Nonactin,monactin, dinactin,trinactin, and tetranactin. A Raman spectroscopic study

Raman spectra are reported for crystalline nonactin, monactin, dinactin, trinactin, and tetranactin and their solutions in CCl₄, CHCl₃, CH₃OH, and 4:1 (v/v) CH₃OH:CHCl₃. The macrotetrolide nactins selectively bind a wide variety of cations, and are important model compounds for the study of ion complexation. The conformations of nonactin, monactin, and dinactin in solution are similar. Their conformations are found to be sufficiently open to permit the ester carbonyl groups to form hydrogen bonds with CH₃OH; this gives rise to characteristic changes in the vibration frequencies associated with the ester groups. Nonactin, which is the least soluble of the nactins in CH₃OH, is also the least effective at forming hydrogen bonds with CH₃OH. The greater ability of the higher nactins to form hydrogen bonds with CH₃OH may be due to the increased inductive effect of ethyl over methyl side chains, which may increase the dipole moment of the ester carbonyl groups. Spectra of crystalline nonactin, monactin, and tetranactin are fairly similar, while the spectra of dinactin and trinactin comprise a second, distinct family. This is consistent with X-ray crystallographic studies, which show that nonactin and tetranactin form monoclinic crystals, while trinactin is triclinic.     

Raman and infrared spectroscopic study of gramicidin A conformations

Raman scattering and infrared spectroscopic techniques were used to study the vibrational spectrum and conformation of the membrane channel protein gramicidin A in the solid state, in organic solutions and, using Raman scattering only, in a phospholipid environment. The investigation also includes measurements on head- and tail-group-modifled gramicidin A and a potassium thiocyanate-gramicidin A complex. Tentative identification of the molecular vibrations is proposed on the basis of the data on model compounds. The existence of four distinct conformations of the gramicidin A chain is established: conformation I present in the solid state, and CH₃OH and CD₃OD solutions; conformation II present in films cast from CHCl₃ solution; conformation III present in (CH₃)₂SO and (CD₃)₂SO solutions at concentrations below 0.5 m gramicidin A; and conformation IV present in the potassium thiocyanate-gramicidin A complex. The data obtainable on a gramicidin A-phospholipid suspension indicate a gramicidin A conformation in this environment corresponding either to the conformation I or II. The details of the spectra in the amide I region are shown to be consistent with a β-parallel hydrogen-bonded π_LD helix for conformational I, in terms of the polypeptide vibrational calculations of Nevskaya and co-workers. Conformation II is found to be consistent with an antiparallel double-stranded π_LD helix, while conformations III and IV probably have π-helical structures with larger channel diameters. The data on head- and tail-modified gramicidin A molecules indicate that their conformations are only slightly different from that of gramicidin A in conformation I.     

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.     

Vibrational spectroscopic study of glutathione complexation in aqueous solutions

A spectroscopic study of glutathione (GSH) and glutathione disulfide (GSSG) has been performed using Fourier-transformed infrared absorption and Raman scattering in order to pinpoint the sites of complexation of these two species with water and particularly with H2O2. Molecules of GSH and GSSG were studied in KBr pellets, and in aqueous solutions of H2O, D2O, and H2O with H2O2 (1 mol L(-1)) to characterize the specific influence of the solvent molecules. A time-resolved Raman study was performed for GSH/H2O2, in aqueous solution at 1:1 molar ratio in order to observe the formation of GSSG and to discuss the mechanism of this redox reaction.     

A new tripodal poly-imine indole-containing ligand: Synthesis, complexation, spectroscopic and theoretical studies

A novel flexible tripodal ligand derived from 3-methylindole, (“InTREN” L), and its mononuclear Zn(II), Cu(II), Ni(II), Hg(II) and Pd(II) complexes are described. All compounds gave analytically pure solid samples. Characterisation of the compounds was accomplished by ¹H NMR, IR and absorption spectroscopies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and elemental analysis and their geometry optimized using density functional theory (DFT).Time-dependent-density functional theory (TD-DFT) calculations have been used to assign the lowest energy absorption bands of the free ligand and the Zn(II) complex. The system is a very good candidate for in situ recognition/coordination effects by MALDI-TOF-MS spectrometry and absorption spectroscopy. The presence of three indole groups in InTREN opens up the possibility to synthesize new three-dimensional self-assembly supramolecular structures.     

Raman spectroscopic study of tropomyosin denaturation

Raman spectra of the pH denaturation of tropomyosin are presented. In the native state tropomyosin has an alpha-helical content of nearly 90%, but this value drops rapidly as the pH is raised above 9.5. The Raman spectrum of the native state is characterized by a strong amide I line appearing at 1655 cm^?1, very weak scattering in the amide III region around 1250 cm^?1, and a medium-intensity line at 940 cm^?1. When the protein is pH-denatured, a strong amide III line appears at 1254 cm^?1 and the 940 cm^?1 line becomes weak. The intensities of the latter two lines are a sensitive measure of the alpha-helical and disordered chain content. These results are consistent with the helix-to-coil studies of the polypeptides. The Raman spectra of α-casein and prothrombin, proteins thought to have little or no ordered secondary structure, are investigated. The amide III regions of both spectra display strong lines at 1254 cm^?1 and only weak scattering is observed at 940 cm^?1, features characteristic of the denatured tropomyosin spectrum. The amide I mode of α-casein appears at 1668 cm^?1, in agreement with the previously reported spectra of disordered polypeptides, poly-L -glutamic acid and poly-L -lysine at pH 7.0 and mechanically deformed poly-L -alanine.     

Raman spectroscopic study of left-handed Z-RNA

The solvent conditions that induce the formation of a left-handed Z form of poly[r(G-C)] have been extended to include 6.5 M NaBr at 35 degrees C and 3.8 M MgCl2 at room temperature. The analysis of the A----Z transition in RNA by circular dichroism (CD), 1H and 31P NMR, and Raman spectroscopy shows that two distinct forms of left-handed RNA exist. The ZR-RNA structure forms in high concentrations of NaBr and NaClO4 and exhibits a unique CD signature. ZD-RNA is found in concentrated MgCl2 and has a CD signature similar to the Z form of poly[d(G-C)]. The loss of Raman intensity of the 813-cm-1 A-form marker band in both the A----ZR-RNA and A----ZD-RNA transitions parallels the loss of intensity at 835 cm-1 in the B----Z transition of DNA. A guanine vibration that is sensitive to the glycosyl torsion angle shifts from 671 cm-1 in A-RNA to 641 cm-1 in both ZD- and ZR-RNA, similar to the B----Z transition in DNA in which this band shifts from 682 to 625 cm-1. Significant differences in the glycosyl angle and sugar pucker between Z-DNA and Z-RNA are suggested by the 16-cm-1 difference in the position of this band. The Raman evidence for structural difference between ZD- and ZR-RNA comes from two groups of bands: First, Raman intensities between 1180 and 1600 cm-1 of ZD-RNA differ from those for ZR-RNA, corroborating the CD evidence for differences in base-stacking geometry. Second, the phosphodiester stretching bands near 815 cm-1 provide evidence of differences in backbone geometry between ZD- and ZR-RNA.     

Chemometric prediction of alginate monomer composition: A comparative spectroscopic study using IR, Raman, NIR and NMR

The potential of using infrared (IR), Raman and near infrared (NIR) spectroscopy combined with chemometrics for reliable and rapid determination of the ratio of mannuronic and guluronic acid (M/G ratio) in commercial sodium alginate powders has been investigated. The reference method for quantification of the M/G ratio was solution-state ¹H nuclear magnetic resonance (NMR) spectroscopy. For a set of 100 commercial alginate powders with a M/G ratio range of 0.5–2.1 quantitative calibrations using partial least squares regression (PLSR) were developed and compared for the three spectroscopic methods. All three spectroscopic methods yielded models with prediction errors (RMSEP) of 0.08 and correlation coefficients between 0.96 and 0.97. However, the model based on extended inverted signal corrected (EISC) Raman spectra stood out by only using one PLS component for the prediction. The results are comparable to that of the experimental error of the reference method estimated to be between 0.01 and 0.08.     

The complexation of metal cations by D-galacturonic acid: a spectroscopic study

Solid complexes of D-galacturonic acid (GalA) with cobalt(II), copper(II), nickel(II) and oxovanadium(IV) (1-4) were prepared and characterised. The metal-to-ligand molar ratio was 1:2 for complexes 1-3 and 1:1 for complex 4. The alpha- and beta-anomers of GalA were detected in all the complexes in solid state and in solutions. An addition of small amounts of the paramagnetic complexes to the D2O solution of pure ligand led to NMR line broadening of some 1H and 13C nuclei. This broadening was sensitive to the anomeric state of GalA in the case of complexes 1 and 4. NMR and vibrational spectroscopic data indicate the formation of carboxylate complexes of all the cations, while noncarboxylic oxygens are also involved into the metal bonding in some cases. VCD spectra of complexes 1-4 in D2O and Me2SO-d6 solutions confirm that GalA carboxylic group may participate in the formation of optically active species around the metal cation. Possible ways of GalA coordination by metal cations of this study were proposed and discussed.     

Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.     

Raman spectroscopic study of the valinomycin--KSCN complex

This paper reports the first Raman spectroscopic study of the potassium complex of the cation-specific antibiotic valinomycin. Complete Raman spectra (140 to 3600 cm^?1) of crystalline valinomycin-KSCN and its CCl₄, CHCl₃ and C₂H₅OH solutions are presented and used to probe the structure of the complex in these environments. In all cases a single, narrow peak is observed in the ester CO stretch region (1750 to 1775 cm^?1) which contrasts strongly with the broad bands observed in solutions of uncomplexed valinomycin. This is consistent with the presence of a single conformation in which all six ester CO groups co-ordinate an enclosed potassium ion. We find that although the ester CO stretch frequencies of the complex are similar in the solid state and in non-polar solution (~1770 cm^?1) they are considerably different in the presence of polar solvents (~1756 cm^?1); this may indicate that the complexed potassium ion is still free to interact with nearby solvent ions (and possibly its counterion) through gaps in the hydrophobic “shield” provided by the hydrocarbon residues of valinomycin. In contrast the amide CO frequencies of the complex (~1650 cm^?1) are solvent-independent. These groups are apparently strongly hydrogen-bonded to provide a rather rigid, compact framework for the complex conformation.     

Oxygen binding to hemocyanin: a resonance Raman spectroscopic study

Oxygenation of hemocyanin gives rise to resonance Raman peaks at 742 and 282 cm^?1. The 742 cm^?1 peak which is in resonance with the 575 nm charge transfer band shifts to 704 cm^?1 when ¹⁸O₂ is substituted for ¹⁶O₂. Our results establish that the bound oxygen is in the form of peroxide (O₂^2?). The 282 cm^?1 peak which is in resonance with the 340 nm optical transition is insensitive to isotopic substitution, suggesting that the 282 cm^?1 peak corresponds to a vibration involving the magnetically-coupled Cu(II)··Cu(II) centers.     

Molecular structure of carrageenans and kappa oligomers: a Raman spectroscopic study

The Raman spectra of kappa and iota carrageenan, a desulphated furcellaran and a series of oligomers have been compared in the region 700-1500 cm-1. Spectral differences depending on the amount and the location of the sulphate group on the ring, the chain length, the nature of the counterion and the conformation are discussed. Indications that the ionic interactions in the Na+ salts of the oligomers are different from those in K+ and Rb+ salts are given. On the macromolecular level it is found that the vibrational movements of the skeleton are related to the chain flexibility and the conformation. In gels of K+ and Rb+ kappa carrageenan spectral evidence is given for the existence of structural order.     

Calcium-induced lateral phase separations in phosphatidylcholine-phosphatidic acid mixtures. A Raman spectroscopic study

The effects of calcium ions on mixed membranes of dimyristoylphosphatidic acid (DMPA) and dimyristoylphosphatidylcholine (DMPC) with either the PA or the PC component deuterated have been studied by Raman spectroscopy. The spectra of the pure components show that the acyl chains of hydrated DMPA bilayers are less tightly packed and have more trans bonds than those of DMPC. This behavior appears to be due to the particular arrangement of the polar head groups of DMPA for which the glycerol chain is oriented parallel to the bilayer surface. In agreement with the calorimetrically determined phase diagram [Graham, I., Gagné, J., & Silvius, J. R. (1985) Biochemistry (preceding paper in this issue)], the Raman results show that, in the absence of calcium, DMPA and DMPC are completely miscible at an equimolar ratio but undergo extensive phase separation in the presence of excess calcium. DMPC in phase-separated DMPC-DMPA (Ca2+) mixtures has a conformation that is very similar to that of pure DMPC bilayers, but it is packed more tightly since, depending on the temperature, it is at least partly incorporated into either a solid solution in DMPA or a DMPA-Ca2+-rich "cochleate" phase. This latter shows the same characteristics as the cochleate phase of pure DMPA-Ca2+ which is highly ordered and does not give rise to a thermotropic transition between 5 and 100 degrees C. However, the cochleate phase in DMPA (Ca2+)-DMPC mixtures contains some 20 mol % of DMPC trapped in small domains. These clusters do not melt cooperatively but become as fluid as pure DMPC at 50 degrees C.