Decavanadate induces mitochondrial membrane depolarization and inhibits oxygen consumption

Decavanadate induced rat liver mitochondrial depolarization at very low concentrations, half-depolarization with 39 nM decavanadate, while it was needed a 130-fold higher concentration of monomeric vanadate (5 microM) to induce the same effect. Decavanadate also inhibits mitochondrial repolarization induced by reduced glutathione in vitro, with an inhibition constant of 1 microM, whereas no effect was observed up to 100 microM of monomeric vanadate. The oxygen consumption by mitochondria is also inhibited by lower decavanadate than monomeric vanadate concentrations, i.e. 50% inhibition is attained with 99 M decavanadate and 10 microM monomeric vanadate. Thus, decavanadate is stronger as mitochondrial depolarization agent than as inhibitor of mitochondrial oxygen consumption. Up to 5 microM, decavanadate does not alter mitochondrial NADH levels nor inhibit neither F(O)F(1)-ATPase nor cytochrome c oxidase activity, but it induces changes in the redox steady-state of mitochondrial b-type cytochromes (complex III). NMR spectra showed that decameric vanadate is the predominant vanadate species in decavanadate solutions. It is concluded that decavanadate is much more potent mitochondrial depolarization agent and a more potent inhibitor of mitochondrial oxygen consumption than monomeric vanadate, pointing out the importance to take into account the contribution of higher oligomeric species of vanadium for the biological effects of vanadate solutions.     

Solution structural studies of molybdate-nucleotide polyanions

The complexation of molybdate with the nucleotides adenosine-5'-monophosphate (5'-AMP), adenosine-3'-monophosphate (3'-AMP) and guanosine-5'-monophosphate (5'-GMP) has been investigated by (1)H and (31)P NMR and Mo K-edge X-ray absorption near edge (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy. Acidification of aqueous solutions containing molybdate and each of the nucleotides resulted in the formation of a single species characterized by (1)H resonances which are deshielded relative to those of free nucleotide. Analysis of the two-component systems indicated a Mo/nucleotide ratio of 2.5:1 for the complexation species. White compounds, characterized as Na(2)[Mo(5)O(15)(HB)(2)] (B=5'-AMP, 5'-GMP), have been isolated from the acidified molybdate/H(2)B solutions. Dissolution in D(2)O replicates the NMR spectra of the solution species observed prior to precipitation. Solution and solid state Mo K-edge XAS and EXAFS spectroscopy of Na(2)[Mo(5)O(15)(HAMP)(2)] and Na(6)[Mo(5)O(15)(PO(4))(2)] provide convincing evidence for the presence of a pentamolybdodiphosphate core in the molybdate-nucleotide complexes in both the solid and solution states.     

Spectroscopic study of the structure of sucrose in the amorphous state and in aqueous solution

The structure of noncrystalline sucrose in the amorphous, solid state and in aqueous solution was investigated. Differences of structure of amorphous solid samples, the quenched-melt, and freeze-dried sucrose, are revealed by differential thermal analysis (d.t.a.) and from the Fourier-transform infrared (F.t.-i.r.) spectra. Factor analysis of the F.t.-i.r. spectra of aqueous solutions of sucrose shows the existence of at least two forms of the sucrose molecule. Analysis of ¹³C-n.m.r. spectra of amorphous and crystalline sucrose reveals a sensitivity of the fructosyl moiety to the morphology of the sample.     

Polyvanadate-stimulated NADH oxidation by plasma membranes--the need for a mixture of deca and meta forms of vanadate.

Polyvanadate solutions obtained by extracting vanadium pentoxide with dilute alkali over a period of several hours contained increasing amounts of decavanadate as characterized by NMR and ir spectra. Those solutions having a metavanadate:decavanadate ratio in the range of 1-5 showed maximum stimulation of NADH oxidation by rat liver plasma membranes. Reduction of decavanadate, but not metavanadate, was obtained only in the presence of the plasma membrane enzyme system. High simulation of activity of NADH oxidation was obtained with a mixture of the two forms of vanadate and this further increased on lowering the pH. Addition of increasing concentrations of decavanadate to metavanadate and vice versa increased the stimulatory activity, reaching a maximum when the metavanadate:decavanadate ratio was in the range of 1-5. Increased stimulatory activity can also be obtained by reaching these ratios by conversion of decavanadate to metavanadate by alkaline phosphate degradation, and of metavanadate to decavanadate by acidification. These studies show for the first time that both deca and meta forms of vanadate present in polyvanadate solutions are needed for maximum activity of NADH oxidation.     

Recent advances into vanadyl, vanadate and decavanadate interactions with actin

Although the number of papers about "vanadium" has doubled in the last decade, the studies about "vanadium and actin" are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate (51)V NMR signals, at -516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at -560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1:1 binding stoichiometry and a K(d) of 7.5 μM(-1). Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC(50) of 68 and 300 μM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 μM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Both vanadium species increased the ε-ATP exchange rate (k = 6.5 × 10(-3) s(-1) and 4.47 × 10(-3) s(-1) for decavanadate and vanadyl, respectively). Finally, (1)H NMR spectra of G-actin treated with 0.1 mM decavanadate clearly indicate that major alterations occur in protein structure, which are much less visible in the presence of ATP, confirming the preventive effect of the nucleotide on the decavanadate interaction with the protein. Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl. By affecting actin structure and function, vanadium can regulate many cellular processes of great physiological significance.     

Conformations in solution of the fuscopeptins. Phytotoxic metabolites of Pseudomonas fuscovaginae.

Fuscopeptins are phytotoxic amphiphilic lipodepsipeptides containing 19 amino acid residues. They are produced by the plant pathogenic bacterium Pseudomonas fuscovaginae in two forms, A and B, which differ only in the number of methylene groups in the fatty acid chain. Their covalent structure and biological properties have been reported previously. CD and NMR spectroscopy investigations in solution revealed the absence of identifiable elements of secondary and tertiary structure for these molecules. Fuscopeptin B appears to be completely unstructured in aqueous solution, and has a large molecular flexibility. A dramatic conformational change was observed upon addition of trifluoroethanol. This study reports the complete interpretation of the two-dimensional NMR spectra and the NOE results obtained for fuscopeptin B in water/trifluoroethanol solutions; the signals relative to the peptidic moiety are identical to those observed for fuscopeptin A. The results of this investigation were used to determine the solution structure of fuscopeptin B by computer simulations applying distance geometry and simulated annealing procedures. In water/trifluoroethanol solutions the peptidic region appears to have a partly helical structure. The lactonic ring assumes defined conformations very similar to those already reported for other lipodepsipeptides. The structure for fuscopeptin B in solution is also valid for fuscopeptin A because of the negligible structural difference between the two metabolites.     

Binomial frequency response to non-binomial pulse sequences for efficient water suppression

Summary This article reports on the use of short-hard pulse and spin-lock pulse combinations giving a binomial-like frequency response for the measurement of NMR spectra in aqueous solutions of quite dilute samples. The pulse sequence proposed provides excellent water suppression and does not introduce any linear or higher order phase errors. Application to the measurement of 2D NOESY data of a 0.25 mM solution of a double-stranded DNA fragment is presented.     

Investigation of the solution structure of a DNA octamer [d(GGAATTCC)]2 using two-dimensional nuclear Overhauser enhancement spectroscopy

Proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra in the pure absorption phase were obtained at 500 MHz for [d(GGAATTCC)]2 in aqueous solution at a series of mixing times. The experimental data were analyzed by comparison with theoretical spectra calculated using the complete 70 X 70 relaxation matrix including all proton dipole-dipole interactions and spin diffusion [Keepers, J. W. & James, T. L. (1984) J. Magn. Reson. 57, 404-426]. The theoretical spectra at each mixing time were calculated using two structures: a standard B-form DNA structure and an energy-minimized structure based on the similarity of the six internal residues of the title octamer with those of the dodecamer [d(CGCGAATTCGCG)]2, for which the crystal structure has been determined. Neither the standard B-form nor the energy-minimized structure will yield theoretical 2D NOE spectra which accurately reproduce all peak intensities in the experimental spectra. However, many features of the experimental spectra can be represented by both the B-form and the energy-minimized structure. Sequence-dependent structural characteristics are manifest in the 2D NOE spectra, in particular at the purine-pyrimidine junction as noted previously in the crystal structure. On the whole, the energy-minimized structure appears to yield theoretical 2D NOE spectra which mimic many, if not all, aspects of the experimental spectra. All 2D NOE data were consistent with nanosecond correction times as implied by proton spin-lattice relaxation time measurements. But better fits of some of the 2D NOE data using small variations in an effective isotropic correlation time suggest that there may be some local variations in mobility within the octamer duplex structure in solution.     

Raman spectra of L-alanine oligomers

The Raman spectra have been obtained of di-, tri-, tetra-, penta-, and hexa-L -alanine in the solid state. Raman spectra of the dimer and trimer in aqueous solution are also reported. The oligomers of alanine exist as zwitterions in the solid state and aqueous solution. Spectral differences between the dipeptide, and other oligomers arise primarily from the conformationally sensitive amide modes. The dipeptide exists as a nonplanar structure in the solid state and the other oligomere as β conformations. Comparison of Raman spectra of tri-L -alanine in the solid state and in aqueous solution suggests a conformational change to a random coil upon dissolution.     

Composition of the aqueous phase of chromaffin granules

Nuclear magnetic resonance spectroscopy has been used to determine the composition of the aqueous phase of bovine chromaffin granules. Relative concentrations of catecholamines (epinephrine plus norepinephrine), ATP and chromogranins have been measured from integrated intensities in the proton spectra using computer simulation techniques. Most or all of the catecholamines (97 ± 8%) are present in the aqueous phase and contribute to the high resolution spectrum. The catecholamine: ATP molar ratio (4.41 ± 0.45) determined by NMR is close to the value (4.45) derived from biochemical assay indicating that most or all of the ATP is present with catecholamine in the aqueous phase. Catecholamine: protein ratios show that approximately 45% of the soluble protein freed by lysis is not NMR visible. Intensity from this fraction does not appear under highly denaturing conditions (8 M urea) but reappears after hydrolysis. This behavior is similar to that of recently isolated soluble lipoprotein complexes. Variations in the NMR spectra associated with (1) different preparative procedures; (2) different suspension media, and (3) increasing osmolality are described. The fact that high concentrations of epinephrine and ATP (approximately 700 mM total) are dissolved in the aqueous phase implies that solution phase interactions at least partially ionic in nature are responsible for the low internal osmolality of chromaffin granules in vivo. Ordered phases containing a substantial fraction of the total catecholamine in an osmotically inactive form are not present.     

Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions

Summary A novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aqueous solutions (WATERGATE, WATER suppression by GraAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180° radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2. mM solution of a double-stranded DNA fragment in 90% H₂O at 5 °C is presented.     

Conformations of P2 Protein of Peripheral Nerve Myelin by Nuclear Magnetic Resonance Spectroscopy

High-resolution 1H NMR spectra of P2 protein from bovine peripheral nerve myelin indicate that the protein contains a high degree of tertiary structure in aqueous solution. Denaturation of the protein in urea solutions is a multi-step process. Binding of lysophosphatidylcholine micelles to the protein causes a conformational change and a broadening of NMR peaks from side chains of aromatic amino acid and methionine residues, with much less effect on upfield methyl resonances.     

Fourier transform infrared spectra of the polypeptide alamethicin and a possible structural similarity with bacteriorhodopsin

FTIR spectra of alamethicin have been obtained in KBr disk, methanol and in aqueous lipid dispersion (above and below the lipid phase transition). The solution structure of this polypeptide in methanol has been shown by recent studies (Esposito et al. (1987) Biochemistry 26, 1043-1050) using NMR spectroscopy to be predominantly alpha-helical in content. It may therefore be regarded as a model structure for the interpretation of the spectra of certain biomembrane proteins. A comparison of the spectra with that obtained with bacteriorhodopsin shows spectral similarities, e.g. the presence of a high-frequency amide I maximum at 1661-1663 cm-1 and shoulders near 1640 cm-1 and 1620 cm-1.     

NMR on-line monitoring of esterification catalyzed by cutinase

A nuclear magnetic resonance (NMR) method has been developed to monitor on-line lipase-catalyzed esterification reactions without the need to sample the reaction medium. The technique, through (1)H NMR, measures the concentrations of alcohol, ester, hydroxylic hydrogens in the organic phase, and hydroxylic hydrogens in the aqueous phase, if any. Also, the chemical shift evolution of the two types of hydroxylic hydrogens has been followed, providing information on water content of the organic phase and on the appearance of a distinct aqueous phase. As far as (13)C NMR is concerned, it has been possible to measure, first the acid and the ester concentrations in the carbonyl region, and second, the alcohol and the ester concentrations in the methylene region. All (1)H and (13)C results are in agreement with one another. Furthermore, NMR allows for the choice of detection zone. Preliminary studies on the solid phase proved the presence of much more water in the solid phase than in the organic phase, and also gave evidence of the existence of two types of esters, one in the organic phase, mainly associated with the acid, and the other one not associated with the acid, most probably entrapped within the solid enzyme.     

VirtualSpectrum,a tool for simulating peak list for multi-dimensional NMR spectra

NMR spectroscopy is a widely used technique for characterizing the structure and dynamics of macromolecules. Often large amounts of NMR data are required to characterize the structure of proteins. To save valuable time and resources on data acquisition, simulated data is useful in the developmental phase, for data analysis, and for comparison with experimental data. However, existing tools for this purpose can be difficult to use, are sometimes specialized for certain types of molecules or spectra, or produce too idealized data. Here we present a fast, flexible and robust tool, VirtualSpectrum, for generating peak lists for most multi-dimensional NMR experiments for both liquid and solid state NMR. It is possible to tune the quality of the generated peak lists to include sources of artifacts from peak overlap, noise and missing signals. VirtualSpectrum uses an analytic expression to represent the spectrum and derive the peak positions, seamlessly handling overlap between signals. We demonstrate our tool by comparing simulated and experimental spectra for different multi-dimensional NMR spectra and analyzing systematically three cases where overlap between peaks is particularly relevant; solid state NMR data, liquid state NMR homonuclear ¹H and ¹⁵N-edited spectra, and 2D/3D heteronuclear correlation spectra of unstructured proteins. We analyze the impact of protein size and secondary structure on peak overlap and on the accuracy of structure determination based on data of different qualities simulated by VirtualSpectrum.     

The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from Desulfovibrio gigas

The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe-4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80?Å), with the majority of φ/ψ angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdII_ox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdII_int), for which no X-ray structure is available.     

Evidence of dithionite contribution to the low-frequency resonance Raman spectrum of reduced and mixed-valence cytochrome c oxidase.

The resonance Raman spectra of deoxygenated solutions of mixed-valence cyanide-bound and fully reduced cytochrome oxidase derivatives that have been reduced in the presence of aqueous or solid sodium dithionite exhibit two new low-frequency lines centered at 474 and 590 cm-1. These lines were not observed when the reductant system was changed to a solution containing ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). Under enzyme turnover conditions, the addition of dithionite to the reoxidized protein (the 428-nm or "oxygenated" form) increases the intensity of these lines, while reoxidation and rereduction of the enzyme in the presence of ascorbate/TMPD resulted in the absence of both lines. Our data suggest that both lines must have contributions from species formed from aqueous dithionite, presumably the SO2 species, since these two lines are also observed in the Raman spectrum of a solution of aqueous dithionite, but not in the spectrum of an ascorbate/TMPD solution. Since heme metal-ligand stretch vibrations are expected to appear in the low-frequency region from 215 to 670 cm-1, our results indicate that special care should be exercised during the interpretation of the cytochrome a3 resonance Raman spectrum.     

Conformational flexibility of luteinizing hormone-releasing hormone in aqueous solution. A carbon-13 spin-lattice relaxation time study.

The carbon-13 nuclear magnetic resonance (13C NMR) spectra of luteinizing hormone-releasing hormone (LH-RH) and lower homologous peptides have been assigned in aqueous solutions at various pH values. 13C spin-lattice relaxation times (T1) have been measured for all proton-bearing carbons at 25.2 and 67.9 MHz. From the T1 data the rates of overall molecular motion and intramolecular motion of side chains have been estimated. LH-RH is a flexible molecule in solution, having segmental motion along the backbone as well as in the nonaromatic side chains.     

Conformational analysis of peptide analogues of silkmoth chorion protein segments using CD, NMR and molecular modelling.

Silkmoth proteins secreted from the follicular cells that surround the oocyte form a large extracellular assembly which is important for protecting and sustaining the structure of the oocyte and the developing embryo. These proteins have been classified into two major families (A and B). Sequence analysis showed conservation of a central domain containing long stretches of six amino acid residue repeats in both families, which have been suggested to be organized in beta-sheet structures. In this work NMR and CD spectra, as well as molecular calculations, have been used to investigate the conformational properties of two synthetic peptides (A and B), analogues of parts of the central domain of silkmoth chorion proteins of the A and B families, respectively. These peptides consist of three tandem repeats of the six-residue basic motif. Analysis of CD spectra of the two peptides in aqueous solutions and mixtures with organic solvents revealed beta-sheet and turn structural elements with a percentage higher than 40%. NOESY spectra at low temperatures (263-273 K) show sequential nOe connectivities (i, i + 1), indicative of a relative flexibility. The presence of HNi-HNi+1 cross-peaks and medium Halphai-HNi+1 connectivities, chemical shift deviations and temperature coefficient data provide, for the first time, experimental evidence that local folded structures around Gly residues occur in peptide segments of chorion proteins in solution. Simulated annealing calculations were used to examine the conformational space of the peptides and to probe the initial steps of amyloid fibril formation in the case of chorion proteins.     

Study of bradykinin conformation in the presence of model membrane by Nuclear Magnetic Resonance and molecular modelling

The conformation of bradykinin (BK), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Monte Carlo simulation in two different media, i.e. in pure aqueous solution and in the presence of phospholipid vesicles. Monolamellar liposomes are a good model for biological membranes and mimic the environment experienced by bradykinin when interacting with G-protein coupled receptors (GPCRs). The NMR spectra showed that lipid bilayers induced a secondary structure in the otherwise inherently flexible peptide. The results of ensemble calculations revealed conformational changes occurring rapidly on the NMR time scale and allowed for the identification of different families of conformations that were averaged to reproduce the NMR observables. These structural results supported the hypothesis of the central role played by the peptide C-terminal domain in biological environments, and provided an explanation for the different biological behaviours observed for bradykinin.