Ceruloplasmin-anion interaction. A circular dichroism spectroscopic study.

The effect of anion binding to ceruloplasmin has been studied using absorption and cirbular dichroism spectral data. At anion to ceruloplasmin molar ratios approaching infinite, OCN-, N3- and SCN- bind to ceruloplasmin giving rise to similar alterations in circular dichroism and absorption spectra. The positive bands at 610 and 520 nm in circular dichroism spectra disappear, a negative one apperars at 600 nm and the peak at 450 nm is only slightly modified. There is a new negative band at 410 nm well-defined in OCN- ceruloplasmin spectra. The decrease in absorption at 610 nm is ascribed to the disruption of one type I Cu-S(cysteine) bond owing presumably to the changes induced by anions in the protein secondary structure. The new band at 410 nm is assigned to a charge transfer transition from the ligand replacing cysteine at its binding site. Both absorption and circular dichroism spectra show isobestic points indicating that anion binding to the enzyme, disruption of one of the two type I Cu-S bonds and coordination of this Cu to another protein residue take place simultaneously.     

Conformational features of bradykinin. A circular dichroism study of the aromatic side-chains

The circular dichroism (CD) of the peptide hormone bradykinin and its analogues, [Phe(H4)5]-bradykinin, [Phe(H4)8]bradykinin, [Phe(H4)5,8]bradykinin, [TyrOMe5]bradykinin, [TyrOMe8]bradykinin and [TyrOMe5.8]bradykinin, is described. The comparison of the CD spectra of these analogues with each other, recorded under a variety of conditions (pH, solvent, temperature), allows the monitoring of the behaviour of the aromatic side-chains (phenylalanine, tyrosine) and an estimation of their respective spectral contributions in both spectral regions (320-250 nm, 250-190 nm) with good precision. Conformational non-equivalence of the residues Phe-5 and Phe-8 together with some overall conformational features of bradykinin are thus established.     

Conformational studies on parvalbumins by circular dichroism.

Structural variations of two parvalbumins, Whiting III and Pike III, in various denaturing conditions, have been studied by circular dichroism. CD signals are depressed from 4 urea. For Pike III, acidic pH, sodium dodecyl sulfate or complete removal of Ca2+ show little effect in the far ultraviolet region but rather strong effects in the near ultraviolet. For Whiting III similar results are obtained at acidic pH. Carboxymethylated Whiting III (0.15 Ca2+/mol) shows, on the contrary, decreased CD signals in the far and in the near ultraviolet spectra. Addition of Ca2+ fully restores the native CD spectra in both proteins. Ca2+ binding produces structural modifications which are found to vary according to parvalbumin and which seem in any case different from those described for troponin C.     

Variability of light-induced circular dichroism spectra of photosystem I complexes of cyanobacteria

Circular dichroism (CD) spectra of photosystem I (PSI) complexes of the cyanobacteria Thermosynechococcus elongatus, Arthrospira platensis and Synechocystis sp. PCC 6803 were studied. CD spectra of dark-adapted PSI trimers and monomers, measured at 77 K, show common bands at 669–670(+), 673(+), 680(−), 683–685(−), 696–697(−), 702(−) and 711(−) nm. The intensities of these bands are species specific. In addition, bands at 683–685(−) and 673(+) nm differ in intensity for trimeric and monomeric PSI complexes. CD difference spectra (P700⁺–P700) of PSI complexes at 283 K exhibit conservative bands at 701(−) and 691(+) nm due to changes in resonance interaction of chlorophylls in the reaction center upon oxidation of P700. Additional bands are observed at 671(−), 678(+), 685(−), 693(−) nm and in the region 720–725 nm those intensities correlate with intensities of analogous bands of antenna chlorophylls in dark-adapted CD spectra. It is suggested that the variability of CD difference spectra of PSI complexes is determined by changes in resonance interaction of reaction center chlorophylls with closely located antenna chlorophylls.     

Deconvolution of the circular dichroism spectra of proteins: the circular dichroism spectra of the antiparallel beta-sheet in proteins.

A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure beta-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:669-679, 1991), was improved by the addition of proteins with high beta-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (alpha-helix, antiparallel beta-pleated sheet, beta-turns, and unordered conformation), as well as a curve attributed to the "aromatic contribution" in the wavelength range of 195-240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the alpha-helical structure, but upon analysis yielded a considerable amount of beta-sheet in agreement with the X-ray structure.     

Vibrational circular dichroism spectra of unblocked proline oligomers.

Vibrational Circular Dichroism (VCD) spectra of unblocked L-proline oligopeptides, (Pro)n n = 3 to 7, dissolved in D2O are reported. For these oligomers, the VCD spectra can be attributed to a conformational dominance of the trans amide conformation with subunits interrelated by a left-handed twist, particularly for the longer oligomers. As a function of oligomer length, formation of this conformation starts at n = 3; and by n = 5 a spectrum closely resembling that of the poly-L-proline II helix in shape and magnitude is seen. The VCD data are compared with previous (Pro)n results using IR, CD, Raman and NMR spectroscopies, and reasons for the variations in interpretation are discussed.     

A circular dichroism study of charged polypeptides interaction with salts.

The effect of salts on the experimental circular dichroism spectra of polypeptides is presented using poly-L-lysine as the main model. Salt effects are analyzed into: (a) shielding at low (less than 0.5 M) concentrations of all salts; (b) binding to positively charged and some neutrally charged side-chains by certain anions (e.g., CCl3COO-, CF3C00-, ClO4-), with induction of helicity; (c) binding of these same anions, at high concentration, to the backbone leading toward random structure; (d) binding of high concentration of denaturing cations (La+3, Ca++, Li+) to the backbone, with La+3 and Ca++ leading to collapsed random structure (R) while Li+ tends to leave the polypeptide somewhat extended; (e) indirect interaction of salting-out salts (NaH2PO4, (NH4)2SO4, NH4F), at high concentration, leading toward complete alpha helicity, probably by competition with the polypeptide and the anion for available water. Effects of changing the temperature from 5 degrees to 50 degrees on the circular dishroism spectra of different polypeptide-salt solutions throughout the region from extended (LES) to alpha helical conformation are analyzed in terms of introduction of randomness (R) at high temperature. Applications to effects of salt on protein structures are considered.     

Conformational features of bradykinin. A circular dichroism study of some peptide fragments and structural analogues of bradykinin.

The vasoactive hormone bradykinin, its N-and C-terminal fragments and some structural analogues were studied by Circular Dichroism. Conformational features of the peptide can be detected by comparative analysis of the various CD spectra recorded as a function of aqueous pH, solvent and temperature. It is shown that the two biologically essential arginine residues (Arg1 and Arg9) are important for the specific folded bradykinin conformation. Differences between bradykinin, its fragments and analogues become clearly established in conformational terms, and are discussed in relation to the biological activity of these peptides.     

Determination of protein secondary structure from circular dichroism spectra. III. Protein-derived base spectra of circular dichroism for antiparallel and parallel beta-structures

Protein-derived basic CD spectra for alpha-helix, antiparallel and parallel beta-structures, beta-bends and irregular form of proteins have been determined from the experimental CD spectra of six (myoglobin, lysozyme, ribonuclease A, papain, lactate dehydrogenase, subtilisin BPN') or seven (glyceraldehyde-3-phosphate dehydrogenase added) reference proteins and the analysis of the X-ray data. The secondary structures of thirteen proteins (seven reference and six additional ones) have been analysed using the basic CD spectra thus obtained. The data obtained have been compared with the results of the X-ray data analysis. It is shown that the accuracy of determination of the beta-structure and beta-bends contents using our basic CD spectra is about 2-3 times better than using the basic spectra reported by Chang et al. (Analyt. Biochem. 91, 13-31, 1978).     

Conformational studies of peptide heart stimulant anthopleurin A. Laser Raman, circular dichroism, fluorescence spectral studies, and Chou-Fasman calculations.

Sea anemone contain a number of closely related peptide heart stimulants. In the present investigation, the conformation of anthropleurin A from Anthopleura xanthogrammica was investigated by laser Raman, circular dichroism, and fluorescence spectral methods and by the Chou-Fasman method using sequence data. The recent 13C NMR data of the peptide (Norton, R.S., and Norton, T.R. (1979) J. Biol. Chem., in press) provided useful information for the interpretation of the above-mentioned spectral data. The results from these spectral methods suggested that anthropleurin A and the related sea anemone peptides are roughly spherical in shape due to the presence of some beta-bends, possibly due to a beta-pleated sheet region and due to the 3 cystine residues in the peptide which exist in the gauche-gauche-gauche configuration. The sole tyrosine residue is exposed to the solvent, a finding which has now been confirmed by 13C NMR. The laser Raman and fluorescence spectral procedures showed that one or more of the tryptophan residues are buried. Interestingly, the reduction of the native protein with dithioerythritol did not change the spherical shape even in the presence of 5 M guanidine HCl and the carboxymethylcysteine derivative of the peptide was folded even in the presence of the denaturing agent, guanidine HCl.     

A comparative Raman spectroscopic study of cholinesterases.

We report Raman spectra of various cholinesterases: lytic tetrameric forms (G4) obtained by tryptic digestion of asymmetric acetylcholinesterase (AChE) from Torpedo californica and Electrophorus electricus, a PI-PLC-treated dimeric form (G2) of AChE from T marmorata, and the soluble tetrameric form (G4) of butyrylcholinesterase (BuChE) from human plasma. The contribution of different types of secondary structure was estimated by analyzing the amide I band, using the method of Williams. The spectra of cholinesterases in 10 mM Tris-HCl (pH 7.0) indicate the presence of both alpha-helices (about 50%) and beta-sheets (about 25%), together with 15% turns and 10% undefined structures. In 20 mM phosphate buffer (pH 7.0), the spectra indicated a smaller contribution of alpha-helical structure (about 35%) and an increased beta-sheet content (from 25 to 35%). This shows that the ionic milieu profoundly affects either the conformation of the protein (AChE activity is known to be sensitive to ionic strength), or the evaluation of secondary structure, or both. In addition, we analyzed vibrations corresponding to the side chains of aromatic and aliphatic amino acids. In particular, the analyses of the tyrosine doublet (830-850 cm-1) and of the tryptophan vibration at 880 cm-1 indicated that these residues are predominantly 'exposed' on the surface of the molecules.     

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.