Towards a protocol for solution structure determination of copper(II) proteins: the case of Cu(II)Zn(II) superoxide dismutase

We have developed an optimized protocol to solve the solution structure of copper(II) proteins. After assignment, proton-proton NOEs are used for the shell where 1H spectra are conveniently observed. In a shell closer to the metal ion, 13C NMR spectra with band-selective homonuclear decoupling provide the assignment of all nuclei except for those of the metal ligands. A convenient method for the measurement of 13C longitudinal-relaxation rates (R1) of carbonyls and carboxylate moieties is proposed. 1H NOEs and 1H and 13C R1 data are sufficient to produce a good/reasonable solution structure, as demonstrated for a monomeric species of superoxide dismutase, a 153-residue protein.     

The effects of ligand exchange and mobility on the peroxidase activity of a bacterial cytochrome c upon unfolding

The effect on the heme environment upon unfolding Paracoccus versutus ferricytochrome c-550 and two site-directed variants, K99E and H118Q, has been assessed through a combination of peroxidase activity increase and one-dimensional NMR spectroscopy. At pH 4.5, the data are consistent with a low- to high-spin heme transition, with the K99E mutation resulting in a protein with increased peroxidase activity in the absence of or at low concentrations of denaturant. Furthermore, the mobility of the polypeptide chain at pH 4.5 for the wild-type protein has been monitored in the absence and presence of denaturant through heteronuclear NMR experiments. The results are discussed in terms of local stability differences between bacterial and mitochondrial cytochromes c that are inferred from peroxidase activity assays. At pH 7.0, a mixture of misligated heme states arising from protein-based ligands assigned to lysine and histidine is detected. At low denaturant concentrations, these partially unfolded misligated heme forms inhibit the peroxidase activity. Data from the K99E mutation at pH 7.0 indicate that K99 is not involved in heme misligation, whereas histidine coordination is proven by the data from the H118Q variant.     

Millisecond Allosteric Dynamics of Activated Ras Reproduced with a Slowly Hydrolyzable GTP Analogue

The millisecond timescale dynamics of activated Ras transiently sample a low-populated conformational state that has distinct surface property from the major state and represents a promising target for binding of small-molecule compounds. To avoid the complications of hydrolysis, dynamics and other properties of active Ras have so far been routinely investigated by using non-hydrolyzable GTP analogues, which, however, were previously reported to alter both the kinetics and distribution of the conformational exchange. In this study, we quantitatively measured and validated the internal dynamics of Ras complexed with a slowly hydrolyzable GTP analogue, GTPγS, which increases the lifetime of active Ras by 23 times relative to that of native GTP. It was found that GTPγS, in addition to its better mimicking of the exchange kinetics than the commonly used non-hydrolyzable analogues GppNHp and GppCH₂p, can rigorously reproduce the natural dynamics network in active Ras, thus indicating its fitness for use in the development of allosteric inhibitors.     

Effect of the solvent on the conformation of a depsipeptide: NMR-derived solution structure of hormaomycin in DMSO from residual dipolar couplings in a novel DMSO-compatible alignment medium

The macrocyclic compound hormaomycin has been investigated by NMR spectroscopy and by restrained molecular-dynamics simulations. Measurement of residual dipolar couplings induced by dissolving the depsipeptide in a polyacrylamide gel compatible with DMSO and their incorporation into the structure calculation of the title compound improved the precision of the family of structures. In DMSO the macrocyclic ring shows two beta-turns, whose positions in the sequence differ from those found in the CDCl3 solution structure and in the crystal structure obtained from hexylene glycol/H2O (50:50). The bulky side chain consisting of a 3-(2-nitrocyclopropyl)alanine and a chlorinated N-hydroxypyrrole moiety is flexible in DMSO.     

Evidence for the combined participation of a C10 and a C15 precursor in the biosynthesis of moenocinol, the lipid part of the moenomycin antibiotics

Upon feeding of [2-(13)C,4-(2)H]-1-deoxy-D-xylulose to Streptomyces ghanaensis, the deuterium label was retained exclusively at positions C-7 and C-17 in the moenocinol part of the moenomycin antibiotics. This result vindicates the hypothesis that the C(25) structure of moenocinol is assembled from a C(10) and a C(15) precursor, each of which requires for its formation the involvement of a dimethylallyl diphosphate starter unit.     

AC impedance study of a synthetic hydrotalcite-like compound modified electrode in aqueous solution

This paper deals with the electrochemical characterisation of Ni/Al-Cl hydrotalcite modified electrodes. The electrochemical impedance spectroscopy technique has been used in order to study the electronic and ionic conduction, both inside and on the surface of the material. The electronic and ionic contributions have been separated and the behaviour of the respective parameters has been studied as a function of the potential. In order to determine the kinetic limiting step of the overall electrochemical process we performed experiments at different temperatures, and calculated the activation energies of the electron hopping process and ion transport process. In addition we studied the behaviour of the system at different OH⁻ concentrations (pH 9.7-12.8) with the aim of clarifying the role of OH⁻ ions in the electrochemical process.     

The Dynamics of Lysozyme from Bacteriophage Lambda in Solution Probed by NMR and MD Simulations

¹⁵N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and H^N–H^α coupling constants, and molecular dynamics (MD) simulations have been used to characterise the behaviour of lysozyme from bacteriophage lambda (λ lysozyme) in solution. The lower and upper lip regions in λ lysozyme (residues 51–60 and 128–141, respectively) show reduced ¹H–¹⁵N order parameters indicating mobility on a picosecond timescale. In addition, residues in the lower and upper lips also show exchange contributions to T₂ indicative of slower timescale motions. The chemical shift, RDC, coupling constant and NOE data for λ lysozyme indicate that two fluctuating β‐strands (β3 and β4) are populated in the lower lip region while the N terminus of helix α6 (residues 136–139) forms dynamic helical turns in the upper lip region. This behaviour is confirmed by MD simulations that show hydrogen bonds, indicative of the β‐sheet and helical secondary structure in the lip regions, with populations of 40–60 %. Thus in solution λ lysozyme adopts a conformational ensemble that will contain both the open and closed forms observed in the crystal structures of the protein.     

Solution NMR Studies of Recombinant Aβ(1–42): From the Presence of a Micellar Entity to Residual β‐Sheet Structure in the Soluble Species

Amyloid‐β (Aβ) peptide is the major component found in senile plaques of Alzheimer's disease patients. The 42‐residue fragment Aβ(1–42) is proposed to be one of the most pathogenic species therein. Here, the soluble Aβ(1–42) species were analyzed by various liquid‐state NMR methods. Transient formation of a micelle species was observed at the onset of the aggregation kinetics. This micelle is dissolved after approximately one day. Subsequent loss of this species and the formation of protofibrils are proposed to be the route of fibril formation. Consequently, the observed micelle species is suggested to be on an off‐pathway mechanism. Furthermore, characterization of the NMR‐observable soluble species shows that it is a random‐coil‐like entity with low propensities for four β‐strands. These β‐strands correlate with the β‐strand segments observed in Aβ fibrils. This finding indicates that the 3D structure of the fibrils might already be predisposed in the soluble species.     

Infrared spectroscopic studies of poly(methyl methacrylate) doped with a new sulfur‐containing ligand and its cobalt(II) complex during γ‐radiolysis

Infrared (IR) spectroscopy studies were performed for poly(methyl methacrylate) (PMMA) samples doped with an organic ligand or its cobalt(II) complex after the extraction of the dopant during γ‐radiolysis. There were no drastic changes in the IR absorption band position, but noticeable changes in the intensities were found. The relative transmission of IR absorption bands, such as those at 750, 840, 1065, and 1388 cm^?1, were measured according to the transmission of the carbonyl group band at 1717 cm^?1. The degradation and recombination mechanism of different groups in the polymeric chain or backbone during radiolysis could be explained by the behavior of the relative transmission data for each group with increasing exposure dose. The tacticity of the PMMA samples was unchanged during radiolysis, and they were found to be syndiotactic. This was confirmed by the IR J values for different PMMA samples before and after irradiation. The protection efficiency of the organic ligand and its cobalt(II) complex was also investigated, and it was found that the organic ligand was more protective than the cobalt(II) complex for PMMA samples against γ‐rays. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 1937–1950, 2004     

The Influence of a Continuum Background on Carrier Relaxation in InAs/InGaAs Quantum Dot

We have investigated the ultra-fast carrier dynamics in Molecular Beam Epitaxy (MBE)-grown InAs/InGaAs/GaAs quantum dots (QDs) emitting at 1.3 μm by time resolved photoluminescence (TRPL) upconversion measurements with a time resolution of about 200 fs. Changing the detection energies in the spectral region from the energy of the quantum dots excitonic transition up to the barrier layer absorption edge, we have found that, under high excitation intensity, the intrinsic electronic states are populated mainly by carriers directly captured from the barrier.     

The electroreduction of molybdenum(VI) complex with tartrate ion in a neutral solution

Hexavalent molybdenum was reduced electrochemically in a neutral solution containing tartrate ion. The formation of a complex of Mo(VI) with tartrate ion was confirmed by means of uv spectroscopy and potentiometry. The polarographic wave was kinetic at higher pH of lower concentration of tartrate ion, and the electrochemical reaction was proceeded by the protonation reaction in which the electro-active species MoO₄ (Htar)^4?₂ was produced.     

The study of aluminium corrosion in acidic solution with nontoxic inhibitors

The inhibitory activity of some substituted N-arylpyrroles on aluminium corrosion in hydrochloric acid was studied in relation to inhibitor concentration, using potentiodynamic and impedance spectroscopy techniques. All investigated compounds were found to act as cathodic-type inhibitors and inhibition was ascribed to the adsorption of inhibitor onto the electrode surface. The inhibiting efficiency of the additives depended on the inductive power of the groups attached to the benzene and/or pyrrole ring. The carbaldehyde group showed better inhibiting power due to additional condensation on the electrode surface. The impedance results analysed in terms of the polarization resistance showed that the EIS technique can be successfully applied in the determination of corrosion resistance in systems where the corrosion kinetics are not simple.     

The Conserved Glu in the Cyclotide Cycloviolacin O2 Has a Key Structural Role

Cyclotides are a large family of plant peptides that are characterised by a head‐to‐tail circular backbone and three disulfide bonds that are arranged in a cystine knot. This unique structural feature, which is referred to as a cyclic cystine knot, gives the cyclotides remarkable stability against chemical and biological degradation. In addition to their natural function as insecticides for plant defence, the cyclotides have a range of bioactivities with pharmaceutical relevance, including cytotoxicity against cancer cell lines. A glutamic acid residue, aside from the invariable disulfide array, is the most conserved feature throughout the cyclotide family, and it has recently been shown to be crucial for biological activity. Here we have used solution‐state NMR spectroscopy to determine the three‐dimensional structures of the potent cytotoxic cyclotide cycloviolacin O2, and an inactive analogue in which this conserved glutamic acid has been methylated. The structures of the peptides show that the glutamic acid has a key structural role in coordinating a set of hydrogen bonds in native cycloviolacin O2; this interaction is disrupted in the methylated analogue. The proposed mechanism of action of cyclotides is membrane disruption and these results suggest that the glutamic acid is linked to cyclotide function by stabilising the structure to allow efficient aggregation in membranes, rather than in a direct interaction with a target receptor.     

Mechanistic in situ High‐Pressure NMR Studies of Benzene Hydrogenation by Supramolecular Cluster Catalysis with [(η6‐C6H6)(η6‐C6Me6)2Ru3(μ3‐O)(μ2‐OH)(μ2‐H)2][BF4]

In situ high‐pressure NMR spectroscopy of the hydrogenation of benzene to give cyclohexane, catalysed by the cluster cation [(η⁶‐C₆H₆) (η⁶‐C₆Me₆)₂Ru₃(μ₃‐O)(μ₂‐OH)(μ₂‐H)₂]⁺ 2 , supports a mechanism involving a supramolecular host‐guest complex of the substrate molecule in the hydrophobic pocket of the intact cluster molecule.     

The alpha-to-beta conformational transition of Alzheimer's Abeta-(1-42) peptide in aqueous media is reversible: a step by step conformational analysis suggests the location of beta conformation seeding

Current views of the role of beta-amyloid (Abeta) peptide fibrils range from regarding them as the cause of Alzheimer's pathology to having a protective function. In the last few years, it has also been suggested that soluble oligomers might be the most important toxic species. In all cases, the study of the conformational properties of Abeta peptides in soluble form constitutes a basic approach to the design of molecules with "antiamyloid" activity. We have experimentally investigated the conformational path that can lead the Abeta-(1-42) peptide from the native state, which is represented by an alpha helix embedded in the membrane, to the final state in the amyloid fibrils, which is characterized by beta-sheet structures. The conformational steps were monitored by using CD and NMR spectroscopy in media of varying polarities. This was achieved by changing the composition of water and hexafluoroisopropanol (HFIP). In the presence of HFIP, beta conformations can be observed in solutions that have very high water content (up to 99 % water; v/v). These can be turned back to alpha helices simply by adding the appropriate amount of HFIP. The transition of Abeta-(1-42) from alpha to beta conformations occurs when the amount of water is higher than 80 % (v/v). The NMR structure solved in HFIP/H2O with high water content showed that, on going from very apolar to polar environments, the long N-terminal helix is essentially retained, whereas the shorter C-terminal helix is lost. The complete conformational path was investigated in detail with the aid of molecular-dynamics simulations in explicit solvent, which led to the localization of residues that might seed beta conformations. The structures obtained might help to find regions that are more affected by environmental conditions in vivo. This could in turn aid the design of molecules able to inhibit fibril deposition or revert oligomerization processes.     

A novel whitening test method of a sulfur-cured EPDM composite filled with carbon black in calcium cation solution

A simple and efficient whitening test method of a carbon black-filled sulfur-cured EPDM composite by formation of calcium stearate was established using Ca²⁺ solution and convenient analytical techniques. The sample was aged in 0.1 M CaCl₂ solution at 30–90 °C for 1 and 3 days. ATR-FTIR spectra of the aged sample surfaces displayed unique calcium stearate peaks. Level of the whitening of a rubber article can be determined by analysis of the cross section of the aged sample using an image analyzer and by analysis of calcium stearate on the aged sample surface using an ATR-FTIR.     

The reaction in water of UF resins with isocyanates at short curing times: A 13C NMR investigation

CP MAS ¹³C NMR spectra of hardened resins have shown that urethane bridges derived from the reaction of the isocyanate group with the hydroxymethyl group of urea do form even at fast curing times comparable to what was used in the wood panels industry, in lower proportions than what was shown earlier. Polyureas and biurets obtained from the reaction of isocyanate with water are the predominant crosslinking reactions of pMDI alone and in UF/pMDI resin systems under fast curing conditions. Residual, unreacted isocyanate groups in the hardened network are consistently observed. Their proportion markedly decreases when the original proportion of urea–formaldehyde (UF) resin is high and that of pMDI is low. Under these fast curing conditions, the UF resin appears to self‐condense through an unusually high proportion of methylene ether links rather than methylene bridges alone. A marked proportion of residual, unreacted hydroxymethyl groups is also noticeable, initially, in the UF self‐condensation network. Direct NMR tests on thin hardboard bonded under fast pressing conditions with different proportions of UF/pMDI confirmed that crosslinking due to polyureas and biurets formation are predominant in the crosslinking of pMDI when alone and in UF/pMDI resin systems. They confirmed that residual, unreacted isocyanate groups are present in the finished panel. Their proportion is higher when the proportion of pMDI in the system is high. The presence or absence of urethanes could not be confirmed directly on the panels as the relevant peaks are masked by the wood carbohydrates signals of wood cellulose and hemicelluloses. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 1624–1632, 2006     

Fast NMR Methods for Measuring in the Direct and/or Competition Mode the Dissociation Constants of Chemical Fragments Interacting with a Receptor

Ligand-based NMR screening represents a powerful method in fragment-based drug discovery for the identification of chemical matter interacting with the receptor of interest. The large dynamic range of these methods allows the detection of weakly binding ligands. However, the methodology has not been extensively used for quantifying the strength of these interactions. This knowledge is important for ranking fragments according to their binding strength and for prioritizing structure-based and medicinal chemistry activities. Rapid NMR methods for measuring the dissociation constant in direct and competition modes are presented here. The theory underpinning these methods are presented, along with their application to the measurement of the binding affinities of several ligands of the heat shock protein 90.     

The Scaffold Protein IscU Retains a Structured Conformation in the FeS Cluster Assembly Complex

IscU and IscS are two essential proteins in the machine responsible for the biogenesis of iron–sulfur clusters, prosthetic groups that are involved in several essential functions. The scaffold protein IscU is the temporary acceptor of the cluster that results when the protein forms a 110 kDa complex with the desulfurase IscS. In the absence of zinc, which stabilises the folded state, IscU is present in solution in equilibrium between a structured and an unstructured form. It has been suggested that IscS preferentially binds unstructured IscU, although crystal structures indicate otherwise. To learn more about the IscS–IscU complex, we have used advanced solution NMR techniques to observe directly the state of fold of IscS‐bound IscU. We present unambiguous evidence that IscU is folded in the complex and that the unstructured form does not bind to IscS. Our data correlate with several observations and explain an IscU‐related pathology.