BiospectroscopyVolume 2, Issue 5 p. 339-340 Biospectroscopists' Calendar Biospectroscopists' calendar R. K. Dukor, R. K. DukorSearch for more papers by this author R. K. Dukor, R. K. DukorSearch for more papers by this author First published: 1996 https://doi.org/10.1002/bspy.350020503AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume2, Issue51996Pages 339-340 RelatedInformation
BiospectroscopyVolume 2, Issue 6 p. 413-414 Biospectroscopists' Calendar Calendar editor R. K. Dukor, R. K. DukorSearch for more papers by this author R. K. Dukor, R. K. DukorSearch for more papers by this author First published: 1996 https://doi.org/10.1002/bspy.350020602AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume2, Issue61996Pages 413-414 RelatedInformation
The ba3 cytochrome oxidase from Thermus thermophilus was studied by resonance Raman spectroscopy. The component spectra of both heme groups were determined by using different excitation wavelengths. In the ferric state the heme a3 group reveals resonance Raman marker bands characteristic for two high spin species with the heme iron in an in-plane and an out-of-plane configuration that reflects a coordination equilibrium. This equilibrium obviously results from protonation of one of the axial ligands that is ascribed to a hydroxide. Coordination by its protonated form, a water molecule, may be too weak to keep the heme iron in the porphyrin plane. The corresponding Fe-OH2 stretching mode was attributed to a weak H/D-sensitive band at 464 cm(-1). The coordination equilibrium not only depends on the pH but is also affected by the buffer, the salt concentration, and the binding of the natural redox partner cytochrome c552. These changes of the coordination equilibrium are attributed to the perturbation of the hydrogen bonding network at the catalytic center that is connected to the protein surface via a relay of hydrogen bonds. Environmental changes at the catalytic site are sensitively reflected by the formyl stretching of heme a3. The unique structural properties of the ba3 oxidase may be related to the unusual proton pump efficiency and heme a3 redox potential.
Membrane lipid composition varies in different tissues and species. Since a defined lipid composition is essential to the function of many membranes, the relationship between membrane lipid composition and structure was determined using infrared and Raman spectroscopy in four membranes containing a calcium pump: rabbit fast and slow twitch muscle sarcoplasmic reticulum and human and bovine lens fiber cell membranes. We found that membrane sphingolipid and phosphatidylcholine content were correlated to a decrease and increase, respectively, in the infrared lipid CH2 symmetric stretching band frequency. We interpret the change in frequency as a change in lipid hydrocarbon chain structural order. This was confirmed by Raman order parameters. The high degree of hydrocarbon chain saturation found in the variable amide chains of sphingolipids is likely to account for this correlation. Lipid phase transition temperature and cooperativity also correlated to sphingolipid and phosphatidylcholine content, and are the forces defining the order in at physiological temperature in the samples studied. Ca(2+)-ATPase caused an increase in the CH2 symmetric stretching frequency in fast twitch muscle sarcoplasmic reticulum (interpreted as an increase in hydrocarbon chain disorder), but had no effect on slow twitch muscle sarcoplasmic reticulum lipid hydrocarbon chain structure. In the natural systems studied, we find that it is the lipid hydrocarbon chain saturation that defines lipid hydrocarbon chain order.
We report our detailed electronic MCD, CD, and optical spectroscopic measurements and analysis of the porphyrin Soret (B(o)) region of four-coordinate 5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrinatopalladium( II), PdP(4), and its bound states with B-DNA duplexes poly(A-T)2, poly(G-C)2, and calf thymus DNA (CT DNA). For system PdP(4)/poly(A-T)2 it was possible to conclude that the porphyrin is bound edge-on in the major groove, specifically at the 5'AT3' site. For this orientation the porphyrin's electric dipole transition moments (edtm), mu(x) (most perturbed direction) and mu(y) (least perturbed direction), have tilt angles alpha approximately 90 degrees and approximately 45 degrees , respectively, relative to the helix axis. It was further concluded from the small shifts of B(o) optical and MCD band intensities and wavelengths and from the net MCD (+) A-term sign retention upon binding that the porphyrin's frontier ppi MOs (1a(1u) 3a(2u) 4eg) are only weakly perturbed by the heterocyclic bases of poly(A-T)2, and therefore that the LUMO (4eg) splitting is less than the |1a(1u)-3a(2u| energy separation, deltaHOMO, that is, deltaLUMO < deltaHOMO for the bound state in PdP(4)/poly(A-T)2. For intercalation systems PdP(4)/ poly(G-C)2 and /CT DNA, with PdP(4) centered in the intercalation "pocket" and having two of its 4-N-methylpyridyls extending into each of the major and minor grooves, the edtms mu(x) and mu(y) were determined to be oriented perpendicular (gamma approximately 0 degrees) and parallel (gamma approximately 90 degrees) to the hydrogen bonds of the base pairs, respectively. Intercalation is characterized by a much stronger binding interaction, viz., the B(o) optical band and net MCD extrema wavelength shifts are relatively large, and the net MCD (+) A-term of PdP(4) is substantially quenched as it becomes the (-) pseudo-A-term of intercalated PdP(4)/poly(G-C)2. This A-term sign reversal informs that the porphyrin MOs are so strongly perturbed by the GC base pairs that deltaLUMO > deltaHOMO, which gives rise to a (-) pseudo-A-term. Also, the findings demonstrate (1) the potential of PdP(4) as a sensitive, discriminating analytical probe of DNA sequences and (2) the diagnostic capability of the composite of five spectra [net MCD, CD, and optical of free and bound PdP(4)] in differentiating the site and sequence selectivity and preferred binding mode of this porphyrin.
A new fluorescence method has been developed to measure simultaneously and independently the release of fluorophores from two vesicle populations. Calcein and sulforhodamine B were used as a probe couple: the leakage of these probes from vesicles can be recorded independently since they can be excited simultaneously at 510 nm, and their individual fluorescence can be isolated by measuring the fluorescence signal at 525 and 590 nm, using a T-shape fluorometer. Controls show that both probes are suitable for the leakage assay based on fluorescence self-quenching, that they do not interact physically or chemically at the concentrations used in the method, and that they leak in a similar fashion from a given vesicle type. This dual-probe technique is applied to examine the specificity of the release relative to the cholesterol content of the vesicles for melittin, a toxin. This new approach shows in a straightforward manner that melittin-induced release for a given population can be modulated by the presence of vesicles with another lipid composition and this competitive release is associated with a preferential distribution of the peptide on the targeted vesicles.
The results of a Raman and solid state 13C-NMR spectroscopic investigation aimed at studying the conformation of piroxicam (P) and its interaction with beta-cyclodextrin (betaCD) in 1 : 1 amorphous PbetaCD inclusion compound are reported. The 1700-1200 cm(-1) FT-Raman and the 13C CP/MAS NMR spectra of 1 : 1 PbetaCD inclusion compound are discussed and assigned in comparison with those of the three main modifications of piroxicam (alpha, beta, and monohydrate). The FT-Raman and 13C-NMR results show that in 1 : 1 PbetaCD inclusion compound piroxicam mainly assumes the zwitterionic structure typical of monohydrate, even if the presence of a different structure, that is, beta form, is not excluded. Piroxicam monohydrate, differently from alpha and beta forms, is characterized by a zwitterionic structure with an internal proton transfer and an increased charge delocalization, as shown by our spectroscopic results. The charge delocalization characteristic of this zwitterionic structure gives rise to the interaction with betaCD via electrostatic and hydrogen bonds. The possibility of a host-guest interaction between piroxicam and betaCD is not excluded; the guest molecule can be accommodated in betaCD cavity by interaction via hydrophobic bonds.
Qy-excitation resonance Raman (RR) spectra are reported for two mutant reaction centers (RCs) from Rhodobacter capsulatus in which the photoactive bacteriopheophytin (BPhL) is replaced by a bacteriochlorophyll (BChl) molecule, designated beta. The pigment change in both mutants is induced via introduction of a histidine residue near the photoactive cofactor. In one mutant, L(M212)H, the histidine is positioned over the core of the cofactor and serves as an axial ligand to the Mg+2 ion. In the other mutant, F(L121)H/F(L97)V, the histidine is positioned over ring V of the cofactor, which is nominally too distant to permit bonding to the Mg+2 ion. The salient observations are as follows: (1) The beta cofactor in F(L121)H/F(L97)V RCs is a five-coordinate BChl molecule. However, there is no evidence for the formation of a Mg-His bond. This bond is either much weaker than in the L(M212)H RCs or completely absent, the latter implying coordination by an alternative ligand. The different axial ligation for beta in the F(L121)H/F(L97)V versus L(M212)H RCs in turn leads to different conformations of the BChl macrocycles. (2) The C9-keto group of beta in F(L121)H/F(L97)V RCs is free of hydrogen bonding interactions, unlike the L(M212)H RCs in which the C9-keto of beta is hydrogen bonded to Glu L104. The interactions between other peripheral substituents of beta and the protein are also different in the F(L121)H/F(L97)V RCs versus L(M212)H RCs. Accordingly, the position and orientation of beta in the protein is different in the two beta-containing RCs. Nonetheless, previous studies have shown that the primary electron transfer reactions are very similar in the two mutants but differ in significant respects compared to wild-type RCs. Collectively, these observations indicate that changes in the conformation of a photoactive tetrapyrrole macrocycle or its interactions with the protein do not necessarily lead to significantly perturbed photochemistry and do not underlie the altered primary events in beta-type RCs.
The hydrated complex of gramicidin A with Na+ cations was studied by FTIR spectroscopy. The water bands observed in the FTIR spectrum are identical with those obtained for the hydrated gA–Li+ complexes. In the far infrared spectrum of the gA–Na+ complex an intense continuum was found indicating large Na+ polarizability of the gA channels. This Na+ polarizability arises, similarly as in the case of the Li+ complex, due to the fast fluctuation of the Na+ ions between two water molecules and four CO groups of the gA backbone. The much smaller barriers in the six minima Na+ potentials explain the much larger mobility of the Na+ when compared with the Li+ ions. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 284–288, 1999
In this work, we have examined, using Fourier-transform Raman (FT-R) spectroscopy, the bacteriochlorophyll a (BChl a) binding sites in light-harvesting (LH) antennae from different species of the Proteobacteria that exhibit unusal absorption properties. While the LH1 complexes from Erythromicrobium (E.) ramosum (RC-B871) and Rhodospirillum centenum (B875) present classic FT-R spectra in the carbonyl high-frequency region, we show that in the blue-shifted LH1 complex, absorbing at 856 nm, from Roseococcus thiosulfatophilus, as well as in the B798-832 LH2 from E. ramosum, or in the B830 complex from the obligate phototrophic bacterium Chromatium purpuratum, some H-bonds between the acetyl carbonyl of the BChl a and the surrounding protein are missing. The molecular mechanisms responsible for the unusual absorption of these complexes are thus similar to those responsible for tuning of the absorption of the LH2 complexes between 850 and 820 nm. Furthermore, our results suggest that the binding pocket of the monomeric BChl in the LH2 from E. ramosum is different from that of Rps. acidphila or Rb. sphaeroides. The FT-R spectra of Chromatium purpuratum indicate that, in contrast with every LH2 complex previously studied by FT-R spectroscopy, no free-from-interaction keto groupings exist in this complex.
Enzyme-substrate intermediates involving the acyl group 5-methyl thiophene acryloyl (5-MTA) bound to the active site of an enzyme via a sulfur or selenium atom have been characterized by Raman spectroscopy (e.g., J. D. Doran and P. R. Carey, Biochemistry 1996, 35, 12495-12502, and M. J. O'Connor et al., J Amer Chem Soc 1996, 118, 239-240). Raman difference spectra reveal the Raman spectrum of the acyl group in the active site and, in turn, these can be used to probe acyl group conformation and active site forces and interactions. In order to improve the understanding of the relationship between conformational states and vibrational spectra of 5-MTA thiolesters, calculations based on a density functional theory analysis are undertaken for 5-methyl thiophene acryloyl ethyl ester. The calculations provide the precise geometries and energies of rotomers of 5-MTA ethyl thiolester involving rotational isomerism about the C--C single bonds flanking the ethylenic linkage and the S--C bond linking the ethyl group to the sulfur atom. The calculations also provide the vibrational spectrum for each conformer and these predictions are compared with the experimental Raman an IR data for the thiolester in carbon tetrachloride. Modes are identified that can act as conformational markers for isomerism about the C--C and S--C2H5 single bonds. These findings are used to identify the two conformational states giving rise to the Raman spectrum of the 5-MTA-S-enzyme formed by the viral cysteine protease HAV-3C.
Fourier transform infrared (FTIR) spectroscopy was used as a convenient and easy-to-run method to monitor radical-induced damage on the radiation-resistant Deinococcus radiodurans strain. Increasing concentrations of ascorbic acid added to the culture medium during the stationary phase produced striking changes in the infrared spectra. These changes especially occurred in the 1700–900 cm−1 region, which is spectroscopically assigned to the amide I and II components, nucleotide bases, phosphodiester backbone and sugar rings, and were correlated with the oxidant effect of ascorbic acid. Thus, FTIR analysis allows a rapid characterization of the changes induced by ascorbic acid in the cell environment, which can be correlated in part with the generation of free radicals. Beyond a critical ascorbic acid concentration of 40 mM, these free radicals can cause severe damage to the biomolecular components, as soon as the antioxidant defenses of the bacterium are overwhelmed. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 229–236, 1999
Hemoglobin I from the clam Lucina pectinata has the unusual ability to bind hydrogen sulfide. This sulfide-reactive hemoglobin has a high content of phenyl residues in the heme pocket that may account for its ligand binding properties. To confirm this, resonance Raman spectroscopy was used to determine the heme structure of deoxy, oxy, carbon monoxy, metaquo, metcyano, and methydrogen sulfide hemoglobin I (HbI) complexes. The oxidation (ν4), spin (ν3), and coordination (ν2) markers were identified in the high-frequency spectra of all the HbI complexes. The data indicated that the aromatic environment near the heme does not affect the iron oxidation state, coordination state, spin state, and core size marker vibrational modes. The marker bands also revealed that metsulfide HbI complex has an Fe(III), six-coordinate, and low-spin structure. The low-frequency vibrational frequencies for the methydrogen sulfide, metcyano, oxy, and carbon monoxy HbI derivatives showed νFe–S at 374 cm−1, νFe–C at 448 cm−1, νFe–O at 563 cm−1, and νFe–C 516 cm−1, respectively. These results suggest a model where the phenyl residues in the Phe29(B10) and Phe68(E11) positions have strong electrostatic interactions with the metcyano, oxy, and carbon monoxy ligands of the HbIO2, HbICO, and HbICN complexes. The multipolar interaction explains the higher νFe–C frequency for the carbon monoxy HbI complex, and the lower νCO, νFe–O2 and νFe–C frequencies for the HbICO, HbIO2, and HbICN complexes, respectively. The repulsion between the carbonyl group of Gln64(E7) and oxygen or nitrogen of the oxy, carbon monoxy, and metcyano HbI complexes would also contribute to the above behavior. This model implies that Gln64(E7), in HbI Lucina pectinata, does not rotate from its original position to stabilize, by means of hydrogen bonding, the coordination of the other ligands, for example, the metcyano, oxy, and carbon monoxy heme complexes. Instead the electronic interaction between the phenylalanine in B10 and E11 positions stabilize these HbI complexes. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 289–301, 1999
1H- and 13C-NMR spectroscopy is applied to investigate the CU(A) and type 1 active sites of copper proteins in solution. The analysis of hyperfine shifted 1H resonances allows the comparison of the electron spin density delocalization in the CU(A) site of the wild-type soluble domains of various cytochrome c oxidases (Thermus thermophilus, Paracoccus denitrificans, and Paracoccus versutus) and genetically engineered constructs (soluble domain of quinol oxidase from Escherichia coli and Thiobacillus versutus amicyanin). Comparable spin densities are found on the two terminal His ligands for the wild-type constructs as opposed to the engineered proteins where the spin is more unevenly distributed on the two His residues. A reevaluation of the Cys H(beta) chemical shifts that is in agreement with the data published for both the P. denitrificans and the P. versutus Cu(A) soluble domains confirms the thermal accessibility of the 2B(3u) electronic excited state and indicates the existence of slightly different spin densities on the two bridging Cys ligands. The 13C-NMR spectrum of isotopically enriched oxidized azurin from Pseudomonas aeruginosa reveals six fast relaxing signals, which can be partially identified by 1- and 2-dimensional (1-D, 2-D) direct detection techniques combined with 3-D triple resonance experiments. The observed contact shifts suggest the presence of direct spin density transfer and spin polarization mechanisms for the delocalization of the unpaired electron.
BiospectroscopyVolume 5, Issue 3 p. 131-132 Alessandro Bertoluzza Giancarlo Fini, Giancarlo Fini University of BolognaSearch for more papers by this author Giancarlo Fini, Giancarlo Fini University of BolognaSearch for more papers by this author First published: 10 June 1999 https://doi.org/10.1002/(SICI)1520-6343(1999)5:3<131::AID-BSPY2>3.0.CO;2-SAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume5, Issue31999Pages 131-132 RelatedInformation
Colicins are killer proteins that use envelope proteins from the outer and the inner membranes to reach their cellular target in susceptible cells of Escherichia coli. Each group A colicin uses a combination of Tol proteins to cross the outer membrane of gram-negative bacteria and to exert their killing activity. The TolA protein, necessary for the import of all the group A colicins, is a 421-amino acid residue protein composed of three domains (TolAI, TolAII, and TolAIII). TolAIII interacts with the N-terminal domain of colicin A (AT1). Analytical ultracentrifugation reveals that TolAII and TolAIII are monomer structures, TolAII has an elongated structure, and TolAIII is rather globular. Circular dichroism (CD) spectra were done with TolAII-III, TolAII, TolAIII, AT1, and the AT1-TolAII-III complex. TolA CD spectra reveal the presence of alpha-helix structure in aqueous solution and the intensity of the a-helix signal is the highest with TolAII. Few structural changes are observed with the complex AT1-TolAII-III. Molecular modeling was done for TolAII-III, taking into account CD and ultracentrifugation data and show that domain II can adopt a barrel structure made of three twisted alpha-helices similar to coiled coil helices while domain III can adopt a globular structure.
A series of oligopeptides containing aromatic and basic residues were synthesized and their interactions with double-stranded nucleic acids studied by proton and phosphorus NMR, viscometry, and DNA melting temperature (Tm). The oligopeptides prepared contain two aromatic amino acids (phenylalanine or p-nitrophenylalanine) as well as one or two lysyl residues. The nucleic acids studied were calf thymus DNA, poly(dA-dT)2, poly(dA) · poly(dT), poly(dG-dC)2, poly(dG) · poly(dC), and d(ATGCAT)2. The results obtained show stacking of both aromatic residues of the oligopeptides with the nucleic acids. Higher upfield shifts of the aromatic amino acid residues were always observed with alternating nucleic acids and were higher with poly(dA-dT)2 in all cases. Evidence for two types of complexes of Lys-Phe-Gly-Gly-p-NO2Phe-LysNH2 with DNA was obtained by NMR, one attributed to a purely electrostatic complex and another involving stacking interactions. Studies with d(ATGCAT)2 indicate that the aromatic residues of the oligopeptides were stacked with the terminal AT base pairs preferentially binding at the ends of the hexanucleotide. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 313–322, 1999
The vibrational circular dichroism (VCD) and absorption spectra of gramicidin D in different organic solvents are presented in the amide I and II regions. The absorption and VCD spectra suggest that gramicidin structures are similar in dioxane and chloroform. However, the structures adopted by gramicidin in chloroform are due to the trace amount of protective ethanol present in chloroform solvent. In the absence of this protective ethanol, gramicidin appears to aggregate in chloroform. The gramicidin structures appear to be similar in propanol and ethanol, but the composition of these structures appear to be different from those in dioxane and chloroform. In methanol-d4, a different composition of structures, including monomers, appears to be present. The structures in dimethyl-d6-sulfoxide and 2,2,2-trifluoroethanol are entirely different from those in all other solvents. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 276–283, 1999
In this article, the assignment of the nu(C-H) stretching region of lipid molecules is revisited. This region is extensively used to follow lipid phase transitions, and especially the frequency shifts and bandwidth alterations in the nu(sym)CH2 band have been utilized in this respect. Here, we propose and prove that behind these phenomena there are pairs of component bands in the cases of both the nu(sym)CH2 and the nu(as)CH2 bands. The lower-frequency components of the pairs are assigned to the vibrations of CH2 groups on trans segments of the fatty acyl chains, while the higher-frequency components of the pairs are assigned to CH2 groups on gauche segments. To prove these assignments, we have shown that the nuCH2 frequencies are characteristic of the conformation of the lipid fatty acyl chain itself, and not the state of the whole lipid matrix. Curve fitting in fact revealed the conformer-specific components. With the use of singular value decomposition analysis we have demonstrated that the relative intensity changes in the components, and not the shifts in the whole bands, cause the observed shifts in the nuCH2 bands upon lipid phase transition. The results of this approach are presented for deuterium-saturated dioleoyl-phosphatidylcholine mixtures, for the gel --> liquid-crystalline phase transition of dipalmitoyl-phosphatidylcholine multilayers, and for a biological membrane, barley thylakoid. This refined assignment offers physically plausible reasoning for the observed phenomena and is able to explain frequency shifts and bandwidth changes observed previously upon lipid phase transitions, including their nonconcerted temperature dependences. In biological membranes, this interpretation allows the separation of protein- and membrane-dynamics-induced lipid conformational changes.
