The electrochemically induced conformational transition of disulfides in bovine serum albumin studied by thin layer circular dichroism spectroelectrochemistry

The conformational transition of disulfides in bovine serum albumin (BSA) induced by electrochemical redox reaction of disulfides were monitored by in-situ circular dichroism (CD) spectroelectrochemistry, with a long optical path thin layer cell and analyzed by a singular value decomposition least square (SVDLS) method. Electrochemical reduction of disulfides drives the left-handed conformation of disulfides changed into the right-handed. At open circuit, eight of the 17 disulfides were of left-handed conformation. Four of the 17 disulfides took part in the electrochemical reduction with an EC mechanism. Only one-fourth of the reduced disulfides returned to left-handed conformation in the re-oxidation process. Some parameters of the electrochemical reduction process, i.e. the number of electrons transferred and electron transfer coefficient, n = 8, alpha n = 0.15, apparent formal potential, E1(0') = -0.65(+/-0.01) V, standard heterogeneous electron transfer rate constant, k1(0) = (2.84 +/- 0.14) x 10(-5) cm s(-1) and chemical reaction equilibrium constant, Kc = (5.13 +/- 0.12) x 10(-2), were also obtained by double logarithmic analysis based on the near-UV absorption spectra with applied potentials.     

Conformational transition of DNA in electroreduction studied by in situ UV and CD thin layer spectroelectrochemistry

Electrochemically induced three conformational transitions of calf thymus DNA from B10.4 to Z10.2-DNA and from B10.2 to B10.4 and to C-DNA in 10 mM phosphate buffer solution (pH 7.21) at glassy carbon electrode are found and studied by in situ circular dichroism (CD) thin layer spectroelectrochemistry with singular value decomposition least square (SVDLS) analysis. It indicates that the so-called B10.2 form and the C-form of DNA may be composed of B10.4 and left-A DNA and of B10.4 and right-A DNA, respectively. The irreversible electrochemical reduction of adenine and cytosine groups in the DNA molecule is studied by UV-Vis spectroelectrochemistry. Some electrochemical parameters alpha n = 0.17, E0' = -0.70 V (vs. Ag/AgCl), and the standard heterogeneous electron transfer rate constant, k0 = 1.8 x 10(-5) cm s(-1), are obtained by double logarithmic analysis and non-linear regression.     

Determination of the formal reduction potential of Lumbricus terrestris hemoglobin using thin layer spectroelectrochemistry

The formal reduction potential (Eo') of Lumbricus terrestris hemoglobin was determined using thin layer spectroelectrochemistry as 0.073 (+/-0.005) V vs Ag/AgCl (0.281 V vs SHE, standard hydrogen electrode). Nernst plots of Lumbricus terrestris hemoglobin with tris-bipyridinecobalt(II) as a mediator titrant have similar linear slopes as Nernst plots of horse heart myoglobin with hexaamineruthenium(II) as a mediator titrant.     

Magnetic circular dichroism of hemoproteins with in situ control of electrochemical potential: "MOTTLE"

Hemoproteins have been recognized for nearly a century and are ubiquitous components of cellular organisms. Despite our familiarity with these proteins, defining the functional role of a given heme can still present considerable challenges. In this situation, magnetic circular dichroism (MCD) is a technique of choice because it has the capacity to define heme oxidation, spin, and ligation states in solution and at ambient temperature. Unfortunately, the resolving power of MCD rarely has been brought to bare on the intermediate redox states accessible to multiheme proteins. This is due in large part to the time-consuming procedure of magnetic field cycling required each time a sample is introduced into the magnet and the risk that control over, and knowledge of, the potential will be lost between sample preparation and spectral acquisition. Here we present a solution to this problem in the form of MCD-compatible optically transparent thin-layer electrochemistry (MOTTLE). MOTTLE defines redox behavior for cytochrome c in good agreement with the literature. In addition, MOTTLE reproduces the redox-driven transformation of heme ligand sets reported for cytochrome bd. Thus, MOTTLE provides a robust analytical tool for the dissection of heme properties with resolution across the electrochemical potential domain.     

Soret circular dichroism of cobalt-substituted myoglobin and hemoglobin derivatives

Circular dichroic spectra in the Soret region were obtained for the following cobalt-substituted hemoproteins: ^CoMb³, ^CoMbO₂, ^CoMbNO, ^CoMb⁺, ^CoHb, ^CoHbO₂, ^CoHbNO and ^CoHb⁺ and compared with the corresponding spectra of the native species to delineate the sensitivity of Soret circular dichroism to ligation, quaternary structures, metal ion substitution and its magnetic moment. Soret rotational strengths, R, were calculated, and dissymmetry ratios were used to reveal hidden transitions. The results indicate that Soret circular dichroism is sensitive to the metal ion, its oxidation state, ligation and local environment but neither to quaternary structural changes as proposed by Ferrone &; Topp (1975), nor to the magnetic moment of the metal ion as suggested by Li &; Johnson (1969).     

How to study proteins by circular dichroism

Circular dichroism (CD) is being increasingly recognised as a valuable technique for examining the structure of proteins in solution. However, the value of many studies using CD is compromised either by inappropriate experimental design or by lack of attention to key aspects of instrument calibration or sample characterisation. In this article, we summarise the basis of the CD approach and its application to the study of proteins, and then present clear guidelines on how reliable data can be obtained and analysed.     

Structural studies of vinblastine alkaloids by exciton coupled circular dichroism

SCF-CI-dipole velocity MO calculations have shown that the bisignate circular dichroic curves of vinblastine/vincristine alkaloids at ca 210 and 220–230 nm are due to exciton coupling between the indoline and indole moieties. Furthermore, a combination of X-ray crystal structure data with MM2 local energy minimization provides a convenient means for estimation of the preferred solution conformation.     

Structural conformation of spidroin in solution: a synchrotron radiation circular dichroism study

Spider silk is made and spun in a complex process that tightly controls the conversion from soluble protein to insoluble fiber. The mechanical properties of the silk fiber are modulated to suit the needs of the spider by various factors in the animal's spinning process. In the major ampullate (MA) gland, the silk proteins are secreted and stored in the lumen of the ampulla. A particular structural fold and functional activity is determined by the spidroins' amino acid sequences as well as the gland's environment. The transition from this liquid stage to the solid fiber is thought to involve the conversion of a predominantly unordered structure to a structure rich in beta-sheet as well as the extraction of water. Circular dichroism provides a quick and versatile method for examining the secondary structure of silk solutions and studying the effects of various conditions. Here we present the relatively novel technique of synchrotron radiation based circular dichroism as a tool for investigating biomolecular structures. Specifically we analyze, in a series of example studies on structural transitions induced in liquid silk, the type of information accessible from this technique and any artifacts that might arise in studying self-assembling systems.     

Structural similarity of chymopapain forms as indicated by circular dichroism.

Four chymopapain forms were isolated by high-resolution liquid chromatography on a cation-exchange column. The three major forms possess nearly identical secondary and tertiary structures, as judged from their c.d. spectra; these components showed similar proteolytic activity and Mr values close to that of papain. The fourth isolated component seems to be a mixture of modified proteins.     

Structural properties of arrestin studied by chemical modification and circular dichroism.

A unique conformation of arrestin is crucial for its interaction with phosphorylated photolyzed rhodopsin. Conformational changes in arrestin were investigated using chemical modification and circular dichroism. We studied the kinetics of sulfhydryl modification of bovine arrestin in order to determine whether its conformation is altered by the presence of ligands or salts at different ionic strengths. We found that all three cysteines (stoichiometry was 2.7 +/- 0.06 3-carboxy-4-nitrophenyl sulfide (NbS)/arrestin) are accessible for modification by NbS2. Under pseudo-first-order conditions (30-100-fold excess of NbS2 over arrestin), the modifications of the 3 cysteines are indistinguishable. At higher concentrations of NbS2 (150-300-fold excess), the pseudo-first-order plot is not linear, and the reaction can be resolved into two processes that involve two classes of sulfhydryl groups. Addition of CaCl2, MgCl2, inorganic phosphate, MgATP, or MgGTP had little effect on the rate of modification of the cysteine residues; however, heparin and inositol hexakisphosphate, which have been shown to induce conformational changes in arrestin, block modification of one sulfhydryl group of arrestin and accelerate the modification of the remaining two. Analysis of CD spectra revealed that arrestin has virtually no alpha-helical structure, about 40% beta-structure, about 18% beta-turns, and about 40% other structure. The CD spectrum for arrestin did not change in the presence of heparin. These studies suggest that arrestin exists in equilibrium between two or more conformational states. However, it is proposed that conversion between these conformations occur without altering significantly the secondary structure of arrestin.     

Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy

Magnetic circular dichroism (MCD) spectra, at ultraviolet–visible or near-infrared wavelengths (185–2000 nm), contain the same transitions observed in conventional absorbance spectroscopy, but their bisignate nature and more stringent selection rules provide greatly enhanced resolution. Thus, they have proved to be invaluable in the study of many transition metal-containing proteins. For mainly technical reasons, MCD has been limited almost exclusively to the measurement of static samples. But the ability to employ the resolving power of MCD to follow changes at transition metal sites would be a potentially significant advance. We describe here the development of a cuvette holder that allows reagent injection and sample mixing within the 50-mm-diameter ambient temperature bore of an energized superconducting solenoid. This has allowed us, for the first time, to monitor time-resolved MCD resulting from in situ chemical manipulation of a metalloprotein sample. Furthermore, we report the parallel development of an electrochemical cell using a three-electrode configuration with physically separated working and counter electrodes, allowing true potentiometric titration to be performed within the bore of the MCD solenoid.     

Vacuum-ultraviolet circular dichroism study of saccharides by synchrotron radiation spectrophotometry

Vacuum-ultraviolet circular dichroism (VUVCD) spectra of five monosaccharides (D-glucose, D-mannose, D-galactose, D-xylose, and D-lyxose) and five disaccharides (maltose, isomaltose, cellobiose, gentiobiose, and lactose) were measured to 160 nm using a synchrotron-radiation VUVCD spectrophotometer in aqueous solution under high vacuum at 25 degrees C. Most of the saccharides show a positive peak with some shoulders at around 170 nm, except for D-galactose and lactose, which show two distinct negative peaks at around 165 and 177 nm. These spectra are influenced by such structural factors as alpha and beta anomers at C-1, axial and equatorial hydroxyl groups at C-2 and C-4, trans (T) and gauche (G) conformations of the hydroxymethyl group at C-5, and the type of glycosidic linkage. Deconvolution of the VUVCD spectra of D-glucose, D-mannose, and D-galactose into six independent Gaussian components for alpha-GG, alpha-GT, alpha-TG, beta-GG, beta-GT, and beta-TG conformations suggests that the alpha anomer has red-shifted spectra relative to the beta anomer, and that GG and GT conformations have positive and negative circular dichroism signs, respectively, while the sign for TG conformation is anomer dependent. These speculations from the deconvolution analyses are also supported by the VUVCD spectra of disaccharides. These results give new insight into the equilibrium conformations of saccharides, demonstrating the usefulness of synchrotron-radiation VUVCD spectroscopy.     

A study of tubulin dimer conformation by near-UV circular dichroism

The near-UV circular dichroism properties of tubulin dimer have been measured for different preparative methods. Tubulin dimer was obtained from assembly compenent microtubule protein by gel filtration, or phosphocellulose ion-exchange chromatography in the presence of magnesium. Tubulin dimer prepared by the protocol of Weisenberg R.C. and Timasheff, S.N. (1970) Biochemistry 9, 4110–4116, was found to be markedly different due to some apparently irreversible change in conformation. We conclude that the removal of microtubule-associated proteins by phosphocellulose ion-exchange chromatography in the presence of magnesium can be performed without affecting the conformation of native tubulin dimer as judged by near-UV circular dichroism.     

Some examples of the use of thin layer spectroelectrochemistry in the study of electron transfer between metals and enzymes

Some examples of the use of optically transparent thin layer electrodes to study electron transfer between platinum or carbon and enzymes are given. With respect to the biomolecule, thin layer spectroelectrochemistry has been used to choose the best electrochemical pretreatment necessary to promote the heterogeneous electron transfer (in the case of lactate dehydrogenase and glucose oxidase), to visualize intermediates in electrochemical reactions (in the case of glucose oxidase and horseradish peroxidase), to determine the electron transfer rate constant between oxidized, intermediate and reduced forms, and to prepare intermediates compounds (in the case of compound III of horseradish peroxidase), or to regenerate by electrochemical means enzymatic cofactors (in the case of hydrogenase).     

A circular dichroism study of modified nucleosides

The conformation of 5-methoxycarbonylmethyluridine and 5-methoxycarbonylmethyl-2-thiouridine was studied by means of circular dichroism in various solvents. In order to calculate the accurate spectral parameters of the Cotton effects, the circular dichroism spectra were resolved into component Gaussian functions which simultaneously fit the adsorption spectra. On the basis of circular dichroism and proton magnetic resonance spectra, these nucleosides were found to occur in the β-configuration with the ³E-gg-anti conformation preferred. Due to the fact that the long-wavelength Cotton effect of mcm⁵s²U is not masked by the Cotton effects of the other nucleic acid monomers, the molecular parameters of this band may be useful for the conformational analysis of tRNA segments.