions are inserted between the −N + of the side groups shielding the positive charges repelling one another. The insertion of the anions requires that the water molecules surrounding the ions can be stripped off, which is easily possible if they are water-structure-breaking ones. At higher perchlorate concentrations, the c.d. spectrum changes. It is characterized by a negative shoulder near 208 nm and a pronounced minimum at ≈ 226 nm. With increasing temperature, the c.d. spectrum of the α-helix occurs. Finally the α-helix undergoes a conformational change to the random coil. The apparent transition enthalpy ΔH vH is remarkably lower than that of the homologue (Me 3Lys) n , obviously due to a lower cooperativity of the transition. In contrast to poly( l-ornithine), (Orn) n , the c.d. spectrum of (Me 3Orn) n remains almost unchanged after adding anionic surfactants such as sodium octyl sulphate (SOS) or sodium dodecyl sulphate (SDS). In organic solvents like methanol or isopropanol, in contrast to (Orn) a and (Lys) n , no α-helix formation occurs. However, in mixtures of these alcohols or dioxane with water, α-helix formation is induced by perchlorate, as in pure water. The thermal stability of the α-helix in these systems is increased.