Ellipticity Bands Research Articles

Influence of ouabain and dihydroouabain on the circular dichroism of cardiac plasmalemmal microsomes.

1. The influence of ouabain on the tertiary structure of cardiac plasmalemmal proteins was investigated by means of circular dichroism measurement. Purified plasmalemmal microsomes were obtained by sucrose gradient centrifugation. The CD-spectra of the membranal proteins were shifted to the red and the amplitudes were smaller than those of the same proteins after solubilization. 2. Ouabain induced an increase of the ellipticity bands at 210 and 222 nm of about 50% above the level yielded with microsomes after sonication. At 222 nm ouabain exhibited the half maximum effect at a concentration of 5 X 10(-9) M. The effect could, however, only be exerted if the inside of the microsomes was exposed to ouabain by sonication, thus reflecting the inside-out nature of the plasmalemmal microsomes. 3. The high specificity of the ouabain effect was underlined by the following experiments: a) Dihydroouabain, a much less cardioactive derivative of ouabain proved to be ineffective in corresponding concentrations, b) ouabain had no influence upon the CD spectrum of microsomes derived from cardiac sarcoplasmic reticulum, c) a detergent-like action of ouabain underlying the observed effect can be excluded since highly active tensides, i.e. desoxycholate and dodecylsulfate, only influence the CD spectra at concentrations exceeding 10(-3) M, d) electronmicrographs of microsomes exposed to ouabain demonstrated no alteration of either the appearance or size of the microsomes. 4. The magnitude of the observed ouabain effect indicates that a large portion of the membrane-bound proteins is involved. The number of binding sites and their isolated structural alteration induced by ouabain are not sufficient to account quantitatively for the enhanced amplitudes of the CD spctra. This suggests that ouabain evokes structural changes of membrane proteins different from actual binding sites. It seems, therefore highly improbable that changes of the Na-K-ATPase present in the plasmalemmal microsomes are responsible for the observed effect.

Conformation of Lysozyme Derivatives Modified at Two Carboxyl Groups

Abstract Conformational studies have been carried out on two lysozyme derivatives in which the carboxyl groups of aspartic acid 119 and the COOH-terminal leucine 129 were coupled, after carbodiimide activation, with glycine methyl ester (i.e. (GlyMe)2-Lysoz) or with histidine methyl ester (i.e. (HisMe)2-Lysoz). No changes were observed in the spectral and sedimentation behaviors of the two derivatives relative to those of lysozyme. In ORD measurements the rotatory behaviors of lysozyme and (GlyMe)2-Lysoz were identical both at the negative minimum at 233 nm and at the positive 199-nm extremum. Also, their b0 values were identical. On the other hand, (HisMe)2-Lysoz showed a decrease in the ORD parameters of [m']233, [m']199, and b0 values. The rotatory behaviors of the derivatives were also determined over a wide pH range. It was significant that the ORD parameters of (GlyMe)2-Lysoz and lysozyme were equal throughout the pH range 2 to 11. At a given pH, the corresponding parameters of (HisMe)2-Lysoz were lower. In circular dichroism measurements, the [θ'] values at the negative ellipticity bands at 208 and 220 nm for lysozyme and (GlyMe)2-Lysoz were equal. The ellipticity values for (HisMe)2-Lysoz were suppressed appreciably relative to lysozyme. Although no conformational differences between lysozyme and (GlyMe)2-Lysoz were observed by ORD and CD measurements, some differences were detectable by chemical monitoring of the conformation. Accessibility to tryptic hydrolysis was appreciably increased in (GlyMe)2-Lysoz (1.15 bonds per mole of protein), and more greatly in the (HisMe)2-Lysoz derivative (3.83 bonds) relative to lysozyme (0.14 bond). Also, there was an appreciable increase in the reducibility of the disulfide bonds in (GlyMe)2-Lysoz (0.29 bond) and (HisMe)2-Lysoz (1.1 bonds) relative to native lysozyme (0.03 bond). The enzymic activity of each derivative retained the same pH optimum as native lysozyme but was decreased drastically. The results are valuable for understanding the immunochemistry of the derivatives. Also, the contribution of the interactions of the modified carboxyl groups to the three-dimensional structure in solution and its relation to the crystalline structure are discussed.

Magnetic circular dichroism of non-heme iron proteins

The magnetic circular dichroism (MCD) at 45 kgauss has been determined for a group of non-heme iron proteins. Both transferrin and conalbumin exhibit a single, positive ellipticity band at 330 nm ([θ] M = 560). Oxy- and methemerythrin, spinach and clostridial ferredoxins and rubredoxin all display distinctive multibanded spectra which may reflect such factors as coordination of the metal, its ligands, metal bridging by other atoms, and varying degrees of metalmetal coupling. The MCD spectra of both ferredoxins and rubredoxin undergo dramatic change upon oxidoreduction providing a potential means for relating the electronic structure of the iron to protein function. In contrast to the plant ferredoxins, the magnetic field does not significantly affect the CD spectra of adrenodoxin and putidaredoxin.

Some physicochemical and chemical properties of light chains isolated from rabbit cardiac myosin.

Cardiac light chains released from myosin at alkaline pH were isolated by ammonium sulfate fractionation. The isolated light chains were separated on a diethylaminoethylcellulose column using a phosphate gradient. Sodium dodecyl sulfate acrylamide gel electrophoresis indicated the presence of two light chains, LC1 and LC2 of molecular weight 27 000 and 18 000, respectively. Densitometer traces of the gels gave a molar ratio of 2 to 1 for the two respective protein components. Molecular weights of 24 000 ± 1000 and 16 000 ± 500 were obtained for these two components by high-speed sedimentation equilibrium studies. Amino acid analyses of the two fractions revealed a high ratio of phenylalanine to tyrosine (4.0 and 6.7 for LC1 and LC2, respectively), which also dominates the aromatic circular dichroic (CD.) spectrum. The C.D. spectra of LC1 and LC2 exhibited ellipticity bands at 220 and 206.5 nm, positions typical of the α-helical conformation, but the low ellipticity values at these positions indicated that only a small portion of their polypeptide chains are in the α-helical conformation consistent with their large content of proline.

Biopolymer — Small Molecule Interactions. Optical Properties of the System Biliverdin‐Serum Albumin in Aqueous Medium

AbstractCircular dichroism (CD) and light‐absorption measurements in the wavelength range 300 to 650 nm were carried out for complexes of biliverdin (∼ 2.5 × 10−5 M) with either bovine or human serum albumin (charcoal treated) in aqueous solution (25–27°C) at several protein concentrations and pH values in the range 4 to 10. In all cases investigated, two large ellipticity bands were measured near 385 nm and about 650 nm, respectively, which were of opposite sign. In addition, two smaller bands near 300 and 535 nm were resolved by computer analysis into Gaussian curves of the CD spectrum of biliverdin‐human serum albumin complexes. The main CD bands were inverted in their sign in biliverdin‐bovine serum albumin complexes when compared at pH 5 and 10. Under the conditions used, free biliverdin did not show significant optical activity. The optical activity observed for the complexes is attributed to electric transition dipole coupling of biliverdin with protein transitions and to possible formation of skewed conformations of the biliverdin chromophore upon binding, leading to inherent dissymmetry. Unlike the corresponding bilirubin complexes investigated previously, there appears to be no evidence for substantial band splitting in the small molecule occurring upon binding to the biopolymer. Thus the presence of a center methene bridge instead of an isolating methylene group, the latter allowing internal rotation in the bile pigment, appears largely to determine the mechanism of generation of optical activity. In the case of bilirubin and at excess serum albumin, the observed optical activity is considered to be due mainly to exciton coupling between electric transition dipole moments of the dipyrromethene chromophores within the asymmetrically bound bilirubin. The optical properties investigated may serve as a very sensitive measure of the different types of interaction between the biopolymer and the small conjugated molecule which result from structural and conformational differences of the molecules participating in complex formation.

Effect of diisopropylfluorophosphate, edrophonium and a stabilizing agent on the conformation of acetylcholinesterase.

The far-ultraviolet circular dichroism spectrum of acetylcholinesterase (E.C. 3.1.1.7) contained three extrema at 220, 208 and near 192 nm. The position of the ellipticity bands resembled those reported for α-helical poly-L-glutamic acid.

Magnetic Circular Dichroism of Tryptophanyl Residues in Proteins

The magnetic circular dichroism (MCD) induced in the wavelength region of absorption by the aromatic chromophores of a number of proteins has been examined. The major contribution to the MCD is from the tryptophanyl chromophores and to a much lesser extent the tyrosyl chromophores. The magnetically induced CD of tryptophanyl residues is independent of the environment of the residue as indicated by the insensitivity of the MCD to protein denaturation. These characteristics make MCD a valuable analytical tool for the determination of tryptophan in proteins. Determinations of tryptophan content by this method on 9 proteins containing 0–6 tryptophanyl residues per molecule are reported. Certain features of MCD and natural CD are compared with particular attention to azurin and several mammalian carbonic anhydrases which have large natural ellipticity bands associated with the aromatic chromophores.

Spectropolarimetric studies of binary and ternary complexes of octopine dehydrogenase.

The optical rotatory dispersion and the circular dichroism of octopine dehydrogenase were studied in the presence and in the absence of NADH and of substrate analogues.Calculation based on the parameter b0 yields for the apoenzyme a helix content of approx. 27%. The presence of β‐structure is suggested by the particular shapes of the spectra and the great difference between the values of the helix content calculated from A193 and A225.In the far‐ultraviolet region there is no change in the optical rotatory dispersion and circular dichroic spectra after addition of NADH alone or NADH +dl‐arginine.The binding of NADH to the apoenzyme induces a positive extrinsic Cotton effect centered around the absorption maximum of the reduced coenzyme. It also produced a broad positive ellipticity band in the same spectral region. This change in the molecular ellipticity was used to calculate the number of binding sites for NADH.The attachment of NADH to the apoenzyme also gives rise to a modification of the circular dichroic spectrum in the region of absorption of aromatic chromophores.The binding of a substrate analogue to the holoenzyme also induces spectral changes which might result from the spatial rearrangement of tyrosine and tryptophan. This fact is discussed in relation to earlier spectrophotometric studies which have also indicated environmental changes of aromatic chromophores due to the formation of ternary complexes.

Conformational studies on copolymers of L-lysine and L-leucine: circular dichroism and potentiometric titration studies.

AbstractConformational aspects of a series of copolymers of L‐Leucine and L‐leucine [poly‐(LysxLeuy)] containing 0 to 0.41 mole fraction L‐leucine have been studied by circular dichroism (CD) and potentiometric titration in 0.05M KF solution. CD studies on the α‐helical conformation showed a dependence of the magnitude of the CD ellipticity band at 222 nm on copolymer composition; the [θ]222 decreasing with higher leucine contents. This was interpreted as the result of an increase of the hydrophobicity of the environment of the amide group due to the presence of the leucyl residues. Values of the Zimm‐Rice parameter, σ, for the copolymers were obtained from the potentiometric titrations and used to fit theoretical curves to the experimental data. Using the variation of σ with polymer composition, a value of σ for the leucyl residue was estimated to be 6.3 × 10−2, assuming independence of σ on the amino acid sequence in the copolymer. The free energy change for the conversion of one mole residue from uncharged helix to uncharged coil, ΔGhc°, was also obtained from the titration data for each copolymer up to a leucine mole fraction of 0.16; a value of 385 cal mole−1 was estimated for ΔGhc° for a leucyl residue. These values for σ and ΔGhc° are compared with other values in the literature for various amino acid residues obtained from titration and melting curve data.

The Influence of Lipid on the Conformation of Human Plasma High Density Apolipoproteins

Abstract The interaction of human plasma high density apolipoproteins (apoHDL) with lipids was examined by circular dichroism (CD) of the peptide and aromatic chromophores. By CD criteria native HDL contained about 70% α helical, 5 to 15% β, and 15 to 20% disordered structure. Delipidation decreased the helical content by about 20% with a corresponding increase in disordered structure. The two major apoproteins of HDL (apoA-I and apoA-II), isolated by chromatography in urea, refolded to different extents after removal of urea; apoA-I had more ordered structure than apoA-II (approximately 55% and 35% α helix, respectively). Reconstitution of apoA-I, apoA-II, and apoHDL with both phosphatidylcholine and cholesteryl ester restored, respectively, 119%, 87%, and 100% of the helical structure of the parent HDL. Phosphatidylcholine alone restored 50 to 70% of the increase produced by both phosphatidylcholine and cholesteryl oleate. With each substrate, the increase in helical content on lipid-protein recombination was accompanied by a corresponding decrease in disordered structure. Spectra of HDL in the near-ultraviolet range showed peaks at 258, 264, 283.5, and 290.5 nm. Delipidation reduced and markedly altered the ellipticity bands of the near-ultraviolet CD spectrum. The bands at 283.5 and 290.5 nm, tentatively assigned to one or more of the tryptophan chromophores in apoA-I, were reversed in sign and shifted to 286 and 292 nm, respectively. The near-ultraviolet CD spectrum of the parent HDL was partially restored by recombination of apoHDL with phosphatidylcholine alone and almost completely restored by recombination with both phosphatidylcholine and cholesteryl oleate. Recombination with phosphatidylcholine alone was sufficient to restore the 286 and 292 nm peaks to their original sign and position. These studies indicate that about 20 to 30% of the amino acid residues in the two major HDL apoproteins are involved in a helix-disordered transition on lipid removal or restoration. The near-ultraviolet CD spectrum is especially sensitive to these lipid-induced structural changes. Both phospholipid and cholesteryl esters are required for complete reorganization of the secondary and tertiary structure of HDL.

Circular dichroism of iron, copper, and zinc complexes of transferrin

The absorption banda of copper-transferrin are optically active with well-defined positive dichroic bands in the visible range having ellipticities of [ θ] 620 = 3,620 deg cm 2 dmole −1 and [ θ] 426 = 6,700 deg cm 2 dmole −1 in terms of bound copper. In iron-transferrin there are also two bands; a negative ellipticity band with [ θ] 455 = −16,000 deg cm 2 dmole −1 and a positive ellipticity band with [ θ] 360 = 6,000 deg cm 2 dmole −1 in terms of bound iron. Additional copper binding to iron-transferrin produces an absorption band at 675 nm with an associated negative ellipticity band. Zinc binding to apotransferrin, 2 moles per mole, affects 1 mole of tyrosine per mole of Zn 2+ bound, and the generated tyrosinate spectrum is optically active. There is optical activity in apotransferrin in the 280 nm region attributable to tryptophan, and optical activities in the 270 and 230 nm regions suggest possible disulfide contributions. Neither the tryptophan optical activity nor the optical activities in the region of 270 nm and 230 nm are affected by metal binding. Apotransferrin has an estimated α-helical content of 17–18%, and there is no observable change in this value when 2 moles of Cu 2+ or Zn 2+ are bound per mole of protein.

Circular dichroic titration of dihydrofolate reductase with TPNH

Dihydrofolate reductase from Streptococcus faecium shows a marked aromatic side chain Cotton effect in the 260–310 nm region of its circular dichroic spectrum. This effect consists of three distinct ellipticity bands with maxima centered at 305 nm, 295 nm and 270 nm. Titration of the enzyme with TPNH to a 1:1 stoichiometry results in the generation of an extrinsic Cotton effect at ca. 340 nm and a decrease in the magnitude of the side chain Cotton effect. This is the first such example of a TPNH-generated extrinsic Cotton effect. The data suggest the involvement of tryptophyl residues in coenzyme binding.

Circular dichroism of mammalian cytochrome [formula omitted

The circular dichroic behavior of cardiac cytochrome c 1 was examined between 190 and 600 nm. Both oxidized and reduced forms show a major positive Soret ellipticity band. In the visible region, the reduced cytochrome exhibits a split CD profile which becomes indistinct upon oxidation. These CD spectra are completely different from those of the mammalian cytochrome c despite the resemblance of the absorption spectra and the identity of the prosthetic group (mesoheme) of these two cytochromes.

A circular dichroism study of transketolase from baker's yeast

The circular dichroism of transketolase from baker's yeast was examined in the 400 to 200 nm region in the presence and absence of its coenzyme and substrates. Coenzyme binding caused an extrinsic Cotton effect v pyrophosphate and a tryptophan moiety of the enzyme protein. Addition of donor substrates as D-fructose-6-phosphate and hydroxy-pyruvate abolished the ellipticity band at 235 nm. Steric hindrance might be responsible for the fact that 2-(1,2-dihydroxyethyl) thiamine pyrophosphate (“active glycolaldehyde”) can no longer form a charge transfer complex. On the other hand, acceptor substrates in the transketolase reaction, such as ribose-5-phosphate, did not influence the broad negative dichroism at 325 nm.

The synthesis and circular dichroism of a series of peptides possessing the structure (L-tyrosyl-L-alanyl-L-glutamyl)n.

A series of peptides possessing the structure (l‐Tyr‐l‐Ala‐l‐Glu)n, with n = 1,2,3,4,7,9, and 13 was synthesized. The oligomers up to n = 4 were prepared by stepwise synthesis using the N‐hydroxysuccinimide ester of the tripeptide derivative for the elongation of the chain. The oligomers with n = 7,9, 13 were prepared by a polycondensation technique, followed by gel filtration.The circular dichroism spectra of the above oligopeptides were measured in the wavelength range 200–330 nm in a solution of 0.15 M sodium chloride –0.02 M sodium phosphate, pH 7.4, conditions at which the high molecular weight polytripeptide (Tyr‐Ala‐Glu)n exists in a helical conformation. The circular dichroic spectrum of the high molecular weight polymer exhibits two negative ellipticity bands: an intense band at 220 nm ([Θ]max= 8700) and a weak band at 273 nm ([Θ]max= 360). The tripeptide (n = 1) exhibits a positive ellipticity band at 227 nm ([Θ]max= 4000) and a broad, positive, weak band at 270 nm ([Θ]max= 115). The other oligopeptides (n = 2 to 13) exhibit positive bands in the 227 nm region, with ellipticity values that decrease as the degree of polymerization increases. Except for (Tyr‐Ala‐Glu)13, which has a negative peak centered at 216 nm, circular dichroic spectra of the other oligomers (n = 1 to 9) in the 200–210 nm region resemble that of random coils. In the 260–290 nm region, the circular dichroic curves of the oligopeptides gradually approach the 272 nm band of the polypeptide (Tyr‐Ala‐Glu)n, as the degree of polymerization increases. Under physiological conditions, only the (Tyr‐Ala‐Glu)13 shows an indication of helical content.

Conformational changes in a synthetic antigen induced by specific antibodies.

The high molecular weight copolymer (mol. wt. = 75 000) containing the repeating sequence–(l‐Tyr‐l‐Ala‐l‐Glu)–and denoted as polypeptide (Tyr‐Ala‐Glu)n has previously been shown to possess an α‐helical structure under physiological conditions. Studies were performed to check whether monovalent fragments of antibodies derived from anti‐(Tyr‐Ala‐Glu)n antibodies could convert the partially helical oligopeptide (Tyr‐Ala‐Glu)13 into a more helical conformation.Circular dichroic spectra of mixtures of the monovalent antibody fragments with (Tyr‐Ala‐Glu)13 were measured at two different concentrations of both components. Analogous experiments were done with the same fragments and the polypeptide (Tyr‐Ala‐Glu)n. When anti‐(Tyr‐Ala‐Glu)n antibody fragments were mixed with (Tyr‐Ala‐Glu)13. When the same fragments were mixed with the polypeptide (Tyr‐Ala‐Glu)n, a difference spectrum was observed at the ellipticity band of the polypeptide centered at 275 nm. It was concluded that anti‐(Tyr‐Ala‐Glu)n monovalent antibody fragments could not only convert the (Tyr‐Ala‐Glu)13 into a structure more similar to the polypeptide (Tyr‐Ala‐Glu)n, but could also stabilize the helical content of this polypeptide.

Studies of the protein subunit of pH-inactivated Mengo virus variants: II. Physicochemical properties

Incubation of three variants of Mengo virus at slightly acid pH's in the presence of chloride or bromide ions results in the dissociation of the viral capsid into protein subunits with the release of the intact viral genome. Analytical ultracentrifuge studies show that the protein subunits derived from all three variants are identical and are comprised of a single homogeneous molecular species which has a molecular weight of 407,000 ± 11,000 and an intrinsic sedimentation coefficient ( o 20,w) of 13.4 ± 0.13 S. The frictional ratio of the subunit estimated from the molecular weight and o 20,w is 1.45. On the basis of a hydrodynamically equivalent ellipsoidal model, the protein possesses a maximal axial ratio of eight. The hydrodynamic data, together with those reported recently (O'Callaghan et al., 1970b) suggest that eight to ten molecules of virus polypeptide I (VPI) and four to five molecules of virus polypeptide II (VPII) comprise the molecular anatomy of the subunit. The circular dichroic (cd) spectra of the subunits of all three variants exhibit comparable ellipticity bands at 208 and 225–228 nm with amplitudes of −4000° and −2500°, respectively, suggesting that the protein possesses an apparent α-helical content of approximately 5–10%. The virtual equivalence of the cd spectra for the three subunits suggests that the general orientation and internal arrangement of their polypeptide chains are similar if not identical. Comparison of the calculated and experimental cd profiles for the subunit reveals differences reflected in terms of an underlying positive cd contribution at 223 nm and a negative one at 209 nm. These differences are interpreted in terms of three possibilities: (1) interaction of the protein coat with the nucleic acid; (2) localized melting of the secondary structure of the viral RNA to allow folding into the tightly compact form found within the virion; (3) the absence of structural protein VPIII in the protein subunit.

Evaluation of the proportion of the keto form of D-fructose and related 2-ketohexoses present in aqueous solution

The acyclic forms of D-fructose and some related compounds have been studied by absorption spectroscopy and circular dichroism. These compounds exhibit absorption and ellipticity bands near 275 nm attributed to the keto group of the acyclic form. At mutarotational equilibrium in aqueous solution, D-fructose exists to the extent of 2̃% in the acyclic form, whereas solutions of D-fructose 1-phosphate, D-fructose 6-phosphate, and D-fructose 1,6-diphosphate contain up to 10 times this proportion of the acyclic form. On the basis of circular dichroism spectra, these phosphoric esters, as well as certain disaccharides of D-fructose, could be differentiated with respect to the position of substitution. The differences in position and magnitude of the dichroic bands were related to specific, conformational features of these molecules.

Le dichroisme circulaire de diverses phosphagène phosphotransférases

1. 1.|The conformations of rabbit creatine kinase and some kinases from invertebrates have been studied and compared by circular dichroism between 320 and 185 nm. 2. 2.|All these enzymes present the same dichroic pattern between 250 and 185 nm, suggesting that they have similar secondary and tertiary structures. 3. 3.|The optical activity of their side-chain chromophores is small. However, the comparison of their ellipticity bands between 300 and 250 nm allows us to distinguish a different tyrosine local environment for each enzyme. 4. 4.|In the lobster arginine kinase the ellipticity bands disappear in the region of aromatic chromophores after alkalinisation or nitration of its four accessible tyrosyl residues. This loss of optical activity is discussed in relation to the conformational alterations following such chemical modifications.

Separation of contributions of tryptophans and tyrosines to the ultraviolet circular dichroism spectrum of hen egg-white lysozyme.

Ultraviolet circular dichroism measurements over the wavelength range of 190–300 nm have been carried out on hen egg‐white lysozyme and on an iodine‐oxidized derivative of lysozyme in which Tryptophan‐108 has been selectively modified. The contribution of Tryptophan‐108 to the aromatic circular dichroism profile has been ascertained from a difference spectrum generated between the native and the modified protein. This tryptophyl residue is exceptional in its properties, as it is the principal contributor to the circular dichroism pattern in this wavelength range. The chemically modified protein shows only minor differences from the native molecule in terms of the magnitude of the conformation‐dependent ellipticity bands at 209 and 192 nm. Separation of the partial contributions of tyrosines was obtained from circular dichroism difference spectra constructed from circular dichroism profiles of both the native and iodine oxidized proteins at pH 7 and 11.2. Oligomers of N‐acetyl‐d‐glucosamine, which are inhibitors of lysozyme, markedly affect the circular dichroism spectrum of the enzyme at the aromatic region. It is postulated that this effect on the spectrum is a result of a change in orientation of Tryptophan‐108, which occurs on binding of the inhibitors to the active site of the enzyme.These results are discussed with respect to the proposed three‐dimensional model of lysozyme.