Heat and cold denaturation of yeast frataxin: The effect of pressure.

Yfh1 is a yeast protein with the peculiar characteristic to undergo, in the absence of salt, cold denaturation at temperatures above the water freezing point. This feature makes the protein particularly interesting for studies aiming at understanding the rules that determine protein fold stability. Here, we present the phase diagram of Yfh1 unfolding as a function of pressure (0.1-500 MPa) and temperature 278-313 K (5-40°C) both in the absence and in the presence of stabilizers using Trp fluorescence as a monitor. The protein showed a remarkable sensitivity to pressure: at 293 K, pressures around 10 MPa are sufficient to cause 50% of unfolding. Higher pressures were required for the unfolding of the protein in the presence of stabilizers. The phase diagram on the pressure-temperature plane together with a critical comparison between our results and those found in the literature allowed us to draw conclusions on the mechanism of the unfolding process under different environmental conditions.

Exploiting gold nanoparticles for diagnosis and cancer treatments.

Gold nanoparticles (AuNPs) represent a relatively simple nanosystem to be synthesised and functionalized. AuNPs offer numerous advantages over different nanomaterials, primarily due to highly optimized protocols for their production with sizes in the range 1-150 nm and shapes, spherical, nanorods (AuNRs), nanocages, nanostars or nanoshells (AuNSs), just to name a few. AuNPs possess unique properties both from the optical and chemical point of view. AuNPs can absorb and scatter light with remarkable efficiency. Their outstanding interaction with light is due to the conduction electrons on the metal surface undergoing a collective oscillation when they are excited by light at specific wavelengths. This oscillation, known as a localized surface plasmon resonance, causes the absorption and scattering intensities of AuNPs to be significantly higher than identically sized non-plasmonic nanoparticles. In addition, AuNP absorption and scattering properties can be tuned by controlling the particle size, shape, and the local refractive index near the particle surface. By the chemical side, AuNPs offer the advantage of functionalization with therapeutic agents through covalent and ionic binding, which can be useful for biomedical applications, with particular emphasis on cancer treatments. Functionalized AuNPs exhibit good biocompatibility and controllable distribution patterns when delivered in cells and tissues, which make them particularly fine candidates for the basis of innovative therapies. Currently, major available AuNP-based cancer therapeutic approaches are the photothermal therapy (PTT) or photodynamic therapy (PDT). PTT and PDT rely upon irradiation of surface plasmon resonant AuNPs (previously delivered in cancer cells) by light, in particular, in the near-infrared range. Under irradiation, AuNPs surface electrons are excited and resonate intensely, and fast conversion of light into heat takes place in about 1 ps. The cancer cells are destroyed by the induced hyperthermia, i.e. the condition under which cells are subject to temperature in the range of 41 °C-47 °C for tens of minutes. The review is focused on the description of the optical and thermal properties of AuNPs that underlie their continuous and progressive exploitation for diagnosis and cancer therapy.

Soluble and Nanoporous Silica Gel-Entrapped C. freundii Methionine γ-Lyase.

Methionine γ-lyase is a pyridoxal 5'-phosphate dependent tetramer that catalyzes the α,γ-elimination of methionine in ammonia, methanethiol and α-ketobutyrate. MGL catalytic power has been exploited as a therapeutic strategy to reduce the viability of cancer cells or bacteria. In order to obtain a stable enzyme to be delivered at the site of action, MGL can be encapsulated in a variety of matrices. As a reference encapsulation strategy we have prepared MGL nanoporous wet silica gels. Immobilized MGL gels were characterized with regards to activity, stability, absorption, circular dichroism and fluorescence properties and compared with soluble MGL. We found that MGL gels exhibit (i) spectroscopic properties very similar to MGL in solution, (ii) a higher stability with respect to the soluble enzyme and (iii) catalytic activity six-fold lower than in solution. These findings prove that MGL encapsulation is a suitable strategy for therapeutic applications.

TiO2 nanoparticles in seawater: Aggregation and interactions with the green alga Dunaliella tertiolecta.

Titanium dioxide nanoparticles (TiO2 NPs) have been widely employed in industrial applications, thus rising concern about their impact in the aquatic environment. In this study we investigated the chemical behaviour of TiO2 NPs in the culture medium and its effect on the green alga Dunaliella tertiolecta, in terms of growth inhibition, oxidative stress, ROS (Reactive Oxygen Species) accumulation and chlorophyll content. In addition, the influence of exopolymeric substances (EPS) excreted by the microalgae on the stability of NPs has been evaluated. The physicochemical characterization showed a high propensity of TiO2 NPs to form micrometric-sized aggregates within 30min, large enough to partially settle to the bottom of the test vessel. Indeed, an increasing amount of TiO2 particles settled out with time, but the presence of EPS seemed to mitigate this behaviour in the first 6h of exposure where the main effects in D. tertiolecta were observed. TiO2 NPs did not inhibit the 72-h growth rate of D. tertiolecta, nor affected the cellular chlorophyll concentration in the range 0.01-10mgL-1. The time-course of ROS production showed an initial transient increase of ROS in TiO2 NP-exposed algae compared to the control, concomitant with an enhancement of catalase activity. Interestingly, intracellular ROS was a small fraction of total ROS, the highest amount being extracellular. The occurrence of cell-mediated chemical transformations of TiO2 NPs in the external medium, related to the presence of EPS, has been evaluated. Our results showed that carbohydrates were the major component of EPS, whereas proteins of medium molecular weight (20-80kDa) were preferentially bound to TiO2 NPs, likely influencing their biological fate.

Gene cloning, characterization, and cytotoxic activity of methionine γ-lyase from Clostridium novyi.

The exploitation of methionine-depleting enzyme methionine γ-lyase (MGL) is a promising strategy against specific cancer cells that are strongly dependent on methionine. To identify MGL from different sources with high catalytic activity and efficient anticancer action, we have expressed and characterized MGL from Clostridium novyi and compared its catalytic efficiency with the previously studied MGL from Citrobacter freundii. The purified recombinant MGL exhibits kcat and kcat /Km for methionine γ-elimination reaction that are 2.4- and 1.36-fold higher than C. freundii enzyme, respectively, whereas absorption, fluorescence, and circular dichroism spectra are very similar, as expected on the basis of 87% sequence identity and high conservation of active site residues. The reactivity of cysteine residues with DTNB and iodoacetamide was investigated as well as the impact of their chemical modification on catalytic activity. This information is relevant because for increasing bioavailability and reducing immunogenity, MGL should be decorated with polyethylene glycol (PEG). It was found that Cys118 is a faster reacting residue, which results in a significant decrease in the γ-elimination activity. Thus, the protection of Cys118 before conjugation with cysteine-reacting PEG represents a valuable strategy to preserve MGL activity. The anticancer action of C. novyi MGL, evaluated in vitro against prostate (PC-3), chronic myelogenous leucemia (K562), and breast (MDA-MB-231 and MCF7) cancer cells, exhibits IC50 of 1.3 U mL-1 , 4.4 U mL-1 , 1.2 U mL-1 , and 3.4 U mL-1 , respectively. A higher cytotoxicity of C. novyi MGL was found against cancer cells with respect to C. freundii MGL, with the exception of PC-3, where a lower cytotoxicity was observed. © 2017 IUBMB Life, 69(9):668-676, 2017.

Temperature and pressure effects on GFP mutants: explaining spectral changes by molecular dynamics simulations and TD-DFT calculations.

By combining spectroscopic measurements under high pressure with molecular dynamics simulations and quantum mechanics calculations we investigate how sub-angstrom structural perturbations are able to tune protein function. We monitored the variations in fluorescence output of two green fluorescent protein mutants (termed Mut2 and Mut2Y, the latter containing the key T203Y mutation) subjected to pressures up to 600 MPa, at various temperatures in the 280-320 K range. By performing 150 ns molecular dynamics simulations of the protein structures at various pressures, we evidenced subtle changes in conformation and dynamics around the light-absorbing chromophore. Such changes explain the measured spectral tuning in the case of the sizable 120 cm(-1) red-shift observed for pressurized Mut2Y, but absent in Mut2. Previous work [Barstow et al., Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 13362] on pressure effects on GFP also involved a T203Y mutant. On the basis of cryocooling X-ray crystallography, the pressure-induced fluorescence blue shift at low temperature (77 K) was attributed to key changes in relative conformation of the chromophore and Tyr203 phenol ring. At room temperature, however, a red shift was observed at high pressure, analogous to the one we observe in Mut2Y. Our investigation of structural variations in compressed Mut2Y also explains their result, bridging the gap between low-temperature and room-temperature high-pressure effects.

A SELDI-TOF approach to ecotoxicology: comparative profiling of low molecular weight proteins from a marine diatom exposed to CdSe/ZnS quantum dots.

Quantum dots (QDs), namely semiconductor nanocrystals, due to their particular optical and electronic properties, have growing applications in device technology, biotechnology and biomedical fields. Nevertheless, the possible threat to human health and the environment have attracted increasing attention as the production and applications of QDs increases rapidly while standard evaluation of safety lags. In the present study we performed proteomic analyses, by means of 2D gel electrophoresis and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS). We aimed to identify potential biomarkers of exposure to CdSe/ZnS quantum dots. The marine diatom Phaeodactylum tricornutum exposed to 2.5nM QDs was used as a model system. Both 2DE and SELDI showed the presence of differentially expressed proteins. By Principal Component Analysis (PCA) we were able to show that the differentially expressed proteins can discriminate between exposed and not exposed cells. Furthermore, a protein profile specific for exposed cells was obtained by SELDI analysis. To our knowledge, this is the first example of the application of SELDI technology to the analysis of microorganisms used as biological sentinel model of marine environmental pollution.

Temperature and pressure effects on C112S azurin: volume, expansivity, and flexibility changes.

The pressure-induced unfolding of the mutant C112S azurin from Pseudomonas aeruginosa was monitored both under steady state and dynamic conditions. The unfolding profiles were obtained by recording the spectral shift of the fluorescence emission as well as by phosphorescence intensity measurements. We evaluated the difference in free energy, ΔG, as a function of pressure and temperature. The dependence of ΔG on temperature showed concave profile at all pressures studied. A positive heat capacity change of about 4.3 kJ mol(-1) deg(-1) fitted all the curves. The volume change of the reaction showed a moderate dependence on temperature when compared with other proteins previously studied. The kinetic activation parameters (ΔV*, ΔH*, ΔS*) were obtained from upward and downward pressure-jump experiments and used to characterize the volumetric and energetic properties of the transition state between native and unfolded protein. Our findings suggest that the folding and unfolding reaction paths passed through different transition states. The change in the phosphorescence lifetime with pressure pointed out that pressure-induced unfolding occurred within two steps: the first leading to an increased protein flexibility, presumably caused by water penetration into the protein. Major structural changes of the tryptophan environment occurred in a second step at higher pressures.

Conjugation to gold nanoparticles of methionine gamma-lyase, a cancer-starving enzyme. Physicochemical characterization of the nanocomplex for prospective nanomedicine applications.

The pyridoxal 5'-dependent enzyme methionine γ-lyase (MGL) catalyzes the degradation of methionine. This activity has been profitable to develop an antitumor agent exploiting the strict dependence of most malignant cells on the availability of methionine. Indeed, methionine depletion blocks tumor proliferation and leads to an increased susceptibility to anticancer drugs. Here, we explore the conjugation of MGL to gold nanoparticles capped with citrate (AuNPs) as a novel strategy to deliver MGL to cancer cells. Measurements of Transmission Electron Microscopy, Dynamic Light Scattering, Asymmetrical Flow Field-Flow Fractionation, X-ray Photoelectron Spectroscopy, and Circular Dichroism allowed to achieve an extensive biophysical and biochemical characterization of the MGL-AuNP complex including particle size, size distribution, MGL loading yield, enzymatic activity, and impact of gold surface on protein structure. Noticeably, we found that activity retention was improved over time for the enzyme adsorbed to AuNPs with respect to the enzyme free in solution. The acquired body of knowledge on the nanocomplex properties and this encouraging stabilizing effect upon conjugation are the necessary basis for further studies aimed at the evaluation of the therapeutic potential of MGL-AuNP complex in a biological milieu.

Protein dynamics and pressure: what can high pressure tell us about protein structural flexibility?

After a brief overview of NMR and X-ray crystallography studies on protein flexibility under pressure, we summarize the effects of hydrostatic pressure on the native fold of azurin from Pseudomonas aeruginosa as inferred from the variation of the intrinsic phosphorescence lifetime and the acrylamide bimolecular quenching rate constants of the buried Trp residue. The pressure/temperature response of the globular fold and modulation of its dynamical structure is analyzed both in terms of a reduction of internal cavities and of the hydration of the polypeptide. The study of the effect of two single point cavity forming mutations, F110S and I7S, on the unfolding volume change (ΔV(0)) of azurin and on the internal dynamics of the protein fold under pressure demonstrate that the creation of an internal cavity will enhance the plasticity and lower the stability of the globular structure. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Use of Exogenous Enzymes in Human Therapy: Approved Drugs and Potential Applications.

The development of safe and efficacious enzyme-based human therapies has increased greatly in the last decades, thanks to remarkable advances in the understanding of the molecular mechanisms responsible for different diseases, and the characterization of the catalytic activity of relevant exogenous enzymes that may play a remedial effect in the treatment of such pathologies. Several enzyme-based biotherapeutics have been approved by FDA (the U.S. Food and Drug Administration) and EMA (the European Medicines Agency) and many are undergoing clinical trials. Apart from enzyme replacement therapy in human genetic diseases, which is not discussed in this review, approved enzymes for human therapy find applications in several fields, from cancer therapy to thrombolysis and the treatment, e.g., of clotting disorders, cystic fibrosis, lactose intolerance and collagen-based disorders. The majority of therapeutic enzymes are of microbial origin, the most convenient source due to fast, simple and cost-effective production and manipulation. The use of microbial recombinant enzymes has broadened prospects for human therapy but some hurdles such as high immunogenicity, protein instability, short half-life and low substrate affinity, still need to be tackled. Alternative sources of enzymes, with reduced side effects and improved activity, as well as genetic modification of the enzymes and novel delivery systems are constantly searched. Chemical modification strategies, targeted-and/or nanocarrier-mediated delivery, directed evolution and site-specific mutagenesis, fusion proteins generated by genetic manipulation are the most explored tools to reduce toxicity and improve bioavailability and cellular targeting. This review provides a description of exogenous enzymes that are presently employed for the therapeutic management of human diseases with their current FDA/EMA-approved status, along with those already experimented at the clinical level and potential promising candidates.

A novel hotspot of gelsolin instability triggers an alternative mechanism of amyloid aggregation.

Gelsolin comprises six homologous domains, named G1 to G6. Single point substitutions in this protein are responsible for AGel amyloidosis, a hereditary disease causing progressive corneal lattice dystrophy, cutis laxa, and polyneuropathy. Although several different amyloidogenic variants of gelsolin have been identified, only the most common mutants present in the G2 domain have been thoroughly characterized, leading to clarification of the functional mechanism. The molecular events underlying the pathological aggregation of 3 recently identified mutations, namely A551P, E553K and M517R, all localized at the interface between G4 and G5, are here explored for the first time. Structural studies point to destabilization of the interface between G4 and G5 due to three structural determinants: ß-strand breaking, steric hindrance and/or charge repulsion, all implying impairment of interdomain contacts. Such rearrangements decrease the temperature and pressure stability of gelsolin but do not alter its susceptibility to furin cleavage, the first event in the canonical aggregation pathway. These variants also have a greater tendency to aggregate in the unproteolysed forms and exhibit higher proteotoxicity in a C. elegans-based assay. Our data suggest that aggregation of G4G5 variants follows an alternative, likely proteolysis-independent, pathway.

Interplay between extracellular polymeric substances (EPS) from a marine diatom and model nanoplastic through eco-corona formation.

The occurrence of nanoplastics in oceans' surface waters is no more a hypothesis and it could severely affect marine organisms from different trophic levels. Nanoscale particles interaction with dissolved natural organic matter (NOM) significantly influence their behaviour and consequently bioavailability and toxicity to marine species. Extracellular polymeric substances (EPS) are among the main components of the NOM pool in seawater yet have been so far little investigated for their effect in altering the physical-chemical properties of nanosized objects. Here we employed EPS from marine diatom Phaeodactylum tricornutum to study the evolution of an eco-corona formation upon incubation with 60 nm carboxylated polystyrene nanoparticles (PS-COOH NPs), as proxy for nanoplastics in seawater. EPS significantly reduced PS-COOH NPs aggregation rate compared to biomolecule free natural seawater (NSW) and caused the formation of complexes constituted by both carbohydrate and protein components. Size Exclusion Chromatography (SEC) revealed four main distinct groups of peaks, spanning from high (>100 kDa) to low molecular weight (20 kDa) molecules, characterized by a high chemical heterogeneity. The lowering of the chromatographic signals detected after EPS incubation with PS-COOH NPs, mainly in the eluates at high molecular weight, suggests that an important fraction of EPS remained adsorbed on PS-COOH NPs. In agreement, SDS-PAGE analysis of proteins adsorbed on PS-COOH showed the occurrence of an eco-corona formed by proteins in the range of molecular weight 30-100 kDa. No toxicity to diatoms was observed upon PS-COOH exposure (72 h, 1-100 mg L-1) even by adding a further source of exogenous EPS during exposure. Moreover, the addition of EPS reduced ROS production, even when cells were incubated with PS-COOH NPs at 10 and 50 mg L-1, suggesting an antioxidant scavenging activity of EPS.

Cavity-creating mutations in Pseudomonas aeruginosa azurin: effects on protein dynamics and stability.

Changes in flexibility and structural stability of Pseudomonas aeruginosa azurin in response to cavity-creating mutations were probed by the phosphorescence emission of Trp-48, which was deeply buried in the compact hydrophobic core of the macromolecule, and by measurements of guanidinum hydrochloride unfolding, respectively. Replacement of the bulky side chains Phe-110, Phe-29, and Tyr-108 with the smaller Ala introduced cavities at different distances from the hydrophobic core. The phosphorescence lifetime (tau(0)) of Trp-48, buried inside the protein core, and the acrylamide quenching rate constant (k(q)) were used to monitor local and global flexibility changes induced by the introduction of the cavity. The results of this work demonstrate the following: 1), the effect on core flexibility of the insertion of cavities is not correlated readily to the distance of the cavity from the core; 2), the protein global flexibility results are related to the cavity distance from the packed core of the macromolecule; and 3), the increase in protein flexibility does not correspond necessarily to a comparable destabilizing effect of some mutations.

Engineering methionine γ-lyase from Citrobacter freundii for anticancer activity.

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis.

Dendrimer-protein interactions studied by tryptophan room temperature phosphorescence.

Dendrimers are a relatively new class of materials with unique molecular architectures, which provide promising opportunities for biological applications as DNA carriers and drug delivery systems. Progress in these fields, however, requires knowledge of their potential interactions with biological components at cellular and molecular level. This study utilizes Trp phosphorescence spectroscopy to examine possible perturbations of the protein native fold in solution by neutral, positively and negatively charged fifth generation polyamidoamine (PAMAM) dendrimers. Phosphorescence lifetime measurements, conducted on model proteins varying in the degree of burial of the triplet probe and in quaternary structure, show that dendrimers interact with proteins in solutions forming stable complexes in which the protein structure may be significantly altered, particularly in superficial, flexible regions of the polypeptide. Both electrostatic and non-electrostatic interactions can give rise to stable complexes, whose affinity and limited number of binding sites distinguish them from mere aspecific molecular associations. Of direct relevance for the application of these polymers in the medical field, structural alterations have also been detected in human plasma proteins such as serum albumin and immunoglobulins. The above results suggest that Trp phosphorescence may provide a useful monitor for working out experimental conditions and protocols that help preserve the structural integrity of proteins in the presence of these polymers.

The response of Phaeodactylum tricornutum to quantum dot exposure: Acclimation and changes in protein expression.

Nanotechnology has a great potential to improve life and environmental quality, however the fate of nanomaterials in the ecosystems, their bioavailability and potential toxicity on living organisms are still largely unknown, mainly in the marine environment. Genomics and proteomics are powerful tools for understanding molecular mechanisms triggered by nanoparticle exposure. In this work we investigated the effect of exposure to CdSe/ZnS quantum dots (QDs) in the marine diatom Phaeodactylum tricornutum, using different physiological, biochemical and molecular approaches. The results show that acclimation to QDs reduced the growth inhibition induced by nanoparticles in P. tricornutum cultures. The increase of glutathione observed at the end of the lag phase pointed to cellular stress. Transcriptional expression of selected stress responsive genes showed up-regulation in the QD-exposed algae. A comparison of the proteomes of exposed and unexposed cells highlighted a large number of differentially expressed proteins. To our knowledge, this is the first report on proteome analysis of a marine microalga exposed to nanoparticles.

Efficient biosynthetic incorporation of tryptophan and indole analogs in an integral membrane protein.

Biosynthetic incorporation of tryptophan (Trp) analogs such as 7-azatryptophan, 5-hydroxytryptophan, and fluorotryptophan into a protein can facilitate its structural analysis by spectroscopic techniques such as fluorescence, phosphorescence, nuclear magnetic resonance, and Fourier transform infrared. Until now, the approach has dealt primarily with soluble proteins. In this article, we demonstrate that four different Trp analogs can be very efficiently incorporated into a membrane protein as demonstrated for the mannitol transporter of Escherichia coli (EII(mtl)). EII(mtl) overexpression was under control of the lambdaP(R) promoter, and the E. coli Trp auxotroph M5219 was used as host. This strain constitutively expresses the heat labile repressor protein of the lambdaP(R) promoter. Together with the presence of the repressor gene on the EII(mtl) plasmid, this resulted in a tightly controlled promoter system, a prerequisite for high Trp analog incorporation. A new method for determining the analog incorporation efficiency is presented that is suitable for membrane proteins. The procedure involves fitting of the phosphorescence spectrum as a linear combination of the Trp and Trp analog contributions, taking into account the influence of the protein environment on the Trp analog spectrum. The data show that the analog content of EII(mtl) samples is very high (>95%). In addition, we report here that biosynthetic incorporation of Trp analogs can also be effected with less expensive indole analogs, which in vivo are converted to L-Trp analogs.

Temperature and pressure dependence of azurin stability as monitored by tryptophan fluorescence and phosphorescence. The case of F29A mutant.

The effects of a single-point, F29A, cavity-forming mutation on the unfolding thermodynamic parameters of azurin from Pseudomonas aeruginosa and on the internal dynamics of the protein fold under pressure were probed by the fluorescence and phosphorescence emission of Trp48, deeply buried in the compact hydrophobic core of the macromolecule. Pressure-induced unfolding, monitored by the shift in the fluorescence spectrum, led to a volume change of 70-90mlmol(-1). The difference in the unfolding volume between F29A and wild type azurin was smaller than the volume of the space theoretically created in the mutant, indicating that the cavity is, at least partially, filled with water molecules. The complex temperature dependence of the unfolding volume, for temperatures up to 20°C, suggests the formation of an expanded form of the protein and highlights how the packing efficiency of azurin appears to contribute to the magnitude of internal void volume at any given temperature. Changes in flexibility of the protein matrix around the chromophore were monitored by the intrinsic phosphorescence lifetime. At 40°C the application of pressure in the predenaturation range initially decreases the internal flexibility of azurin, the trend eventually reverting on approaching unfolding. The main difference between wild type and the cavity mutant is the inversion point which happens at 300MPa for wild type and at 150MPa for F29A. This suggests that, for the cavity mutant, pressure-induced internal hydration is more dominant than any compaction of the globular fold at relatively low pressures.

Interaction of CdSe/ZnS quantum dots with the marine diatom Phaeodactylum tricornutum and the green alga Dunaliella tertiolecta: a biophysical approach.

In this study, we investigated the interaction of nanoparticles, such as CdSe/ZnS quantum dots (QDs), with the marine diatom Phaeodactylum tricornutum and the green alga Dunaliella tertiolecta, as biological models in the marine environment. Fluorescence kinetics measurements indicated that 30min after dispersion in seawater QDs lost the 60% of the initial emission intensity, possibly due to the occurrence of aggregation processes. However, the presence of algae seemed to mitigate this effect. By using confocal microscopy, we highlighted the presence of QDs adsorbed on the surface of both algae, but not inside the cells. The toxicity of QDs was evaluated in terms of inhibition of growth rate, oxidative stress, and lipid peroxidation. QDs in the range of 1-2.5nM gradually inhibited the growth rate of P. tricornutum and increased the oxidative stress, as evinced by the increase in lipid peroxidation, reactive oxygen species (ROS) production and activity of two main antioxidant enzymes (superoxide dismutase and catalase). On the contrary, QDs did not inhibit the growth rate of D. tertiolecta, at most a modest stimulation was observed in the range of 0.5-2nM, suggesting a hormetic response. No effect in the parameters indicating oxidative stress was observed in the green alga. In conclusion our results showed that the biological effects were species-specific.