Changes of phage T7 nucleoprotein structure at low ionic strength. A Raman spectroscopic study.

To detect changes in DNA and/or protein structures of phage T7 under different ionic strength, Raman spectra of phage T7 have been recorded in solutions of three different NaCl + Tris concentrations. Iterative Jansson-Van Cittert deconvolution, as well as decomposition methods have been used to quantify changes in DNA structure. Significant modifications in ratios of contributions from 675 and 685 per cm vibrations, as well as in the DNA backbone vibrations, characteristic for B-DNA, near 835 per cm frequency have been found. Changes of the base electronic structure were identified in the interval between 1280 and 1400 cm(-1). Estimation of the overall protein structure suggests predominant beta-sheet content.

Raman spectroscopic study of conjugates of butyrylcholinesterase with organophosphates.

Raman spectra of human butyrylcholinesterase (BuChE; E.C. 3.1.1.8) were analyzed in the native state and after conjugation with organophosphates (soman, DFP and paraoxon). The secondary structure of the native BuChE in Tris-HCl buffer (pH 7.5), determined from analysis of the amide I polypeptide vibration band, indicates 47% alpha-helices, 26% beta-sheets, 16% turns and 12% undefined structure. We obtained the same values for paraoxon-phosphorylated BuChE, but 39% helical structure, 31% beta-sheets, 17% turns and 13% undefined structure for 'aged' DFP-BuChE conjugates and 36% helical structure, 34% beta-sheets, 20% turns and 10% undefined structure for 'aged' soman-BuChE conjugates. The approximately 10% decrease of alpha-helical structure observed upon phosphorylation by DFP and phosphonylation by soman, probably corresponds to the 'aging' process, which does not take place in the case of paraoxon. Considerable differences have been observed between native, paraoxon inhibited and 'aged' BuChE in aromatic ring vibrations, suggesting that the dealkylation of organophosphate conjugates modifies the environment or the interactions of aromatic amino-acid residues. In the aliphatic side chains an increase of the number of gauche configurations has been observed in 'aged' DFP-BuChE and soman-BuChE.

A Raman spectroscopic study of acetylcholine receptor-rich membranes from Torpedo marmorata. Interaction of the receptor with carbamylcholine and (+)-tubocurarine.

Raman spectroscopy is used to determine structural features of alkali-treated subsynaptic membrane fragments from Torpedo marmorata electric organ, rich in native functional AcChR. Distinct vibrations attributable to the membrane proteins and lipids were identified and studied before and after addition of the agonist carbamylcholine and the competitive antagonist (+)-tubocurarine. The protein secondary structure determined by using amide-I polypeptide vibrational analysis, indicates 47% alpha-helices, 25% beta-sheets, 18% turns and 11% undefined structure. The secondary structure of the AcChR molecule was not subject to large modifications upon addition of carbamylcholine. But, the presence of the (+)-tubocurarine leads to detectable changes in the amide-I region which might be interpreted as reflecting different contributions of alpha-helices and turns in the secondary structure. In addition, Raman spectra provide information about the environment of aromatic amino acids (tyrosine and tryptophan), the (C-C) bonds, the CH2 and CH3 groups of aliphatic side chains, as well as the disulfide (S-S) and cystein (C-S) bonds. The tyrosines seem 'exposed' to the aqueous medium. The Raman spectra of the AcChR-carbamylcholine complex suggest 'exposed' tryptophans, while those of the unliganded membrane-bound AcChR or of the receptor with (+)-tubocurarine are shown 'buried'. The disulfide bridges in the AcChR subunits show identical conformation in the absence and presence of carbamylcholine. On the contrary, considerable changes are found in the AcChR-(+)-tubocurarine complex. Carbamylcholine and especially (+)-tubocurarine decrease lipid fluidity.

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.

Interaction of modified neurotoxins from Naja nigricollis with the nicotinic acetylcholine receptor from Torpedo marmorata. A Raman spectroscopy study.

Two derivatives of alpha-toxin from Naja nigricollis venom were used in order to study, by resonance Raman spectroscopy, its interaction with the nicotinic acetylcholine (AcCho) receptor from membranes of Torpedo marmorata electrocytes. The two modified toxins carry either an NO2 group bound to Tyr25 or a nitrophenylthioether (NPS) bound to Trp29. The comparison of the spectra of the free and bound derivatized toxins indicates that the environment of Tyr25 is not perturbed upon binding to the AcCho receptor; but the surroundings of NPS bound to Trp29 are changed. This result indicates that Tyr25 is not involved in binding, while Trp29 of the alpha-toxin may be in contact with the AcCho receptor. Examination of the spectrum of the AcCho receptor membrane after binding of the NPS-Trp toxin discloses some modifications of the vibrations of the tryptophan and cysteine disulfide bridge of the receptor. These residues are possibly involved in toxin binding.

Structure and chemical modifications of neurotoxin from Naja nigricollis studied by Raman spectroscopy.

Raman spectroscopy was used to determine structural features of the native toxin alpha from Naja nigricollis, which contains only one Trp and one Tyr, and of chemically modified toxins having chromophores added to these two conserved aromatic amino acids. The percentages of secondary structure were determined by using amide I polypeptidic vibration analysis and are in agreement with X-ray structure [Low et al. (1976) Proc. Natl. Acad Sci. U.S.A. 73, 2991-2994] as well as with the geometry of the disulfide bridges estimated by using the v(S-S) vibrations. In the native toxin alpha, the single invariant tyrosine 25 appears to be buried in the structure and involved in a strong hydrogen bond. We have chemically modified these two invariant aromatic side chains by addition of chromophores. The presence of a (nitrophenyl)sulfenyl (NPS) chromophore bound to the Trp does not perturb the secondary structure of the toxin as shown by the analysis of the polypeptidic amide I vibrations; however, the environment of this Trp and the geometry of a disulfide bridge seem to be modified. The secondary structure is not affected by the presence of the NPS chromophore; therefore, the decrease in binding affinity observed after modification of Trp-29 by the reagent NPS-Cl [Faure et al. (1983) Biochemistry 22, 2068-2076] is due to an alteration of the environment of this aromatic amino acid and/or a steric hindrance and not to an overall modification of the toxin structure. The binding assays of [nitrotyrosyl]toxin show that after nitration the affinity toward the monoclonal antibody M alpha 1 is unchanged and that the affinity toward the cholinergic receptor (AcChR) from Torpedo marmorata remains high. We concluded that the structure of toxin alpha after adding the NO2 chromophore to Tyr-25 is the same as it is in native toxin.

Raman spectroscopic study on the conformation of 11 S form acetylcholinesterase from Torpedo californica.

Vibrational Raman spectroscopy has been used to study the conformation of the 11 S form of acetylcholinesterase from Torpedo californica. Secondary structure analysis by the method of Williams [(1983) J. Mol. Biol. 166, 581-603] shows 49% alpha-helical structure, 23% beta-sheets, 11% turns and 15% undefined structure. Secondary structure estimates obtained for this enzyme by Raman spectroscopy and circular dichroism have been analyzed.

A Raman spectroscopic study on the interaction of an ion-channel protein with a phospholipid in a model membrane system (gramicidin A/L-alpha-lysophosphatidylcholine).

The interaction of the ion channel polypeptide gramicidin A with the L-alpha-lysophosphatidylcholine micelles in a membrane state association (approximative molar ratio 1:9) was investigated by Raman spectroscopy. Studies were carried out over the spectral ranges of 700-1700 cm-1 and 2800-3100 cm-1 at 10 degrees C. The Raman spectrum of L-alpha-lysophosphatidylcholine micelles indicated a disordered structure of the lipid acyl chains by the high intensities of the gauche conformation vibrations. Changing from the micellar phase to the membrane state of association with gramicidin A, the intensities of all-trans stretching modes increased whereas the intensities of gauche conformation vibrations decreased, reflecting the emergence of ordered lipid chains. Hydrophobic interactions between the acyl chains and the polypeptide side chain residues were demonstrated. The absence of modifications in intensities of the very strong tryptophan vibrations in the complex spectrum indicated that, if the tryptophan-stacking interactions suggested by some authors exist, they are very weak ones.

A Raman spectroscopic investigation of the lipid state in acetylcholine receptor-rich membranes from Torpedo marmorata.

The lipid state in acetylcholine receptor (AcChR)-rich membranes purified from electric organ of Torpedo marmorata was studied in the temperature interval from 0 degrees C to 35 degrees C using the (C-H) stretching and (C-C) skeletal optical vibrations. The Raman spectra of AcChR-rich membranes, recorded immediately after preparation of the samples, indicate that the lipids are in a predominant triclinic crystalline lattice and do not undergo a phase transition when the temperature increases up to 35 degrees C. However, the polar groups of the lipids appear subject to temperature-induced variations. After extraction of 43-kd and other non-receptor proteins, spectra indicate an order-disorder phase transition of lipids at approximately 21 degrees C. This transition appears less cooperative than the transition of the membrane lipid extract. The role of the proteins in preservation of the crystalline state of lipids in AcChR-rich membranes is discussed.

UV-induced small structural changes in the T7 bacteriophage studied by melting methods.

UV optical absorption and circular dichroism (CD) properties (spectra and melting curves) of T7 bacteriophage were investigated to detect "in situ" structural damages which can be related to the biological inactivation due to UV irradiation. UV doses (0.2-1.2 kJ/m2 at 254 nm) near to the biologically effective minimal dose were applied where the initial genetic damage (approximately 10 events/phage) was observed. The decrease of the melting temperature of the helix-coil transition and the broadening of the transition range indicate the destabilization of the intraphage structure due to the presence of about 0.1-0.6% damaged base concentration.

Vibrational spectroscopic approach to the study of acetylcholine and related compounds.

The present review reports the coordinated application of three spectroscopic methods (Raman, infrared(IR) and inelastic electron tunneling spectroscopy (IETS)) in the study of the conformation of Ach and some analogues (beta-MeAch, Mu and Nic) in solid state, aqueous solution and in interaction with a surface. Useful correlated information is obtained by Raman and IR spectroscopies on the conformational possibilities of these molecules in transition from solid state to aqueous solution. With this information in hand as well as on the basis of Raman and IR study of the nonenzymatic hydrolysis of Ach, the first detailed experimental investigation of the interaction of Ach and beta-MeAch adsorbed on a surface (A1203) is realised by the IETS method. The results are used to discuss an interaction analogous to that of Ach with receptor and another one analogous to that of Ach and AchE.

Thermotropic state transition in isolated platelet membranes studied by Raman spectroscopy.

The thermotropic state transition of the lipids in isolated platelet membranes has been studied by Raman spectroscopy in the temperature range from -3 degrees C to +45 degrees C using the (C-H) stretching vibrations, carotenoid(s) vibrations at 1530 cm-1 and 1160 cm-1, as well as the skeletal optical vibrations. 1. The increase of temperature causes a decrease in intensity of the 2885 cm-1 band relative to the 2855 cm-1 one. The evaluation of the ratio I2885/I2855 as a function of temperature indicates a double thermotropic state transition of platelet membrane lipids: the first one near 5 degrees C and the second near 17.5 degrees C. 2. The ratio I1530/I1160 shows that the intensity variations in the carotenoid(s) peaks follow the second lipid transition. Thus, it seems that the platelet membrane carotenoid(s) might be linked to the lipids which undergo transition near 17.5 degrees C. 3. The spectral changes in the skeletal optical range suggest a considerable proportion of all trans chains in the membrane lipids at lower temperatures whereas gauche structures seem to be introduced at higher temperatures.

Inelastic electron tunneling spectroscopic study of interaction of acetylcholine and beta-methyl acetylcholine with alumina surface.

The conformation of acetylcholine (Ach) and its muscarinic analogue beta-methyl acetylcholine (beta-MeAch) on an alumina surface was analyzed by inelastic electron tunneling spectroscopy (IETS). This method detects vibrational modes of organic molecules that are active in both Raman (R) and IR spectroscopies. By using previously recorded and interpreted R and IR spectra of Ach and beta-MeAch in solid-state and aqueous solutions we studied the perturbations due to adsorption. The results were used to interpret the interaction of both molecules with the alumina surface, and a comparison to that with receptors or with acetylcholinesterase was attempted. In the case of nonhydrolytic interaction, the positive trimethylammonium groups of both molecules seemed to be attracted by the negative oxygen ions of the surface. There was evidence that the O--C--C--N skeleton of Ach changed its conformation in aqueous solution and adopted the solid-state conformation, which is very similar to that of beta-MeAch. This conformation once established, Ach appeared to interact with the alumina surface in the same way as did beta-MeAch: both tunneling spectra were very similar. There was also evidence that in the acetyl part of both molecules the C=O double bond was broken and that the oxygen atom coordinated with an Al+ cation. The acetyl skeleton did not show important conformational changes for either molecule. In the case of hydrolytic interaction of Ach or beta-MeAch, the products of the hydrolysis, acetate ion and choline--the latter also adsorbed in ionic form--were found on the alumina surface. In both cases the conformation of the lateral groups bonded to the choline and acetyl skeletons was also analyzed.