Protein structural changes induced by glutathione-coated CdS quantum dots as revealed by Trp phosphorescence.

We evaluated the potential of tryptophan (Trp) phosphorescence spectroscopy for investigating conformational states of proteins involved in interaction with nanoparticles. Characterization of protein-nanoparticle interaction is crucial in assessing biological hazards related to use of nanoparticles. We synthesized glutathione-coated CdS quantum dots (GSH-CdS), which exhibited an absorption peak at 366 nm, indicative of 2.4 nm core size. Chemical analysis of purified GSH-CdS suggested an average molecular formula of GSH18S56Cd60. Investigations were conducted on model proteins varying in terms of isoelectric point, degree of burial of the Trp probe, and quaternary structure. GSH-CdS fluorescence measurements showed improvement in nanoparticle quantum yield induced by protein interaction. Trp phosphorescence was used to examine the possible perturbations in the protein native fold induced by GSH-CdS. Phosphorescence lifetime measurements highlighted significant conformational changes in some proteins. Despite their small size, GSH-CdS appeared to interact with more than one protein molecule. Rough determination of the affinity of GSH-CdS for proteins was derived from the change in phosphorescence lifetime at increasing nanoparticle concentrations. The estimated affinities were comparable to those observed for specific protein-ligand interactions and suggest that protein-nanoparticle interaction may have a biological impact.

Purification, stability and kinetic properties of highly purified adenosine deaminase from Bacillus cereus NCIB 8122.

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Bacillus cereus NCIB 8122 has been purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration through Sephadex G-100, DEAE-Sephadex A-50 chromatography and ion-exchange HPLC on DEAE-Polyol. The enzyme activity is stabilized (at temperatures from 0 degrees C to 40 degrees C) by 50 mM NH4+ or K+, while it is irreversibly lost in the absence of these or a few other monovalent cations. Glycerol (24% by volume) helps the cation in stabilizing the enzyme activity above 40 degrees C, but also exerts per se a noticeable protecting effect at room temperature. B. cereus adenosine deaminase displays the following properties: Mr on Sephadex G-200, 68,000; Mr in SDS-polyacrylamide gel electrophoresis, 53,700; optimal pH-stability (in the presence of 50 mM KCl) over the range 8-11 at 4 degrees C, and maximal catalytic activity at 30 degrees C between pH 7 and 10; Km for adenosine around 50 microM over the same pH range and Km for 2'-deoxyadenosine around 400 microM.

Pressure effects on protein flexibility monomeric proteins.

Alterations in flexibility of monomeric proteins induced by hydrostatic pressure in the predenaturational range (< or = 3 kbar) were probed through the decay kinetics of tryptophan phosphorescence. With apoazurin, ribonuclease T1, wild-type and V67G mutant and phosphoglycerate kinase, pressure effects on the triplet lifetime (tau) and the amplitudes of multicomponent decays emphasize that subtle changes in conformation are ubiquitous. With apoazurin the increase in tau attests to a tightening of the protein core that is enhanced at high temperature. On the contrary, tau decreases with ribonuclease T1, wild-type and mutant, and with phosphoglycerate kinase, indicating that pressure induces a greater flexibility to protein regions in proximity to the surface of the macromolecule. For phosphoglycerate kinase the decrease in tau and the parallel increase in fluorescence intensity and red-shift of the fluorescence spectrum unveil an "unfolding" like transition with midpoint pressures of 1.1 kbar at 5 degrees C and 1.6 kbar at 25 degrees C. Evidence that unfolding of the C-domain of this protein is, however, less than complete is provided by a delta G zero that is about half of that obtained by denaturation in guanidine hydrochloride and also by the ability of this structure to undergo conformational drift. In 70% glycerol, pressure effects on tau of apoazurin are attenuated while for ribonuclease T1 there is a reversal of the tendency with a pronounced increase in tau. With phosphoglycerate kinase glycerol abolishes entirely the "unfolding" transition and all hysteresis effects. A consistent picture of these findings is provided in terms of the location of the probe and of the opposing effects that pressure exerts on protein flexibility by reducing internal cavities and increasing the hydration of the polypeptide.

Pressure/temperature effects on protein flexibilty from acrylamide quenching of protein phosphorescence.

Pressure is an effective modulator of protein structure and biological function. The influence of hydrostatic pressure (

Pressure effects on the structure of oligomeric proteins prior to subunit dissociation.

In studies of pressure-induced subunit dissociation of protein aggregates, now widely used to evaluate the association free energy, entropy and enthalpy of very stable complexes, it is assumed that high pressure does not influence their structure/thermodynamic parameters and that some peculiarities of these equilibria, such as the decrease in subunit affinity at larger degrees of dissociation (alpha) and hysteresis in alpha/pressure diagrams are imputable to the slow conformational drift of isolated subunits. To test this premise, the conformation of dimeric alcohol dehydrogenase from horse liver and alkaline phosphatase from Escherichia coli was monitored as a function of pressure (up to 3 kbar) and temperature (0 to 50 degrees C) by means of the intrinsic Trp fluorescence and phosphorescence emission and binding of the 1-anilinonaphatalene-8-sulphonic acid (ANS) fluorophore. The results show a distinct influence of high pressure on the native dimers whose changes in conformation may, depending on whether or not these alterations are promptly reversed, be distinguished in elastic and inelastic changes. Elastic changes are ubiquitous and refer to pronounced modulations of the phosphorescence lifetime which is a monitor of the internal flexibility of the macromolecules. They attest to a tightening of the globular structure in the lower pressure range (below 1.5 kbar) as opposed to an increased fluidity in the higher range. The trend is similar between the two proteins and the tightening/loosening effect is fully consistent with the role that internal cavities and hydration of polypeptide is expected to play in determining the compressibility of these biopolymers. Inelastic perturbations reveal a more profound loosening of the globular fold and were observed only with alcohol dehydrogenase under conditions (low temperature (t < 10 degrees C) and high pressure (p > 2.5 kbar)) that favour protein hydration. They involve slow consecutive reactions that produce drastic reductions in phosphorescence lifetime, spectral red shifts, quenching of fluorescence and phosphorescence emission and modulation of ANS binding. Judging from the full protection afforded by glycerol as cosolvent, or the remarkable enhancement caused by modest concentrations of urea, the driving force of these perturbations appears to be pressure-induced hydration of the protein. Inelastic conformational changes are accompanied by a slow and often incomplete recovery of enzymatic activity. The characteristic times of these processes, their pressure dependence and the slow, thermally activated, reversibility are discussed in the light of hysteresis phenomena and changes of subunit affinity in dissociation equilibria.

Dynamical structure of glutamate dehydrogenase as monitored by tryptophan phosphorescence. Signal transmission following binding of allosteric effectors.

Changes in conformation of glutamate dehydrogenase from beef liver as a result of interactions with allosteric effectors have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas activators ADP and L-leucine enhance considerably the rigidity of the protein structure surrounding the chromophore, inhibitors GTP, Zn2+ and Ag+ act in an opposite manner increasing the flexibility of this region of the macromolecule. Such changes in dynamical structure of the protein are confirmed independently for the ADP and GTP complexes by oxygen diffusion studies. Phosphorescence lifetime measurements at various protein concentrations and with the enzyme crosslinked by glutaraldehyde demonstrate that ADP and GTP exert the same effect on the structure of the protein regardless of its degree of polymerization. The connection between changes in protein structure and regulatory function is strengthened by the finding that (1) ligands with no regulatory function (Eu3+) do not affect protein structure; (2) pairs of opposite effectors which neutralize each other's influence on catalytic activity do restore an apparent native-like structure in the enzyme. Mutual neutralization and the observation that ADP and GTP display maximum activity at partial saturation of the binding sites has been interpreted in terms of a model which assumes asymmetry in the hexameric enzyme at the trimer level. Evidence for the existence of conformational heterogeneity among the subunits of the enzyme has been provided.

Active-site copper and zinc ions modulate the quaternary structure of prokaryotic Cu,Zn superoxide dismutase.

The influence of the constitutive metal ions on the equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase has been studied for the wild-type and for two mutant protein forms bearing a negative charge in the amino acid clusters at the dimer association interface. Depletion of copper and zinc dissociates the two mutant proteins into monomers, which reassemble toward the dimeric state upon addition of stoichiometric amounts of zinc. Pressure-dependent dissociation is observed for the copper-depleted wild-type and mutated enzymes, as monitored by the fluorescence shift of a unique tryptophan residue located at the subunit association interface. The spectral shift occurs slowly, reaching a plateau after 15-20 minutes, and is fully reversible. The recovery of the original fluorescence properties, after decompression, is fast (less than four minutes), suggesting that the isolated subunit has a relatively stable structure, and excluding the presence of stable intermediates during the dimer-monomer transition. The dimer dissociation process is still incomplete at 6.5 kbar for the copper-depleted wild-type and mutated enzymes, at variance with what is generally observed for oligomeric proteins that dissociate below 3 kbar. Measurement of the degree of dissociation, at two different protein concentrations, allows us to calculate the standard volume variation upon association, Delta V, and the dissociation constant K(d0), at atmospheric pressure, (25 ml/mol and 3 x 10(-7)M, respectively). The holoprotein is fully dimeric even at 6.5 kbar, which allows us to evaluate a lower Delta G degrees limit of 11.5 kcal/mol, corresponding to a dissociation constant K(d0)<10(-9)M.

Oxygen and acrylamide quenching of protein phosphorescence: correlation with protein dynamics.

Oxygen quenching of protein phosphorescence and activation enthalpies for the structural fluctuations underlying O2 and acrylamide diffusion were determined for RNase T1, glyceraldehyde-3-phosphate dehydrogenase and beta-lactoglobulin, which have the phosphorescing residues located in relatively solvent-exposed and flexible regions of the polypeptide. The results, compared with those obtained for proteins characterised by a very rigid environment, established that kqO2 was directly correlated to the flexibility of the protein matrix surrounding the chromophore. While the migration of acrylamide was characterised by delta H(double dagger), which was strongly dependent on the fluidity of the structure about the Trp residue, the values of the activation enthalpies for the oxygen migration of all the proteins studied were rather similar, approximately 10 kcal mol(-1), in spite of the depth of the chromophore and the rigidity of its environment. The implications of these findings for the migration of small solutes inside proteins have been discussed.

Tryptophan phosphorescence as a monitor of the solution structure of phosphoglycerate kinase from yeast.

The enzyme phosphoglycerate kinase from yeast possesses two tryptophan residues whose phosphorescence spectrum in low-temperature glasses is resolved into two distinct components with 0-0 vibronic bands centered at 408 and 412.5 nm. The thermal profile of the phosphorescence intensity and lifetime shows that the red (longer wavelength) component is quenched in fluid solutions so that the long-lived phosphorescence observed at ambient temperature in buffer is due entirely to the blue (shorter wavelength) component. The remarkable heterogeneity in flexibility of the two chromophores' sites inferred from the thermal behaviour, when analyzed in terms of the crystallographic structure, allows to make a straightforward assignment of the long-lived emission to internal Trp-333. Because in buffer the phosphorescence is due to only one Trp residue the biphasic nature of the decay reveals the presence of stable, slowly interconverting, conformers with profound differences in the internal fluidity of the C-domain. Further, according to the triplet lifetime, complex formation with substrates affect the protein structure in a very selective way. Thus, while 3-phosphoglycerate has practically no influence on the average lifetime, Mg ATP and Mg ADP increases tau by a factor of 1.9 and 5.3, respectively. The change in lifetime implies a remarkable stiffening of the C-domain which is partly relaxed in ternary complexes with 3-phosphoglycerate. These findings are discussed in terms of ligand-induced "closed" conformations of the protein.

Heterogeneity of protein conformation in solution from the lifetime of tryptophan phosphorescence.

The decay of Trp phosphorescence of proteins in fluid solutions was shown to provide a sensitive tool for probing the conformational homogeneity of these macromolecules in the millisecond to second time scale. Upon examination of 15 single Trp emitting proteins multiexponential decays were observed in 12 cases, a demonstration that the presence of slowly interconverting conformers in solution is more the norm rather than an exception. The amplitude of preexponential terms, from which the conformer equilibrium is derived, was found to be a sensitive function of solvent composition (buffer, pH, ionic strength and glycerol cosolvent), temperature, and complex formation with substrates and cofactors. In many cases, raising the temperature, a point is reached at which the decay becomes practically monoexponential, meaning that conformer interconversion rates have become commensurate with the triplet lifetime. Estimation of activation free energy barriers to interconversion shows that the large values of DeltaG* are rather similar among polypeptides and that the protein substates involved are sufficiently long-lived to display individual binding/catalytic properties.

Early cognitive and communication development in children with focal brain lesions.

Early cognitive and language development of children with congenital focal brain lesions, documented by magnetic resonance imaging, was studied in 18 cases, 9 with left-hemisphere damage and 9 with right-hemisphere damage, at about 2 (Time 1) and 4 years of age (Time 2). All of the children showed normal cognitive development, but their global Griffiths Developmental Scales scores were lower at Time 2, and developmental profiles across individual subscales revealed side-specific effects, resembling the adult left/right cerebral hemisphere lesion model. Expressive lexicon and grammar were delayed, more often in left-hemisphere-damaged than in right-hemisphere-damaged children, at Time 1 and Time 2. Functional findings were not related to the size and location of the brain lesion, whereas the presence of epilepsy was a highly significant predictor of cognitive and language outcome, irrespective of the side of the lesion. The stable disadvantage in the verbal domain shown by left-hemisphere-damaged children within the age range of this study might suggest that the left hemisphere has some initial bias for language learning. The effects of right-hemisphere damage were more variable and emerged at a later stage of language development.

Alteration of the intramolecular dynamics of glycogen phosphorylase b by allosteric ligands.

Phosphorylase b (E.C. 2.4.1.1), prepared from rabbit skeletal muscle, was used to study whether the binding of allosteric ligands modifies the intramolecular dynamics of the protein matrix. Protein dynamics were monitored through the fluorescence and phosphorescence parameters of the 12 tryptophan (Trp) residues (one monomer) of the enzyme. The phosphorescence lifetime was measured at room temperature both in the absence and the presence of ligands. The addition of an allosteric inhibitor (ATP) decreased the lifetime, while the presence of activator (AMP) and/or substrate (G-1-P) had no detectable effect. The lifetime data allow us to conclude that the environment of the buried tryptophans becomes more flexible upon the binding of ATP, while the other ligands did not induce such change. The ATP-induced perturbation was also examined by the quenching of Trp fluorescence by acrylamide. The quenching parameters did not show any change, suggesting that the effect of ATP is localized to the vicinity of the phosphorescent Trp residues.

Acaricidal activity of pine essential oils and their main components against Tyrophagus putrescentiae, a stored food mite.

Some essential oils obtained from the branches of four Pinus species (P. pinea L., P. halepensis Mill., P. pinaster Soil in Ait., and P. nigra Arnold) have been evaluated for their acaricidal activity by aerial diffusion against the stored food mite Tyrophagus putrescentiae (L.). All the essential oils showed a good efficacy, but P. pinea oil and its two constituents 1,8-cineole and limonene were the most effective compounds, showing 100% acaricidal activity at 8 microL; 1,8-cineole showed the same activity at 6 microL.

In vitro antimicrobial activity of extracts and isolated constituents of Rubus ulmifolius.

The antimicrobial activity on bacteria and fungi of increasing polarity extracts of Rubus ulmifolius and that of some isolated constituents, quercetin-3-O-beta-D-glucuronide; kaempferol-3-O-beta-D-glucuronide, gallic acid, ferulic acid and tiliroside was evaluated. The phenolic and tannins fractions showed an high antimicrobial activity.

Antimicrobial activity of extracts of Mutisia acuminata var. acuminata.

The biological activity of methanol and water extracts of Mutisia acuminata Ruiz et Pavon var. acuminata (Asteraceae) against bacteria and fungi (spores and mycelia) was evaluated. The role of pH of the medium in antimicrobial activity was investigated.

Composition and antimicrobial properties of essential oils of four Mediterranean Lamiaceae.

Essential oils from Satureja montana L., Rosmarinus officinalis L., Thymus vulgaris L., and Calamintha nepeta (L.) Savi, were chemically analysed and their antimicrobial and fungicide activities evaluated on the basis of their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). All four oils have a biotoxic effect, the most active being those from Calamintha and Thymus.

Acaricidal agents of natural origin against Psoroptes cuniculi.

Lavandula angustifolia Miller essential oil and some of its main constituents, i.e. linalool, linalyl acetate, and camphor were tested in vitro for their bioactivities against Psoroptes cuniculi (Delafond, 1859), mite of the rabbit. The essential oil and linalool were found to have very powerful miticidal activities.

[Goltz syndrome]. / La sindrome di Goltz.

Goltz syndrome or focal dermal hypoplasia is a hereditary disorder, is a rare mesodermal hypoplasia found primarily in females. It is characterized by linear hypoplasia of the skin and tumors of fat or lipomatous lesions. There are significant defects of the skeleton, dental structures, eyes, soft tissues and skin. In our work an example of new-born female with this syndrome is reported, and a review of 136 cases from the literature is presented.

Experimental and simulative dissociation of dimeric Cu,Zn superoxide dismutase doubly mutated at the intersubunit surface.

The equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase mutant bearing two negative charges in the amino acid clusters at the association interface has been studied, experimentally and computationally, and compared to those of the native enzyme. Pressure-dependent dissociation is observed for the mutant, as observed by the fluorescence shift of the unique tryptophan residue located at the intersubunit surface. The spectral shift occurs slowly, reaching a plateau after 15-20 min, and is fully reversible. Measurement of the degree of dissociation allows us to calculate the standard volume variation upon association and the dissociation constant at atmospheric pressure. On the other hand the native protein is undissociable at any pressure. In the simulative approach, the dissociation free energy has been calculated through the blue moon calculation method for the case of a multidimensional reaction coordinate, corrected for the rotational contribution within the semiclassical approximation for a free rigid-body rotor. The scheme permits to define a definite path for the rupture of the dimer and to calculate the effective force involved in the process. The calculated free energy difference is close to the experimental one, and the value obtained for the mutant is well below that obtained for the native protein, indicating that the theoretical reaction scheme is able to reproduce the experimental trend. Moreover, we find that, when the separation distance increases, the protein structure of the monomer is stable in line with the fast recovery of the original fluorescence properties after decompression, which excludes the presence of partly unfolded intermediates during the dimer-monomer transition.