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Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5'-monophosphate decarboxylase, where the rapid (μs timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (βα)

Enzymes are conformationally dynamic, and their dynamical properties play an important role in regulating their specificity and evolvability. In this context, substantial attention has been paid to the role of ligand-gated conformational changes in enzyme catalysis; however, such studies have focused on tremendously proficient enzymes such as triosephosphate isomerase and orotidine 5’-monophosphate decarboxylase, where the rapid (μs timescale) motion of a single loop dominates the transition between catalytically inactive and active conformations. In contrast, the (βα)8-barrels of tryptophan and histidine biosynthesis, such as the specialist isomerase enzymes HisA and TrpF, and the bifunctional isomerase PriA, are decorated by multiple long loops that undergo conformational transitions on the ms (or slower) timescale. Studying the interdependent motions of multiple slow loops, and their role in catalysis, poses a significant computational challenge. This work combines conventional and enhanced molecular dynamics simulations with empirical valence bond simulations to provide rich detail of the conformational behavior of the catalytic loops in HisA, PriA and TrpF, and the role of their plasticity in facilitating bifunctionality in PriA and evolved HisA variants. In addition, we demonstrate that, similar to other enzymes activated by ligand-gated conformational changes, loops 3 and 4 of HisA and PriA act as gripper loops, facilitating the isomerization of the large bulky substrate ProFAR, albeit now on much slower timescales. This hints at convergent evolution on these different (βα)8-barrel scaffolds. Finally, our work highlights the potential of engineering loop dynamics as a powerful tool to artificially manipulate the diverse catalytic repertoire of TIM-barrel proteins.

The cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. An in-depth understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical, and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for the cooperative interplay between the nucleophilic and general-acid catalytic groups in the wild-type enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is similar to 10(4)-fold. Cooperativity with the catalytic acid adds >= 10(2)-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.

Cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. A deepened understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes, and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for cooperative interplay between the nucleophilic and general-acid catalytic groups in the wildtype enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is around 10^4-fold. Cooperativity with the catalytic acid adds ≥10^2-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.

Hyperbolic nanoparticles provide a versatile platform to widely tune light-matter interactions. Active nanophotonics can be realized by controlling the optical properties of materials with external magnetic fields. Here, we explore the influence of optical anisotropy on the magneto-optical response of hyperbolic nanoparticles across the visible and near infrared spectral range. By using a perturbative approach, we establish a model where the magneto-optical activity of the system is described in terms of the coupling of fundamental electric and magnetic dipole modes, which are induced by the hyperbolic dispersion, with a static magnetic field. Finally, an analytical model is established in the framework of Mie theory to describe the magneto-optical response and identify the contribution of electric and magnetic modes to the total spectrum.

We study magneto-optical circular dichroism in type-II hyperbolic nanoantennas. Experiments and numerical simulations reveal a broadband response, which we ascribe to the excitation of electric and magnetic dipole modes coupled to an external magnetic field.

We introduce a novel concept of hybrid metal-dielectric meta-antenna supporting type II hyperbolic dispersion, which enables full control of absorption and scattering of light in the visible/near-infrared spectral range. This ability lies in the different nature of the localized hyperbolic Bloch-like modes excited within the meta-antenna. The experimental evidence is corroborated by a comprehensive theoretical study. In particular, we demonstrate that two main modes, one radiative and one non-radiative, can be excited by direct coupling with the free-space radiation. We show that the scattering is the dominating electromagnetic decay channel, when an electric dipolar mode is induced in the system, whereas a strong absorption process occurs when a magnetic dipole is excited. Also, by varying the geometry of the system, the relative ratio of scattering and absorption, as well as their relative enhancement and/or quenching, can be tuned at will over a broad spectral range, thus enabling full control of the two channels. Importantly, both radiative and nonradiative modes supported by our architecture can be excited directly with far-field radiation. This is observed to occur even when the radiative channels (scattering) are almost totally suppressed, thereby making the proposed architecture suitable for practical applications. Finally, the hyperbolic meta-antennas possess both angular and polarization independent structural integrity, unlocking promising applications as hybrid meta-surfaces or as solvable nanostructures.

Active nanophotonics can be realized by controlling the optical properties of materials with external magnetic fields. Here, we explore the influence of optical anisotropy on the magneto-optical activity in nonmagnetic hyperbolic nanoparticles. We demonstrate that the magneto-optical response is driven by the hyperbolic dispersion via the coupling of metallic-induced electric and dielectric-induced magnetic dipolar optical modes with static magnetic fields. Magnetic circular dichroism experiments confirm the theoretical predictions and reveal tunable magneto-optical activity across the visible and near infrared spectral range. published

We study magnetic circular dichroism of type II hyperbolic nanoparticles. Experiments and numerical simulations reveal a broadband response that is analytically described via coupling of electric and magnetic dipole modes with a static magnetic field.

We report on the fabrication and optical characterization of hyperbolic nanoparticles on a transparent substrate. These nanoparticles enable a separation of ohmic and radiative channels in the visible and near-infrared frequency ranges. The presented architecture opens the pathway towards novel routes to exploit the light to energy conversion channels beyond what is offered by current plasmon-based nanostructures, possibly enabling applications spanning from thermal emission manipulation, theragnostic nano-devices, optical trapping and nano-manipulation, non-linear optical properties, plasmonenhanced molecular spectroscopy, photovoltaics and solar-water treatments, as well as heat-assisted ultra-dense and ultrafast magnetic recording.

This chapter aims to describe the combination of photoactive nanoparticles (NPs) and biomolecules in order to create the so-called smart NPs and their applications in light-based therapies and diagnostics. Several methods of ligand–NP functionalization, providing a cross section of various ligand classes, have been discussed together with the different approaches of complex characterization technique currently in use.

This work presents the development and optimization of two fabrication methods that combine plasma polymerization and electron beam lithography techniques for producing chemical patterns at the sub-micro scale. The first method uses sacrificial resist as mask to produce the functionalization of the bioadhesive areas and the second method consists in the direct EBL writing of adhesive regions on a non-adhesive plasma deposited PEO-like film. The produced patterned surfaces exhibit high binding capacity as tested by Surface Plasmon Resonance Imaging with Human Serum Albumin. Furthermore, we show that adsorption on the structured surfaces is similar to that observed on a flat surface with a direct proportionality to the active area. This work shows the efficiency and suitability of the developed chemical patterning techniques and their high potential applicability for the study of protein surface interactions and miniaturized biosensing device development.

Light assisted molecular immobilization has been used for the first time to engineer covalent bioconjugates of superparamagnetic nanoparticles and proteins. The technology involves disulfide bridge disruption upon UV excitation of nearby aromatic residues. The close spatial proximity of aromatic residues and disulfide bridges is a conserved structural feature in proteins. The created thiol groups bind thiol reactive surfaces leading to oriented covalent protein immobilization. We have immobilized a model carrier protein, bovine serum albumin, onto Fe3O4@Au core–shell nanoparticles as well as arrayed it onto optically flat thiol reactive surfaces. This new immobilization technology allows for ultra high dense packing of different bio-molecules on a surface, allowing the creation of multi-potent functionalized active new biosensor materials, biomarkers identification and the development of nanoparticles based novel drug delivery system. Bioeng. 2011; 108:999–1010. © 2010 Wiley Periodicals, Inc.

Medical interest in nanotechnology originates from a belief that nanoscale therapeutic devices can be constructed and directed towards its target inside the human body. Such nanodevices can be engineered by coupling superparamagnetic nanoparticle to biomedically active proteins. We hereby report the immobilization of a PhEst, a S-formylglutathione hydrolase from the psychrophilic P. haloplanktis TAC125 onto the gold coated surface of modified superparamagnetic core-shell nanoparticles (Fe3O4@Au). The synthesis of the nanoparticles is also reported. S-formylglutathione hydrolases constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes. PhEst was originally annotated as a putative feruloyl esterase, an enzyme that releases ferulic acid (an antioxidant reactive towards free radicals such as reactive oxygen species) from polysaccharides esters. Dynamic light scattering, scanning electron microscopy with energy dispersive X-ray spectroscopy, UV-visible absorption spectroscopy, fluorescence spectroscopy, magnetic separation technique and enzyme catalytic assay confirmed the chemical composition of the gold covered superparamagnetic nanoparticles, the binding and activity of the enzyme onto the nanoparticles. Activity data in U/ml confirmed that the immobilized enzyme is approximately 2 times more active than the free enzyme in solution. Such particles can be directed with external magnetic fields for bio-separation and focused towards a medical target for therapeutical as well as bio-sensor applications. © 2010 Springer Science+Business Media, LLC. Medical interest in nanotechnology originates from a belief that nanoscale therapeutic devices can be constructed and directed towards its target inside the human body. Such nanodevices can be engineered by coupling superparamagnetic nanoparticle to biomedically active proteins. We hereby report the immobilization of a PhEst, a S-formylglutathione hydrolase from the psychrophilic P. haloplanktis TAC125 onto the gold coated surface of modified superparamagnetic core-shell nanoparticles (FeO@Au). The synthesis of the nanoparticles is also reported. S-formylglutathione hydrolases constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes. PhEst was originally annotated as a putative feruloyl esterase, an enzyme that releases ferulic acid (an antioxidant reactive towards free radicals such as reactive oxygen species) from polysaccharides esters. Dynamic light scattering, scanning electron microscopy with energy dispersive X-ray spectroscopy, UV-visible absorption spectroscopy, fluorescence spectroscopy, magnetic separation technique and enzyme catalytic assay confirmed the chemical composition of the gold covered superparamagnetic nanoparticles, the binding and activity of the enzyme onto the nanoparticles. Activity data in U/ml confirmed that the immobilized enzyme is approximately 2 times more active than the free enzyme in solution. Such particles can be directed with external magnetic fields for bio-separation and focused towards a medical target for therapeutical as well as bio-sensor applications. © 2010 Springer Science+Business Media, LLC.

We here report for the first time the creation of prostate specific antigen (PSA) and Fab anti-PSA biosensor arrays using UV light-assisted molecular immobilization (LAMI), aiming at the detection and quantification of PSA, a cancer marker. The technology involves formation of free, reactive thiol groups upon UV excitation of protein aromatic residues located in spatial proximity of disulphide bridges, a conserved structural feature in both PSA and Fab molecules. The created thiol groups bind onto thiol reactive surfaces leading to oriented covalent protein immobilization. Protein activity was confirmed carrying out immunoassays: immobilized PSA was recognized by Fab anti-PSA in solution and immobilized Fab anti-PSA cross-reacted with PSA in solution. LAMI technology proved successful in immobilizing biomedically relevant molecules while preserving their activity, highlighting that insight into how light interacts with biomolecules may lead to new biophotonic technologies. Our work focused on the application of our new engineering principles to the design, analysis, construction, and manipulation of biological systems, and on the discovery and application of new engineering principles inspired by the properties of biological systems.

Small analytes such as glucose, L-glutamine (Gln), and ammonium nitrate are detected by means of optical biosensors based on a very common nanostructured material, porous silicon (PSi). Specific recognition elements, such as protein receptors and enzymes, were immobilized on hydrogenated PSi wafers and used as probes in optical sensing systems. The binding events were optically transduced as wavelength shifts of the porous silicon reflectivity spectrum or were monitored via changes of the fluorescence emission. The biosensors described in this article suggest a general approach for the development of new sensing systems for a wide range of analytes of high social interest.

In this review we explore the advantages deriving from the use of either enzymes or sugar binding proteins isolated from thermophilic organisms to develop stable fluorescence biosensors. We report on a novel approach to address the consumption of the analyte by enzyme-based biosensors, namely the utilization of apo-enzymes as non-active forms of proteins which are still able to bind the ligand but cannot transform it into product. We also report recent studies in which the fluorescence labeling of a naturally thermostable binding protein allows a quantitative determination of glucose.

We developed a method for immobilization of biomolecules onto thiol functionalized surfaces according to UV diffraction patterns. UV light-assisted molecular immobilization proceeds through the formation of free, reactive thiol groups that can bind covalently to thiol reactive surfaces. We demonstrate that, by shaping the pattern of the UV light used to induce molecular immobilization, one can control the pattern of immobilized molecules onto the surface. Using a single-aperture spatial mask, combined with the Fourier transforming property of a focusing lens, we show that submicrometer (0.7 mu m) resolved patterns of immobilized prostate-specific antigen biomolecules can be created. If a dual-aperture spatial mask is used, the results differ from the expected Fourier transform pattern of the mask. It appears as a superposition of two diffraction patterns produced by the two apertures, with a fine structured interference pattern superimposed. (C) 2010 Optical Society of America

S-formylglutathione hydrolases (FGHs) constitute a family of ubiquitous enzymes which play a key role in formaldehyde detoxification both in prokaryotes and eukaryotes, catalyzing the hydrolysis of S-formylglutathione to formic acid and glutathione. While a large number of functional studies have been reported on these enzymes, few structural studies have so far been carried out. In this article we report on the functional and structural characterization of PhEst, a FGH isolated from the psychrophilic bacterium Pseudoalteromonas haloplanktis. According to our functional studies, this enzyme is able to efficiently hydrolyze several thioester substrates with very small acyl moieties. By contrast, the enzyme shows no activity toward substrates with bulky acyl groups. These data are in line with structural studies which highlight for this enzyme a very narrow acyl-binding pocket in a typical α/β-hydrolase fold. PhEst represents the first cold-adapted FGH structurally characterized to date; comparison with its mesophilic counterparts of known three-dimensional structure allowed to obtain useful insights into molecular determinants responsible for the ability of this psychrophilic enzyme to work at low temperature. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 669–677, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The effects of hydrostatic pressure on the structure and stability of porcine odorant-binding protein (pOBP) in the presence and absence of the odorant molecule 2-isobutyl-3-methoxypyrazine (IBMP) were studied by steady-state and time-resolved fluorescence spectroscopy as well as by molecular dynamics simulation. The authors found that the application of moderate values of hydrostatic pressure to pOBP solutions perturbed the microenvironment of Trp(16) and disrupted its highly quenched complex with Met(39). In addition, compared with the protein in the absence of IBMP, the MD simulations experiments carried out at different pressures highlighted the role of this ligand in stabilizing the Trp(16)/Met(39) interaction even at 2000 bar. The obtained results will assist for the tailoring of this protein as specific sensing element in a new class of fluorescence-based biosensors for the detection of explosives.

Psychrophiles produce cold-evolved enzymes that display a high catalytic efficiency, associated with a low thermal stability. In recent years, these enzymes have attracted the attention of scientists because of their peculiar properties that render them particularly useful in investigating the relationship existing between enzyme stability and flexibility on one hand, and enzyme activity on the other hand. Among these enzymes, the esterases, and particularly the feruloyl esterases, have potential uses over a broad range of applications in the agro-food industries. In recent years, the number of microbial feruloyl esterase activities has increased in the growing genome databases. Based on substrate utilization data and supported by primary sequence identity, four subclasses of esterase have been characterized so far. Up to the present, ten genomes from psychrophilic bacteria have been completely sequenced and additional fourteen genomes are under investigation. From the bacteria strains whose genome has been completely sequenced, we analyzed the presence of esterase genes, both the putative genes and the determined experimentally genes, and performed a ClustalW analysis for feruloyl esterases. Major details will be presented for the ORF PSHAa1385 from P. haloplanktis TAC125 that recently has been studied in our research group. In addition, the potential biotechnology applications of this class of enzymes will be discussed.

The complete genome of the psychrophilic bacteria Pseudoalteromonas haloplanktis TAC 125, recently published, owns a gene coding for a putative esterase activity corresponding to the ORF PSHAa1385, also classified in the Carbohydrate Active Enzymes database (CAZY) belonging to family 1 of carbohydrate esterase proteins. This ORF is 843 bp in length and codes for a protein of 280 amino acid residues. In this study we characterized and cloned the PSHAa1385 gene in Escherichia coli. We also characterized the recombinant protein by biochemical and biophysical methodologies. The PSHAa1385 gene sequence showed a significant homology with several carboxyl-esterase and acetyl-esterase genes from gamma-proteobacteria genera and yeast. The recombinant protein exhibited a significant activity towards pNP-acetate, alpha-and beta-naphthyl acetate as generic substrates, and 4-methylumbelliferyl p-trimethylammonio cinnamate chloride (MUTMAC) as a specific substrate, indicating that the protein exhibits a feruloyl esterase activity that it is displayed by similar enzymes present in other organisms. Finally, a three-dimensional model of the protein was built and the amino acid residues involved in the catalytic function of the protein were identified.

Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillum and Aspergillus. It has been mainly isolated from apples and apple products contaminated with the common storage-rot fungus of apples, Penicillum expansum, but it has also been extracted from rotten fruits, moldy feeds, and stored cheese. Human exposure to patulin can lead to serious health problems, and according to a long-term investigation in rats, the World Health Organization has set a tolerable weekly intake of 7 ppb body weight. The content of patulin in foods has been restricted to 50 ppb in many countries. Conventional analytical detection methods involve chromatographic analyses, such as HPLC, GC, and, more recently, techniques such as LC/MS and GC/MS. However, extensive protocols of sample cleanup are required prior to the analysis, and to accomplish it, expensive analytical instrumentation is necessary. An immunochemical analytical method, based on highly specific antigen-antibody interactions, would be desirable, offering several advantages compared to conventional techniques, i.e., low cost per sample, high selectivity, high sensitivity, and high throughput. In this paper, the synthesis of two new derivatives of patulin is described, along with their conjugation to the bovine serum albumin for the production of polyclonal antibodies. Finally, a fluorescence competitive immunoassay was developed for the on-line detection of patulin.

In this work is presented the first attempt to develop a fluorescence assay for detection of traces of gluten in food by utilizing the recombinant glutamine-binding protein (GlnBP) from E. coli. We found that GlnBP specifically binds the sequence of amino acids present both in gliadin and other prolamines classified as toxic for celiac patients. Affinity chromatography experiments together with mass spectrometry experiments demonstrated that GlnBP can bind the following amino acid sequence XXQPQPQQQQQQQQQQQQL. Sequence alignment experiments pointed out that this sequence is exclusively representative of the gliadin and the other prolamines considered toxic for celiac patients. These findings suggest the development of a competitive resonance energy transfer (RET) assay for an easy and rapid detection of this sequence in raw and cooked food.

Diabetes mellitus is increasing rapidly and will double in the next 15 years.1,2 In addition, diabetic patients have a mortality excess for cardiovascular disease up to 2.5-4 times more than non diabetic population.3,4 In the last years became evident a cluster of cardiovascular risk factors like hypertension, central obesity, dyslipidemia with low HDL-cholesterol and high triglycerides, impaired fibrinolysis, hypercoagulation and endothelial dysfunction that has been called metabolic syndrome.5 The underlying defect that shares all these alterations is insulin resistance with compensatory hyperinsulinemia that is associated with increased cardiovascular events and mortality.6,7 Patients with diabetes and/or metabolic syndrome must be treated aggressively about every risk factors to minimize the cardiovascular events. Hyperglycemia is clearly related to microvascular complication of diabetes: retinopathy, nephropathy and neuropathy, while for macrovascular complication other coexisting risk factors are important. Many studies have demonstrated that an intensive treatment of diabetes reduces the macro and microvascular complications and the best results are obtained when every risk factor is aggressively treated.8–10

The effect of the pressure on the structure and stability of the D-galactose/D-glucose binding protein from Escherichia coli in the absence (GGBP) and in the presence (GGBP/Glc) of glucose was studied by Fourier transform infrared (FT-IR) spectroscopy and molecular dynamic (MD) simulations. FT-IR spectroscopy experiments showed that the protein beta-structures are more resistant than alpha-helices structures to pressure value increases. In addition, the infrared data indicated that the binding of glucose stabilizes the protein structure against high pressure values, and the protein structure does not completely unfold up to pressure values close to 9000 bar. MD simulations allow a prediction of the most probable configuration of the protein, consistent with the increasing pressures on the two systems. The detailed analysis of the structures at molecular level confirms that, among secondary structures, alpha-helices are more sensitive than beta-structures to the destabilizing effect of high pressure and that glucose is able to preserve the structure of the protein in the complex. Moreover, the evidence of the different resistance of the two domains of this protein to high pressure is investigated and explained at a molecular level, indicating the importance of aromatic amino acid in protein stabilization.

In this work is presented the first attempt to develop a fluorescence assay for detection of traces of gluten in food by utilizing the recombinant glutamine-binding protein (GlnBP) from E. coli. We found that GlnBP specifically binds the sequence of amino acids present both in gliadin and other prolamines classified as toxic for celiac patients. Affinity chromatography experiments together with mass spectrometry experiments demonstrated that GInBP can bind the following amino acid sequence XXQPQPQQQQQQQQQQQQL. Sequence alignment experiments pointed out that this sequence is exclusively representative of the gliadin and the other prolamines considered toxic for celiac patients. These findings suggest the development of a competitive resonance energy transfer (RET) assay for an easy and rapid detection of this sequence in raw and cooked food.

In this report we show the purification to homogeneity and a partial characterization of a new odorant-binding protein from Canis familiar (CfOBP) nasal mucosa. In addition, we report preliminary data on the utilization of CfOBP as a probe for the development of a refractive index-based biosensor.

Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell-nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study AuNPs effects. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. The strength of this investigation resides in combining the high-resolving power of fluorescence two-dimensional differential gel electrophoresis with protein identification by high-resolution mass spectrometry. Of interest was the difference observed in the protein pattern expression of cells exposed to 5 and 15 nm AuNPs. From 2D gel-based proteomic data, it was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15 nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, inflammatory response., JRC.I.4-Nanobiosciences