Mauro A. Cremonini

The use of pseudocontact shifts to refine solution structures of paramagnetic metalloproteins: Met80Ala cyano-cytochrome c as an example

Abstract The availability of NOE constraints and of the relative solution structure of a paramagnetic protein permits the use of pseudocontact shifts as further structural constraints. We have developed a strategy based on: (1) determination of the χ tensor anisotropy parameters from the starting structure; (2) recalculation of a new structure by using NOE and pseudocontact shift constraints simultaneously; (3) redetermination of the χ tensor anisotropy parameters from the new structure, and so on until self-consistency. The system investigated is the cyanide derivative of a variant of the oxidized Saccharomyces cerevisiae iso-1-cytochrome c containing the Met80Ala mutation. The structure has been substantially refined. It is shown that the analysis of the deviation of the experimental pseudocontact shifts from those calculated using the starting structure may be unsound, as may the simple structure refinement based on the pseudocontact shift constraints only.

Pseudocontact shifts as constraints for energy minimization and molecular dynamics calculations on solution structures of paramagnetic metalloproteins

The pseudocontact shifts of NMR signals, which arise from the magnetic susceptibility anisotropy of paramagnetic molecules, have been used as structural constraints under the form of a pseudopotential in the SANDER module of the AMBER 4.1 molecular dynamics software package. With this procedure, restrained energy minimization (REM) and restrained molecular dynamics (RMD) calculations can be performed on structural models by using pseudocontact shifts. The structure of the cyanide adduct of the Met80Ala mutant of the yeast iso‐1‐cytochrome c has been used for successfully testing the calculations. For this protein, a family of structures is available, which was obtained by using NOE and pseudocontact shifts as constraints in a distance geometry program. The structures obtained by REM and RMD calculations with the inclusion of pseudocontact shifts are analyzed. Proteins 29:68–76, 1997.

PSEUDYANA for NMR structure calculation of paramagnetic metalloproteins using torsion angle molecular dynamics

The program DYANA, for calculation of solution structures of biomolecules with an algorithm based on simulated annealing by torsion angle dynamics, has been supplemented with a new routine, PSEUDYANA, that enables efficient use of pseudocontact shifts as additional constraints in structure calculations of paramagnetic metalloproteins. PSEUDYANA can determine the location of the metal ion inside the protein frame and allows to define a single tensor of magnetic susceptibility from a family of conformers. As an illustration, a PSEUDYANA structure calculation is provided for a metal-undecapeptide complex, where simulated pseudocontact shifts but no NOE restraints are used as conformational constraints.

New isatin derivatives with antioxidant activity

The reaction between isatin and 2,5-dimethoxyaniline is described. The main product was identified as 3,3-bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one. The antioxidant activity of the compounds isolated was evaluated with two methods. Three published antitumor E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones entered the same tests to search whether they are endowed with antioxidant activity too. 3,3-Bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one and the three antitumor agents showed a good chemical antioxidant activity.

The use of sodium bicarbonate for marination of broiler breast meat

This study aimed to evaluate marination performances and the effect on meat quality traits of sodium bicarbonate, used alone or in combination with sodium chloride, when compared with sodium trypolyphosphate by using advanced analytical tools, including low-field nuclear magnetic resonance and differential scanning calorimetry. In total, 140 samples (cylindrical shape of 1 × 4 cm size) were obtained from a batch of 24-h postmortem broiler breast meat (Ross 708, females, 47 d old). Six of the groups were used for subsequent marination treatments, whereas the last group was kept as a nonmarinated control. Samples were subjected to vacuum tumbling in a special equipped laboratory rotary evaporator with a 12% (wt/wt) water:meat ratio using 6 marinade solutions: 7.7% (wt/wt) NaCl (S); 2.3% (wt/wt) Na(4)O(7)P(2) (P); 2.3% (wt/wt) NaHCO(3) (B); S and P; S and B; S, P, and B. Samples marinated with bicarbonate alone or in combination (B, SB, and SPB) significantly increased (P < 0.05) the meat pH by approximately 0.7 units compared with that of the control, whereas phosphate alone or in combination with salt increased (P < 0.05) the pH by 0.2 units. The combination containing all of the ingredients (SPB) produced the highest marinade performances; however, SB was able to guarantee a better marinade uptake and water retention ability with respect to that of SP. According to low-field nuclear magnetic resonance, the combined use of B and P with S determined a remarkable increase in proportion of entrapped water into the myofibrillar spaces, while the extramyofibrillar water fraction was not modified. Moreover, water gain following marination does not correspond to an increase in the freezable water amount, as detected by differential scanning calorimetry. In conclusion, B is a very promising marinating agent, and it can be exploited to develop processed poultry products with no added phosphates to match the request to avoid the nutritional drawbacks recently indicated with the use of phosphates.

The effect of exocyclic conjugation on the inversion of a saturated six-membered ring. A dynamic NMR study of N-substituted morpholines.

Abstract The effect of exocyclic conjugation on the inversion of a saturated six-membered ring is shown in a dynamic nmr study of a series of N-substituted morpholines where the substituents have similar shapes but different conjugating ability. As conjugation increases the ring inversion barrier decreases in contrast to previous observations of an unsaturated ring with conjugating substituents.

Iron-sulfur interconversions in the anaerobic ribonucleotide reductase from Escherichia coli

Abstract The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S]+ form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mössbauer spectroscopies. The [4Fe-4S]+ form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S]2+, [3Fe-4S]+/0, and to the stable [2Fe-2S]2+ form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Futhermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.

Arene hydroxylases: metalloenzymes catalysing dioxygenation of aromatic compounds☆

Abstract Some properties of hydroxylating enzymes able to add the two oxygen atoms of molecular oxygen into aromatic substrates such as benzene and phthalate are reviewed. In particular the non-haem single iron dioxygenases capable of incorporating two hydroxyl groups are presented as well as their up-to-date biophysical characterization.

Effects of heating on virgin olive oils and their blends: focus on modifications of phenolic fraction.

The phenolic profiles of two different virgin olive oils and their admixtures in different percentages have been analyzed after heating treatments by microwave or conventional oven. Changes in the phenolic profile upon heating were evaluated by chromatographic and spectroscopic methods, also monitoring the antioxidant activity by ABTS(*+) test. 3,4-DHPEA-EA, p-HPEA-EA, and EA showed the highest decreases after thermal treatments. The only compounds that showed a clear increase with heating, in particular by conventional oven, were the dialdehydic form of elenolic acid (EDA) and p-hydroxyphenylethanol linked to the dialdehydic form of elenolic acid (p-HPEA-EDA). A comparison between the variations after heating of the sum of monoaldehydic and dialdehydic forms of phenolic compounds obtained by using different analytical approaches (HPLC-DAD/MSD and 1D and 2D NMR spectroscopy) was made. The results showed a good agreement of these two high-resolution techniques.

Structural and Nutritional Properties of Pasta from Triticum monococcum and Triticum durum Species. A Combined 1H NMR, MRI, and Digestibility Study

The aim of the present study was to characterize the structure of two different types of pasta, namely Triticum turgidum ssp. durum (cv. Saragolla) and Triticum monococcum ssp. monococcum (cv. Monlis), under different processing conditions. MRI analysis and NMR spectroscopy (i.e., T1 and T2 NMR relaxation times and diffusion parameters) were conducted on pasta, and (1)H NMR spectroscopic analysis of the chemical compounds released by pasta samples during the cooking process was performed. In addition, starch digestibility (enzimatically determined) was also investigated. The NMR results indicated that Saragolla pasta has a more compact structure, ascribed to pasta network and in particular to different technological gluten properties, that mainly determine the lower ability of Monlis pasta in binding water. These results correlate well with the lower rate of starch hydrolysis measured for Monlis pasta compared to Saragolla when both are dried at high temperature.