Properties of Human Hemoglobins with Increased Polarity in the α- or β-Heme Pocket

The spectroscopic, conformational, and functional properties of mutant carbonmonoxy hemoglobins in which either the β-globin Val67(E11) or the α-globin Val62(E11) is replaced by threonine have been investigated. The thermal evolution of the Soret absorption band and the stretching frequency of the bound CO were used to probe the stereodynamic properties of the heme pocket. The functional properties were investigated by kinetic measurements. The spectroscopic and functional data were related to the conformational properties through molecular analysis. The effects of this nonpolar-to-polar isosteric mutation are: (i) increase of heme pocket anharmonic motions, (ii) stabilization of the A 0 co…

Characterization of protofibrillar aggregates of bovine serum albumin by tryptophans fluorescence lifetime

We report an experimental study on the thermally induced aggregation of Bovine Serum Albumin at basic pH. In these conditions, we observe the growth of simple protofibrillar structures via the formation of intermolecular beta-sheets promoted by the increased electrostatic repulsion. Here we present a study on the time resolved fluorescence of Tryptophans (Trp) along the aggregation kinetics in the above reported conditions. We use the lifetimes distribution approach as a useful tool for the interpretation of the fluorescence decay in terms of protein conformational substates and interconversion dynamics. Trp fluorescence lifetime depends from protein conformations, also in relation with sol…

Metal ions modulate thermal aggregation of betalactoglobulin: a join chemical and physical characterization

Abstract Molecular basis of the role played by Cu 2 + and Zn 2 + ions during the thermal aggregation processes of beta-lactoglobulin (BLG) was studied by using a joint application of different techniques. In particular, Raman spectroscopy was very useful in identifying the different effects caused by the two metals at molecular level (i.e. changes in His protonation state, disulfides bridge conformation, and micro-environment of aromatic residues), evidencing the primary importance of the protein charge distribution during the aggregation process. Both metal ions are able to act on this factor and favor the protein aggregation, but Zn 2 + is able to alter the natural conformational state of…

Oxidative alteration of Human Serum Albumin Amyloid Aggregation Pathway

Thermal aggregation of proteins in presence of metal ions.

The study of the aggregation processes in presence of metal ions is an essential step for understanding the key role of metals in protein-protein and protein-solvent interactions. Indeed, the presence of metal ions can radically change the main features of the standard denaturation/aggregation processes and such effects result to be strongly dependent on the kind of metal and on its concentration. Metal ions have an active role in thermal aggregation and cold set gelation processes. These processes are intrinsically different, but both are based on the proteins ability to form aggregates.

ThT influences Abeta(1-40) aggregation process

Data concerning the proteolytic resistance and oxidative stress in LAN5 cells after treatment with BSA hydrogels

AbstractProteolytic resistance is a relevant aspect to be tested in the formulation of new nanoscale biomaterials. The action of proteolytic enzymes is a very fast process occurring in the range of few minutes. Here, we report data concerning the proteolytic resistance of a heat-set BSA hydrogel obtained after 20-hour incubation at 60°C prepared at the pH value of 3.9, pH at which the hydrogel presents the highest elastic character with respect to gel formed at pH 5.9 and 7.4 “Heat-and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold” (G. Navarra, C. Peres, M. Contardi, P. Picone, P.L. San Biagio, M. Di Carlo, D. Giacomazza, V. Militello, 2016) […

Conformational changes involved in thermal aggregation processes of bovine serum albumin

We report a kinetic study on thermal aggregation process of the model protein bovine serum albumin (BSA) in low concentration regime. Aim of this study is to provide information on relationship between conformational changes and initial step of aggregation. The experimental approach is based on steady-state fluorescence spectra of the two tryptophans located in two different domains, in way to study conformational changes in the surrounding of these residues. We also follow emission spectra of Fluorescein-5-Maleimide dye bound to the single free cysteine of BSA. Complementary information on the extent of aggregation and on the structural changes is obtained by Rayleigh scattering and circul…

Thermal induced conformational changes involved in the aggregation pathways of beta-lactoglobulin.

Aggregation of proteins appears to be associated most often with conformational and structural changes that lead to exposure of some apolar residues. Depending on the native structure of the protein in exam, aggregation is a process that involves different mechanisms, whose time of occurrence and interplay can depend upon temperature. To single out information about the multistages of the aggregation pathway, here we investigate the thermally induced conformational and structural changes of the beta-lactoglobulin (BLG). The experimental approach consists in studying steady-state fluorescence spectra of intrinsic chromophores, two tryptophans, and Anylino-Naphthalene-Sulfonate dye (ANS) mole…

OH-related infrared bands in oxide glasses

Back to the oligomeric state: pH-induced dissolution of concanavalin A amyloid-like fibrils into non-native oligomers

The subtle interplay between long range electrostatic forces, hydrophobic interactions and short range protein-protein interactions regulates the onset/evolution of protein aggregation processes as well as the stability of protein supramolecular structures. Using a combination of FTIR spectroscopy, light scattering and advanced imaging, we present evidence on the main role of electrostatic forces in the formation and stability of amyloid-like fibrils formed from concanavalin A (ConA), a protein showing structural homology with the human serum amyloid protein. At high protein concentration, where protein-protein interactions cannot be neglected, we highlight a thermal-induced aggregation pat…

Detecting Protein Aggregation on Cells Surface: Concanavalin A Oligomers Formation

A number of neurodegenerative diseases involve protein aggregation and amyloid formation. Recently evidence has emerged indicating small-transient prefibrillar oligomers as the primary pathogenic agents. Noteworthy, strict analogies exist between the behaviour of cells in culture treated with misfolded non-pathogenic proteins and in pathologic conditions, this instance together with the observation that the oligomers and fibrils are characterised by common structural features suggest that common mechanisms for cytotoxicity could exists and have to be perused in common interactions involved in aggregation.We here report an experimental study on ConcanavalinA (ConA) aggregation and its effect…

CONCANAVALIN A AGGREGATION AND TOXICITY ON NEUROBLASTOMA LAN5 CELL CULTURES

Thermal aggregation of bovine serum albumin at different pH: comparison with human serum albumin.

We report here a study on thermal aggregation of BSA at two different pH values selected to be close to the isoelectric point (pI) of this protein. Our aim is to better understand the several steps and mechanisms accompanying the aggregation process. For this purpose we have performed kinetics of integrated intensity emission of intrinsic and extrinsic dyes, tryptophans and ANS respectively, kinetics of Rayleigh scattering and of turbidity. The results confirm the important role played by conformational changes in the tertiary structure, especially in the exposure of internal hydrophobic regions that promote intermolecular interactions. We also confirm that the absence of electrostatic repu…

Concanavalin A toxicity on Neuroblastoma LAN5 cell cultures

Uptake of silica covered Quantum Dots into living cells: Long term vitality and morphology study on hyaluronic acid biomaterials

Quantum Dots (QDs) are promising very bright and stable fluorescent probes for optical studies in the biological field but water solubility and possible metal bio-contamination need to be addressed. In this work, a simple silica-QD hybrid system is prepared and the uptake in bovine chondrocytes living cells without any functionalization of the external protective silica shield is demonstrated. Moreover, long term treated cells vitality (up to 14 days) and the transfer of silica-QDs to the next cell generations are here reported. Confocal fluorescence microscopy was also used to determine the morphology of the so labelled cells and the relative silica-QDs distribution. Finally, we employ sil…

Nucleation mechanism of Lysozyme at physiological pH

pH effect on thermal aggregation of concanavalin A

Spectroscopic and microscopic characterization of BSA hydrogels: towards new biomaterials

Bovine Serum Albumin protofibril-like aggregates formation: Solo but not simple mechanism

We report an experimental study on the model protein Bovine Serum Albumin (BSA), with the aim of elucidating the mechanisms by which a fully folded globular protein undergoes different aggregation pathways leading to the formation of amyloid fibrils or amorphous aggregates. We observe thermally induced formation of fibrillar structures at pH far from the protein isoelectric point. The increase of electrostatic repulsion results in protein destabilization and in modifications of inter and intra-molecular interactions leading to the growth of fibril-like aggregates stabilized by inter-molecular-β sheets. The aggregation kinetics is studied by means of fluorescence techniques, light scattering…

Thioflavin T templates amyloid β(1–40) conformation and aggregation pathway

Aβ(1-40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimer's disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies to control the occurring events. To this purpose the exploitation of small molecules interacting with Aβ aggregation represents one of the possible routes. Moreover, the use specific labeling has represented so far one of the most common and effective methods to investigate such a process. This possibility in turn rests on the reliability of the probe/labels involved. Here we present evidences of the effe…

Thermal broadening of the Soret band in heme complexes and in heme-proteins: role of iron dynamics

We report the thermal broadening of the Soret band in heme-CO, heme-OH and protoporphyrin IX in the temperature range 300-20 K. For protoporphyrin IX the temperature dependent Gaussian line broadening follows the behavior predicted by the harmonic approximation in the entire temperature range investigated. In contrast, for heme-CO and heme-OH the harmonic behavior is obeyed only up to about 180 K and an anomalous line broadening increase is observed at higher temperatures. This effect is attributed to the onset of anharmonic motions of the iron atom with respect to the porphyrin plane. Comparison with previously reported analogous data for heme proteins enables us to suggest that the onset …

Pressure effects on α-synuclein amyloid fibrils: An experimental investigation on their dissociation and reversible nature

α–synuclein amyloid fibrils are found in surviving neurons of Parkinson's disease affected patients, but the role they play in the disease development is still under debate. A growing number of evidences points to soluble oligomers as the major cytotoxic species, while insoluble fibrillar aggregates could even play a protection role. In this work, we investigate α–synuclein fibrils dissociation induced at high pressure by means of Small Angle X-ray Scattering and Fourier Transform Infrared Spectroscopy. Fibrils were produced from wild type α–synuclein and two familial mutants, A30P and A53T. Our results enlighten the different reversible nature of α–synuclein fibrils fragmentati…

Thioflavin T Promotes Aβ(1−40) Amyloid Fibrils Formation

Fibrillogenesis of the small peptide Aβ(1-40) is considered to be the hallmark of Alzheimer's disease. Some evidence indicates small oligomers, rather than mature fibrils, as the key cytotoxic agents. The fluorescent dye Thioflavin T (ThT) is often used to detect amyloid deposits in both in vivo and in vitro experiments, and it is used to study kinetic measurements, under the fundamental hypothesis that this probe does not influence the aggregation processes. We report experimental data showing that ThT may promote the Aβ(1-40) peptide amyloid aggregation changing solvent-peptide interactions and stabilizing more ordered β-like conformation. This finding has a two-fold importance: It is a f…

Local dynamic properties of the heme pocket in native and solvent-induced molten-globule-like states of cytochrome c

We report the Soret absorption band, down to cryogenic temperature, of native and molten-globule-like state of horse heart cytochrome c. The band profile is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Franck-Condon approximation. From the temperature dependence of the Gaussian broadening and of the peak position, we obtain information on the 'bath' of low frequency harmonic motions of the heme group within the heme pocket. The reported data indicate that, compared to the native state, the less rigid tertiary structure of the molten globule is reflected in a higher flexibility of the heme pocket and in greater conformat…

Heterogeneity of aggregates in the fibrillation mechanisms of proteins probed by time resolved fluorescence

Under appropriate conditions almost all proteins are able to aggregate to form long, well-ordered and beta-sheet rich arrays known as amyloid fibrils. The formation of such structures involves complex intra and intermolecular interactions modulated by the structure and dynamics of the native protein, and by the physico-chemical properties of the solvent. Multiple interactions and cross-feedback during the aggregation pathway cause different ultimate aggregates’ morphologies and the possible simultaneous occurrence of multiple species. The structural definition of such assemblies is complicated by the polymorphism of the amyloid fibrils. Aim of this study is to inquire on the different natur…

Dynamic properties of some β-chain mutant hemoglobins

The thermal behavior of the Soret band relative to the carbonmonoxy derivatives of some beta-chain mutant hemoglobins is studied in the temperature range 300-10 K and compared to that of wild-type carbonmonoxy hemoglobin. The band profile at various temperatures is modeled as a Voigt function that accounts for homogeneous broadening and for the coupling with high- and low-frequency vibrational modes, while inhomogeneous broadening is taken into account with a gaussian distribution of purely electronic transition frequencies. The various contributions to the over-all bandwidth are singled out with this analysis and their temperature dependence, in turn, gives information on structural and dy…

NANO-EMETTITORI NIR A BASE DI SILICE PER APPLICAZIONI IN-VIVO E RELATIVO PROCESSO DI PRODUZIONE

Realizzazione di nano-sonde paramagnetiche e fosforescenti, ottenibili a partire da particelle nanometriche di silice mediante procedure di sintesi e arricchimento di O2 a seguito di opportuni trattamenti termici in ambiente controllato. Le nano-sonde così ottenute presentano emissioni nel vicino infrarosso (NIR), sono eccitabili nel visibile e nel NIR, e sono caratterizzate da un tempo di vita nell’ordine del secondo. Tali nano-sonde presentano un notevole potenziale scientifico e commerciale nel mercato della spettroscopia confocale per bio-imaging e nel mercato delle applicazioni medico-farmaceutiche di drug-labelling e drug-delivery.

Silica Nanoparticles for Near-Infrared Imaging and Photonics Applications

Insulin Nanogels: a New Strategy for the Treatment of Alzheimer’s Disease

A growing body of evidence shows that Insulin, Insulin Receptor (IR) and IR signaling are involved in brain cognitive functions and their dysfunction is implicated in Alzheimer’s disease (AD) degeneration. Thus, administration of insulin could be a strategy for AD treatment. For this aim we have designed, synthesized and characterized a nanogel system (NG) to deliver insulin to the brain, as a tool for the development of a new therapy for AD. A carboxyl-functionalized poly(N-vinyl pyrrolidone) nanogel system produced by ionizing radiation was chosen as substrate for the covalent attachment of insulin or fluorescent molecules relevant for its characterization. Biocompatibility of the naked c…

Conformational substates and dynamic properties of carbonmonoxy hemoglobin.

Heme pocket dynamics of human carbonmonoxy hemoglobin (HbCO) is studied by Fourier transform infrared spectroscopy. The CO stretching band at various temperatures in the interval 300-10 K is analyzed in terms of three taxonomic A substates; however, in HbCO the band attributed to the A(1) taxonomic substate accounts for approximately 90% of the total intensity in the pH range 8.8-4.5. Two different regimes as a function of temperature are observed: below 160 K, the peak frequency and the bandwidth of the A(1) band have constant values whereas, above this temperature, a linear temperature dependence is observed, suggesting the occurrence of transitions between statistical substates within th…

Lysozyme Aggregation Driven by Unfolding: the Role of Metal Ions

Effects of succinylation on thermal induced amyloid formation in Concanavalin A.

We have recently shown that upon slight thermal destabilization the legume lectin Concanavalin A may undergo two different aggregation processes, leading, respectively, to amyloid fibrils at high pH and amorphous aggregates at low pH. Here we present an experimental study on the amyloid aggregation of Succinyl Concanavalin A, which is a dimeric active variant of Concanavalin. The results show that, as for the native protein, the fibrillation process appears to be favoured by alkaline pH, far from the isoelectric point of the protein. Moreover, it strongly depends on temperature and requires large conformational changes both at secondary and tertiary structure level. With respect to the nati…

Fluctuation Methods To Study Protein Aggregation in Live Cells: Concanavalin A Oligomers Formation

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralle…

Near-Infrared emission of O2 embedded in amorphous SiO2 nanoparticles

We report an experimental study on the emission properties of O2 molecules loaded by a thermal diffusion process at 200 °C into high-purity silica nanoparticles with mean diameters of 7 and 40 nm. The embedded O2 features a singlet to triplet emission band peaked at 1272 nm in agreement with the band observed for bulk silica materials. The photoluminescence excitation spectra have been determined in the visible and in the infrared range and are characterized by narrow bands peaked at 691, 764,and 1069 nm, respectively. By comparison of the transition energies, the vibrational quanta have been determined for the ground and for both the excited states; the values found are lower than the corr…

Ferritin-Coated SPIONs as New Cancer Cell Targeted Magnetic Nanocarrier

Superparamagnetic iron oxide nanoparticles (SPIONs) may act as an excellent theragnostic tool if properly coated and stabilized in a biological environment, even more, if they have targeting properties towards a specific cellular target. Humanized Archaeoglobus fulgidus Ferritin (HumAfFt) is an engineered ferritin characterized by the peculiar salt-triggered assembly-disassembly of the hyperthermophile Archaeoglobus fulgidus ferritin and is successfully endowed with the human H homopolymer recognition sequence by the transferrin receptor (TfR1 or CD71), overexpressed in many cancer cells in response to the increased demand of iron. For this reason, HumAfFt was successfully used in this stud…

Thermal aggregation of glycated bovine serum albumin

International audience; Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose con…

Role of copper and zinc ions on the heat- induced aggregation

THERMALLY INDUCED FIBRILLAR AGGREGATION OF BOVINE SERUM ALBUMIN

Thermal aggregation of two “beta-protein” models at different pH values.

Pre-aggregates characterization and viscoelastic studies of BSA cold gels induced by metal ions

High-throughput screening at the picoliter scale by combining Dip Pen Lithography with Inkjet printing

Drug screening is a complex, expensive and time consuming field consisting of diseasebased target identification in conjunction with high-throughput screening of chemical and natural product libraries. Conventional drug screening technology is usually time and reagent consuming (micro-, nanoliter scale) and is based on complex liquid handling robotics. In this work, we show a low-cost and miniaturized drug screening methodology based on direct bio-printing methodologies like Inkjet Printing and Dip Pen Lithography. We show the possibility to precisely deliver femtoliter scale droplets of protein targets by Dip Pen Lithography by finely tuning deposition parameters. This allows obtaining mic…

Different aggregation pathways of human and bovine Serum Albumin.

Deciphering metal-induced oxidative damages on glycated albumin structure and function

Background: Metal ions such as copper or zinc are involved in the development of neurodegenerative pathologies and metabolic diseases such as diabetes mellitus. Albumin structure and functions are impaired following metal-and glucose-mediated oxidative alterations. The aim of this study was to elucidate effects of Cu(II) and Zn(II) ions on glucose-induced modifications in albumin by focusing on glycation, aggregation, oxidation and functional aspects. Methods: Aggregation and conformational changes in albumin were monitored by spectroscopy, fluorescence and microscopy techniques. Biochemical assays such as carbonyl, thiol groups, albumin-bound Cu, fructosamine and amine group measurements w…

Amyloid Fibrils Formation in Concanavalin A studied by Dynamic Light Scattering and Fluorescence techniques

Molecular mechanisms in thermally induced amyloid formation of Concanavalin A

Concanavalin A aggregation and toxicity on cell cultures

A number of neurodegenerative diseases are known to involve protein aggregation. Common mechanisms and structural properties of amyloids are thought to be involved in aggregation-related cytotoxicity. In this context we propose an experimental study on Concanavalin A (Con A) aggregation and use it as a model to study the relationship between cell toxicity and aggregation processes. Depending on solution conditions, Con A aggregation has been monitored by static and dynamic light scattering, Thioflavin T emission, and FTIR absorption. The morphology of different aggregate species was verified by means of Atomic Force Microscopy and Confocal Microscopy. During the aggregation pathway the nati…

Near-Infrared Emission of O2 Embedded in Amorphous SiO2 Nanoparticles

We report an experimental study on the emission properties of O2 molecules loaded by a thermal diffusion process at 200 °C into high-purity silica nanoparticles with mean diameters of 7 and 40 nm. The embedded O 2 features a singlet to triplet emission band peaked at 1272 nm in agreement with the band observed for bulk silica materials. The photoluminescence excitation spectra have been determined in the visible and in the infrared range and are characterized by narrow bands peaked at 691, 764, and 1069 nm, respectively. By comparison of the transition energies, the vibrational quanta have been determined for the ground and for both the excited states; the values found are lower than the co…

Heat- and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold

Aggregation and gelation of globular proteins can be an advantage to generate new forms of nanoscale biomaterials based on the fibrillar architecture. Here, we report results obtained by exploiting the proteins' natural tendency to self-organize in 3D network, for the production of new material based on BSA for medical application. In particular, at five different pH values the conformational and structural changes of the BSA during all the steps of the thermal aggregation and gelation have been analyzed by FTIR spectroscopy. The macroscopic mechanical properties of these hydrogels have been obtained by rheological measurements. The microscopic structure of the gels have been studied by AFM…

Nouvelles perspectives concernant la structure et la fonction du domaine carboxyl terminal de Hfq

Accumulating evidence indicates that RNA metabolism components assemble into supramolecular cellular structures to mediate functional compartmentalization within the cytoplasmic membrane of the bacterial cell. This cellular compartmentalization could play important roles in the processes of RNA degradation and maturation. These components include Hfq, the RNA chaperone protein, which is involved in the post-transcriptional control of protein synthesis mainly by the virtue of its interactions with several small regulatory ncRNAs (sRNA). The Escherichia coli Hfq is structurally organized into two domains. An N-terminal domain that folds as strongly bent β-sheets within individual protomers to…

EARLY STAGES AND SPATIAL HETEROGENEITY IN INSULIN AMYLOID FIBRILS FORMATION

AMYLOID AGGREGATION IN CONCANAVALIN A AT HIGH PH STUDIED BY LIGHT SCATTERING, FLUORESCENCE AND CIRCULAR DICHROISM SPECTROSCOPY

Oxidation Enhances Human Serum Albumin Thermal Stability and Changes the Routes of Amyloid Fibril Formation

Oxidative damages are linked to several aging-related diseases and are among the chemical pathways determining protein degradation. Specifically, interplay of oxidative stress and protein aggregation is recognized to have a link to the loss of cellular function in pathologies like Alzheimer's and Parkinson's diseases. Interaction between protein and reactive oxygen species may indeed induce small changes in protein structure and lead to the inhibition/modification of protein aggregation process, potentially determining the formation of species with different inherent toxicity. Understanding the temperate relationship between these events can be of utmost importance in unraveling the molecul…

Decoding vibrational states of Concanavalin A amyloid fibrils.

International audience; Amyloid and amyloid-like fibrils are a general class of protein aggregates and represent a central topic in life sciences for their involvement in several neurodegenerative disorders and their unique mechanical and supramolecular morphological properties. Both their biological role and their physical properties, including their high mechanical stability and thermodynamic inertia, are related to the structural arrangement of proteins in the aggregates at molecular level. Significant variations may exist in the supramolecular organization of the commonly termed cross-β structure that constitutes the amyloid core. In this context, a fine knowledge of the structural deta…

Thioflavin T triggers β amyloid peptide (1-40) fibrils formation.

Introduction A general characteristic of aggregation is the multiple interaction and cross-feedback among distinct mechanisms occurring at different hierarchical levels. The comprehension of the different species interconversion during aggregation is very important since emerging evidences indicate intermediate oligomeric aggregates as primary toxic species. In this context, Aβ amyloid peptide provides a challenging model for studying aggregation phenomena both for the complexity of its association process and for the direct implications in Alzheimer’s Disease. Aggregates growth conditions strongly affect the final morphology, the fibrillar molecular structure as well as the aggregation pat…

Ionizing radiation-engineered nanogels as insulin nanocarriers for the development of a new strategy for the treatment of Alzheimer's disease

A growing body of evidence shows the protective role of insulin in Alzheimer's disease (AD). A nanogel system (NG) to deliver insulin to the brain, as a tool for the development of a new therapy for Alzheimer's Disease (AD), is designed and synthetized. A carboxyl-functionalized poly(N-vinyl pyrrolidone) nanogel system produced by ionizing radiation is chosen as substrate for the covalent attachment of insulin or fluorescent molecules relevant for its characterization. Biocompatibility and hemocompatibility of the naked carrier is demonstrated. The insulin conjugated to the NG (NG-In) is protected by protease degradation and able to bind to insulin receptor (IR), as demonstrated by immunofl…

Thermal induced conformational changes involved in the aggregation of beta-lactoglobulin

Aggregation of proteins appears to be associated most often with conformational and structural changes that lead to exposure of some apolar residues. Depending on the native structure of the protein in exam, aggregation is a process that involves different mechanisms, whose time of occurrence and interplay can depend upon temperature. To single out information about the multistages of the aggregation pathway, here we investigate the thermally induced conformational and structural changes of the beta-lactoglobulin (BLG). The experimental approach consists in studying steady-state fluorescence spectra of intrinsic chromophores, two tryptophans, and Anylino-Naphthalene-Sulfonate dye (ANS) mole…

OH-related infrared absorption bands in oxide glasses

EFFECT OF SUCCINYLATION AND SUGAR BINDING ON THERMAL INDUCED AMYLOID FORMATION IN CONCANAVALIN A

HSA Oxidation ImprovesThermal Stability and InhibitsAmyloid Fibril Formation

Active site conformation in the αH87G mutant hemoglobin: An optical absorption and FTIR study

We have studied the active site conformation in the carbonmonoxy derivative of the αH87G mutant hemoglobin by means of optical absorption and FTIR spectroscopies. A red shift (≈30 cm−1) of the Soret band peak frequency, together with a concomitant red shift (≈2 cm−1) of the bound CO stretching frequency has been observed for the mutant protein. This indicates an altered electrostatic environment of the heme group in the mutated subunits. In view of the FTIR data showing that the bound CO molecule experiences an increased positive electrostatic field, we attribute the observed effects to a closer interaction of the CO ligand with the partially positively charged imidazole side chain of the p…

Techniques to Analyze sRNA Protein Cofactor Self-Assembly In Vitro

Post-transcriptional control of gene expression by small regulatory noncoding RNA (sRNA) needs protein accomplices to occur. Past research mainly focused on the RNA chaperone Hfq as cofactor. Nevertheless, recent studies indicated that other proteins might be involved in sRNA-based regulations. As some of these proteins have been shown to self-assemble, we describe in this chapter protocols to analyze the nano-assemblies formed. Precisely, we focus our analysis on Escherichia coli Hfq as a model, but the protocols presented here can be applied to analyze any polymer of proteins. This chapter thus provides a guideline to develop commonly used approaches to detect prokaryotic protein self-ass…

Low temperature optical spectroscopy of cobalt-substituted hemocyanin from Carcinus maenas

In this work we report the optical absorption spectra of three cobalt-substituted derivatives of hemocyanin (He) from Carcinus maenas, in the temperature range 300–20 K. The derivatives studied are the mononuclear (Co2+)-He with a single cobalt ion in the “CuA” site, the binuclear (Co2+)2-He and the binuclear mixed metal (Co2+-Cu1+)-He. At low temperature three main bands are clearly resolved; the temperature dependence of their zeroth, first and second moments sheds light on the stereodynamic properties in the surroundings of the chromophore. Within the limits of the reported analysis, in the binuclear derivatives the motions coupled to the chromophore appear to be “essentially harmonic” i…

High energy radiation processing for the synthesis of insulin nanocarriers for the development of a new strategy for the treatment of Alzheimer's Disease

Nanogels are nanoscalar polymeric networks, characterized by a dynamic internal structure and a flexible adaptive shape. When they are used as drug carriers, their flexibility and shape changing ability can facilitate the bypass of biological barriers, ensure protection of the payload and enable interaction of any attached ligand with its receptors. Poly(N-vinyl pyrrolidone)-co-acrylic acid nanogels (NGs) have been produced by ebeam irradiation of diluted aqueous solutions of a water-soluble polymer in the presence of a small amount of acrylic acid. In particular, industrial accelerators and the typical set-ups and doses applied for sterilization have been used. [1, 2] Particle size, molecu…

THz spectroscopy studies on proteins: exploring collective modes of amyloid fibrils

Application of FTIR Spectroscopy to Analyze RNA Structure

Fourier transform infrared (FTIR) spectroscopy has been widely used for the analysis of both protein and nucleic acid secondary structure. This is one of the vibration spectroscopy methods that are extremely sensitive to any change in molecular structure. While numerous reports describe how to proceed to analyze protein and deoxyribonucleic acid (DNA) structures using FTIR, reports related to the analyses of ribonucleic acids (RNAs) are few. Nevertheless, RNAs are versatile molecules involved in a multitude of roles in the cell. In this chapter, we present applications of FTIR for the structural analysis of RNA, including the analysis of helical parameters and noncanonical base pairing, oft…

Thermal oxidative process in extra-virgin olive oils studied by FTIR, rheology and time-resolved luminescence

Abstract With the aim to characterise the antioxidant properties of different extra-virgin olive oils and to understand in more detail the mechanisms of oil degradation, we have made an experimental study on thermal induced oxidative processes of extra-virgin olive oils by using different techniques: Fourier Transform Infrared (FTIR) spectroscopy, rheology and time-resolved luminescence. The oxidation process was followed at three different heating temperatures (30, 60 and 90 °C) as a function of time up to 35 days. Thermal treatment induced changes in the FTIR spectra in the wavenumbers region 3100–3600 cm −1 : in particular, the absorption profiles show an initial formation of hydroperoxi…

High-throughput drug screening by Printing Biology

Printing biology is our way to define a novel field employing material printing techniques generally used in plastic electronics to solve important issues of biology by miniaturized and high-throughput platforms. In this field, we already showed the possibility to use Dip Pen Lithography to fabricate single-cell biochips [1]. Also,we employed non-contact patterning methods such as inkjet printing methods to fabricate microarrays for drug screening at solid-liquid interfaces [2] or in picoliter-scale liquid droplets [3] so enabling high-throughput screening of chemical libraries onto disease-based targets. In this regard, printing methods would greatly reduce times and costs of standard drug…

Effects of Disaccarides On Thermal Aggregation of Bovine Serum Albumin.

Insulin-activated Akt rescues Aβ oxidative stress-induced cell death by orchestrating molecular trafficking

Increasing evidence indicates that Alzheimer's disease, one of the most diffused aging pathologies, and diabetes may be related. Here, we demonstrate that insulin signalling protects LAN5 cells by amyloid-β42 (Aβ)-induced toxicity. Aβ affects both activation of insulin receptors and the levels of phospho-Akt, a critical signalling molecule in this pathway. In contrast, oxidative stress induced by Aβ can be antagonized by active Akt that, in turn, inhibits Foxo3a, a pro-apoptotic transcription factor activated by reactive oxygen species generation. Insulin cascade protects against mitochondrial damage caused by Aβ treatment, restoring the mitochondrial membrane potential. Moreover, we show t…

Engineered Ferritin with Eu3+ as a Bright Nanovector: A Photoluminescence Study

Ferritin nanoparticles play many important roles in theranostic and bioengineering applications and have been successfully used as nanovectors for the targeted delivery of drugs due to their ability to specifically bind the transferrin receptor (TfR1, or CD71). They can be either genetically or chemically modified for encapsulating therapeutics or probes in their inner cavity. Here, we analyzed a new engineered ferritin nanoparticle, made of the H chain mouse ferritin (HFt) fused with a specific lanthanide binding tag (LBT). The HFt-LBT has one high affinity lanthanide binding site per each of the 24 subunits and a tryptophane residue within the tag that acts as an antenna able to transfer …

Irreversible gelation of thermally unfolded proteins:structural and mechanical properties of lysozyme aggregates

The formation of protein aggregates is important in many fields of life science and technology. The morphological and mechanical properties of protein solutions depend upon the molecular conformation and thermodynamic and environmental conditions. Non-native or unfolded proteins may be kinetically trapped into irreversible aggregates and undergo precipitation or gelation. Here, we study the thermal aggregation of lysozyme in neutral solutions. We characterise the irreversible unfolding of lysozyme by differential scanning calorimetry. The structural properties of aggregates and their mechanisms of formation with the eventual gelation are studied at high temperature by spectroscopic, rheolog…

Glycation alters ligand binding, enzymatic, and pharmacological properties of human albumin.

Albumin, the major circulating protein in blood plasma, can be subjected to an increased level of glycation in a diabetic context. Albumin exerts crucial pharmacological activities through its drug binding capacity, i.e., ketoprofen, and via its esterase-like activity, allowing the conversion of prodrugs into active drugs. In this study, the impact of the glucose-mediated glycation on the pharmacological and biochemical properties of human albumin was investigated. Aggregation product levels and the redox state were quantified to assess the impact of glycation-mediated changes on the structural properties of albumin. Glucose-mediated changes in ketoprofen binding properties and esterase-lik…

Aggregation processes in beta-lactoglobulin studied by FTIR spectroscopy

A Long Journey into the Investigation of the Structure–Dynamics–Function Paradigm in Proteins through the Activities of the Palermo Biophysics Group

An overview of the biophysics activity at the Department of Physics and Chemistry Emilio Segrè of the University of Palermo is given. For forty years, the focus of the research has been on the protein structure–dynamics–function paradigm, with the aim of understanding the molecular basis of the relevant mechanisms and the key role of solvent. At least three research lines are identified; the main results obtained in collaboration with other groups in Italy and abroad are presented. This review is dedicated to the memory of Professors Massimo Ugo Palma, Maria Beatrice Palma Vittorelli, and Lorenzo Cordone, which were the founders of the Palermo School of Biophysics. We all have been, directl…

Multiple aggregation mechanism in Abeta(1-40) fibril formation

A general characteristic of aggregation is the multiple interaction and cross-feedback among distinct mechanisms occurring at different hierarchical levels. The comprehension of the different species interconversion during aggregation is very important since emerging evidences indicate intermediate oligomeric aggregates as primary toxic species. In this context, Aβ amyloid peptide provides a challenging model for studying aggregation phenomena both for the complexity of its association process and for the direct implications in Alzheimer’s Disease. Indeed, aggregates growth conditions strongly affect their final morphology and their molecular structure as well as the time evolution of aggre…

Insulin Nanogel as New Strategy for the Treatment of Alzheimer’s Disease

A growing body of evidence shows that Insulin, Insulin Receptor (IR) and IR signaling are involved in brain cognitive functions and their dysfunction is implicated in neuronal degeneration associated with Alzheimer's disease (AD). Thus, the administration of insulin to the brain could be a strategy for the prevention and treatment of AD disease. With this aim, we have designed, synthesized and characterized a nanogel system (NG) that can be used as substrate for the conjugation of insulin and/or fluorescent molecules relevant for their characterization. In particular, a carboxyl-functionalized poly(N-vinyl pyrrolidone) nanogel system, has been produced by ionizing radiation starting from th…

Optically active and biocompatible polyaniline nanoparticles - hydrogel composites.

Synthesis and characterization of CdS nanoparticles embedded in a polymethylmethacrylate matrix

CdS nanopowder capped with sodium bis(2-ethylhexyl)sulfosuccinate was synthesized by using water-in-oil microemulsions. The CdS nanoparticles of about 5 nm obtained were embedded in polymethylmethacrylate matrix by a photocuring process. The transparent yellow solid compound was characterized by optical absorption and emission spectroscopy, high-resolution transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The properties of this compound were compared with those of the nanopowder dispersed in heptane and in methylmethacrylate. The results obtained indicate that the nanoparticles are homogeneously dispersed in the matrix and do not change in size during the embedding…

Interconnected mechanism in Abeta(1-40) peptide fibril formation

Neutron Scattering Reveals Enhanced Protein Dynamics in Concanavalin A Amyloid Fibrils

Protein aggregation is one of the most challenging topics in life sciences, and it is implicated in several human pathologies. The nature and the role of toxic species is highly debated, with amyloid fibrils being among the most relevant species for their peculiar structural and functional properties. Protein dynamics and in particular the ability to fluctuate through a large number of conformational substates are closely related to protein function. This Letter focuses on amyloid fibril dynamics, and, to our knowledge, it is the first neutron scattering study on a protein (Concanavalin A) isolated in its fibril state. Our results reveal enhanced atomic fluctuations in amyloid fibrils and i…

Vibrational analysis of Ni(II)- and Cu(II)-octamethylchlorin by polarized resonance Raman and Fourier transform infrared spectroscopy

We measured the polarized resonance Raman spectra of Cu(II)-2,2,7,8,12,13,17,18-octamethylchlorin in CS2 at various excitation wavenumbers in a spectral region covering the Qy, Qx and Bx optical absorption bands. Additionally, we measured the FTIR-Raman spectrum of the highly overcrowded spectral region between 1300 and 1450 cm−1. The spectral decomposition was carried out by a self-consistent global fit to all spectra obtained. The thus identified Raman and IR lines were assigned by comparison with the resonance Raman spectra of Cu(II)-octaethylporphyrin, by utilizing their depolarization ratio dispersions and by a normal mode analysis. The latter was based on a modified transferable molec…

An overview on different processes in thermal aggregation of globular proteins

Multistage pathways involved in thermal aggregation processes of proteins.

Secondary nucleation in stirring induced alpha-lactalbumin amyloid fibrils formation

Thermal aggregation of glycated Bovine Serum Albumin

Chemical and physical characterization of thermal aggregation of model proteins modulated by zinc(II) and copper(II) ions

BACKGROUND: Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies, where the protein deposition occurs, and in the biotechnology field like the food technology where many processes in food manufacturing are based on thermal treatments. OBJECTIVE: The influence of Cu2+ or Zn2+ ions on the thermal aggregation process of Bovine beta-lactoglobulin (BLG) and Bovine Serum Albumin (BSA), two protein models, was studied with the aim of delineating the role of these ions in the protein aggregation kinetics and to clarify the related molecular mechanisms. METHODS: The protein structure changes were monitored by Raman spectroscopy, whereas the aggregate gr…

The role of metal ions in the thermal aggregation of Bovine Serum Albumin

Amyloid fibrils formation and amorphous aggregation in Concanavalin A

We here report an experimental study on the thermal aggregation process of concanavalin A, a protein belonging to the legume lectins family. The aggregation process and the involved conformational changes of the protein molecules were followed by means of fluorescence techniques, light scattering, circular dichroism, zeta potential measurements and atomic force microscopy. Our results show that the aggregation process of concanavalin A may evolve through two distinct pathways leading, respectively, to the formation of amyloids or amorphous aggregates. The relative extent of the two pathways is determined by pH, as amyloid aggregation is favored at high pH values ( approximately 9), while th…

Interconnected mechanisms in Abeta(1-40) peptide fibril formation

Metallic Ions Effects on the Heat-aggregation Process of b-Lactoglobulin

Review: "Thermal aggregation of proteins in the presence of metal ions"

Stirring effects in amyloid fibril formation

Development of a biosensor for copper detection in aqueous solutions using an Anemonia sulcata recombinant GFP.

Fluorescent proteins from marine organisms represent potential candidates for biosensor development. In this paper, we described the isolation of a native green fluorescent protein from Anemonia sulcata and the cloning and purification of its equivalent as a recombinant protein in Escherichia coli. Furthermore, the spectroscopic behaviours of the native and recombinant GFPs were investigated as a function of Cu2+, Cd2+, Pb 2+ and Ni2+ concentration. Our results suggest the high selectivity of both proteins at copper than the other metals and, for the recombinant protein, a great sensitivity at a very low concentration (0.1-1 μM). Moreover, starting from these data, using the combination of …

Disaccarides effects on thermal aggregation of Bovine Serum Albumin

Thermal aggregation of human and bovine serum albumin in the low concentration regime

High Fluorescence of Thioflavin T Confined in Mesoporous Silica Xerogels

Trapping of organic molecules and dyes within nanoporous matrices is of great interest for the potential creation of new materials with tailored features and, thus, different possible applications ranging from nanomedicine to material science. The understanding of the physical basis of entrapment and the spectral properties of the guest molecules within the host matrix is an essential prerequisite for the design and control of the properties of these materials. In this work, we show that a mesoporous silica xerogel can efficiently trap the dye thioflavin T (ThT, a molecule used as a marker of amyloid fibrils and with potential drug benefits), sequestering it from an aqueous solution and pro…

Aggregation Kinetics of Bovine Serum Albumin Studied by FTIR Spectroscopy and Light Scattering

To investigate which type of structural and conformational changes is involved in the aggregation processes of bovine serum albumin (BSA), we have performed thermal aggregation kinetics in D(2)O solutions of this protein. The tertiary conformational changes are followed by Amide II band, the secondary structural changes and the formation of beta-aggregates by the Amide I' band and, finally, the hydrodynamic radius of aggregates by dynamic light scattering. The results show, as a function of pD, that: tertiary conformational changes are more rapid as pD increases; the aggregation proceeds through formation of ordered aggregates (oligomers) at pD far from the isoelectric point of the protein;…

Biodistribution of Insulin-Nanogels in Mouse: A Preliminary Study for the Treatment of Alzheimer's Disease

A growing body of evidence shows that Insulin, Insulin Receptor (IR) and IR signaling are involved in brain cognitive functions and their dysfunction is implicated in Alzheimer's disease (AD) neurodegeneration. Thus, administration of insulin could be a strategy for AD treatment. For this aim we have designed, synthesized and characterized a nanogel system (NG) that has been conjugated to insulin molecules (NG-In) to deliver the protein into the brain, as a tool for the development of a new therapy against AD. In our preclinical study in mice, intraperitoneal injection of fluorescent-labeled NG has allowed to determine the biodistribution of NG vs time in the whole body and its clearance th…

Thermal aggregation of beta-lactoglobulin in presence of metal ions.

In this work, we report a study of the effects of zinc and copper ions on the heat-induced aggregation of beta-lactoglobulin (BLG). Kinetics investigations on aggregates growth by light scattering measurements and on secondary structure changes by FTIR absorption measurements show the different role played by the two metals during the whole process. In particular, the presence of zinc in solution promotes the formation of aggregates of BLG at a lower temperature than copper. Then, at fixed temperature, formation of a large amount of aggregates, of large dimension, is observed for Zn-BLG in shorter time; on the contrary, the presence of copper in solution does not affect the aggregation proc…

OH-related Infrared Absorption Bands in Oxide Glasses

We report the infrared activity, in the spectral region of the OH stretching modes, of different composite silicate glasses whose chemical composition is established by X-ray fluorescence measurements. The analysis of the absorption line profiles is made in terms of different spectral contributions, Gaussian in shape. The comparison with analogous spectra obtained in vitreous silica samples with impurity concentrations < 100 part per million moles is evidence of the effects of the different oxides on the vibrational properties of the OH groups. In particular, for oxide glasses a red shift of the composite band at about 3670 cm(-1), assigned to the OH stretching modes of free Si-OH groups an…

Simultaneous Determination of Caffeine and Chlorogenic Acids in Green Coffee by UV/Vis Spectroscopy

A simple method for the simultaneous determination of caffeine and chlorogenic acids content in green coffee was reported. The method was based on the use of UV/Vis absorption. It is relevant that the quantification of both caffeine and chlorogenic acids was performed without their preliminary chemical separation despite their spectral overlap in the range 250–350 nm. Green coffee was extracted with 70% ethanol aqueous solution; then the solution was analyzed by spectroscopy. Quantitative determination was obtained analytically through deconvolution of the absorption spectrum and by applying the Lambert-Beer law. The bands used for the deconvolution were the absorption bands of both caffein…

Sintesi di nanogeli per la veicolazione di insulina al cervello: una strategia per il trattamento della malattia di Alzheimer

I nanogeli sono nanoparticelle polimeriche reticolate idrofile. La struttura polimerica del nanogelo permette la coniugazione di molecole che possano esplicare azioni terapeutiche, diagnostiche e di indirizzamento attivo su siti bersaglio. Le caratteristiche di flessibilità e conformabilità del nanogelo, e delle sue maglie, ne favoriscono l’attraversamento delle barriere biologiche, l’interazione di eventuali ligandi esposti sulla superfice con i recettori target, un’azione protettiva nei confronti del principio attivo incorporato. L’irraggiamento con radiazioni ionizzanti di soluzioni acquose di polimeri è una metodologia sintetica interamente “water-based”, che garantisce rese elevate e c…

Insulin-nanogels: preliminary study in mouse for the treatment of Alzheimer's disease

A growing body of evidence shows that Insulin, Insulin Receptor (IR) and IR signalling are involved in brain cognitive functions and their dysfunction is implicated in Alzheimer’s disease (AD). Thus, administration of insulin could be a strategy for AD treatment. For this aim we have designed, synthesized and characterized a nanogel system (NG) that has been conjugated to insulin molecules (NG-In) (1) as new therapeutic approach against AD. In our preclinical studies in mice, intraperitoneal injection of fluorescent-labeled NG has allowed to determine the biodistribution of NG vs time in the whole body and its clearance through the kidneys and bladder. Furthermore, we have observed that mic…

Two distinct phases characterize thermal aggregation of b-Lactoglobulin at neutral pH

Aggregation of albumin: influence of the protein glycation

Free radicals are a normal component of cellular oxygen metabolism in mammals. However, free radical-associated damage is an important factor in many pathological processes. Aggregation, glycation and oxidative damage cause protein modifications, frequently observed in numerous diseases. Albumin represents the most abundant circulating protein. Many epidemiological studies have established an inverse relationship between the level of serum albumin and the risk of death. Albumin is involved in several biological functions, including the regulation of oncotic pressure, and the binding and transport of many molecules. In addition, albumin displays potent antioxidant and free radical scavenging…

High-Pressure-Driven Reversible Dissociation of α-Synuclein Fibrils Reveals Structural Hierarchy

The analysis of the α-synuclein (aS) aggregation process, which is involved in Parkinson's disease etiopathogenesis, and of the structural feature of the resulting amyloid fibrils may shed light on the relationship between the structure of aS aggregates and their toxicity. This may be considered a paradigm of the ground work needed to tackle the molecular basis of all the protein-aggregation-related diseases. With this aim, we used chemical and physical dissociation methods to explore the structural organization of wild-type aS fibrils. High pressure (in the kbar range) and alkaline pH were used to disassemble fibrils to collect information on the hierarchic pathway by which distinct β-sh…

In Situ Characterization of Hfq Bacterial Amyloid: A Fourier-Transform Infrared Spectroscopy Study

Hfq is a bacterial protein that regulates gene expression at the post-transcriptional level in Gram-negative bacteria. We have previously shown that Escherichia coli Hfq protein, and more precisely its C-terminal region (CTR), self-assembles into an amyloid-like structure in vitro. In the present work, we present evidence that Hfq unambiguously forms amyloid structures also in vivo. Taking into account the role of this protein in bacterial adaptation and virulence, our work opens possibilities to target Hfq amyloid self-assembly and cell location, with potential to block bacterial adaptation and treat infections. Fil: Partouche, David. Centre National de la Recherche Scientifique; Francia. …

Insulin activated Akt rescues Aβ oxidative stress-induced cell death by orchestrating molecular trafficking.

Increasing evidence indicates that Alzheimer's disease, one of the most diffused aging pathologies, and diabetes may be related. Here, we demonstrate that insulin signalling protects LAN5 cells by amyloid-β42 (Aβ)-induced toxicity. Aβ affects both activation of insulin receptors and the levels of phospho-Akt, a critical signalling molecule in this pathway. In contrast, oxidative stress induced by Aβ can be antagonized by active Akt that, in turn, inhibits Foxo3a, a pro-apoptotic transcription factor activated by reactive oxygen species generation. Insulin cascade protects against mitochondrial damage caused by Aβ treatment, restoring the mitochondrial membrane potential. Moreover, we show t…

Silica-Based NIR Nano-Emitters for Applications in Vivo and Process for Production Thereof

Production of paramagnetic and phosphorescent nanoprobes, obtainable starting from nanometric silica particles by methods of synthesis and enrichment with O2 following suitable thermal treatments in a controlled environment. The nanoprobes thus obtained display emission in the near infrared (NIR), are excitable in the visible and in the NIR, and are characterized by a lifetime of the order of one second. These nanoprobes have considerable scientific and commercial potential in the market of the confocal spectroscopy for bio-imaging and in the market of the medical pharmaceutical applications of drug labelling and drug delivery.

Amyloid Fibrils Formation of Concanavalin A at Basic pH

Mechanisms of partial unfolding and aggregation of proteins are of extreme interest in view of the fact that several human pathologies are characterized by the formation and deposition of protein-insoluble material, mainly composed of amyloid fibrils. Here we report on an experimental study on the heat-induced aggregation mechanisms, at basic pH, of concanavalin A (ConA), used as a model system. Thioflavin T (ThT) fluorescence and multiangle light scattering allowed us to detect different intertwined steps in the formation of ConA aggregates. In particular, the ThT fluorescence increase, observed in the first phase of aggregation, reveals the formation of intermolecular β-sheet structure wh…

Caratterizzazione chimico-fisica della denaturazione termica della b-Lattoglobulina, modulata da ioni metallici

Influence of metal ions on thermal aggregation of bovine serum albumin: aggregation kinetics and structural changes

Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies. In this work the effects of Cu(II) and Zn(II) ions on heat-induced structural modifications of bovine serum albumin (BSA) were studied, with the aim of delineating the role of these ions in the early stages of proteins aggregation kinetics. A joint application of different techniques was used. The aggregate growth was followed by dynamic light scattering measurements, whereas the conformational changes occurring in the protein structure were monitored by Raman and IR spectroscopy. Both in absence and in presence of metal ions, heating treatment gave rise to b-structures to the detriment of a…

HFt-LBT with Eu3+: a new nanovector for bioimaging applications

Thermal aggregation of bovine serum albumin close to the isoelectric point.

Different degrees in protein thermal aggregation processes in presence of trealose

Thermal aggregation and ion-induced cold-gelation of bovine serum albumin

Protein cold-gelation has recently received particular attention for its relevance in bio and food technology. In this work, we report a study on bovine serum albumin cold-gelation induced by copper or zinc ions. Metal-induced cold-gelation of proteins requires two steps: during the first one, the heat treatment causes protein partial unfolding and aggregation; then, after cooling the solution to room temperature, gels are formed upon the addition of metal ions. The thermally induced behaviour has been mainly investigated through different techniques: Fourier transform infrared (FTIR) spectroscopy, circular dichroism, dynamic light scattering (DLS) and rheology. Data have shown that the agg…

Characterization of the nucleation process of lysozyme at physiological pH: Primary but not sole process

We report on a kinetic study of the heat-induced aggregation process of lysozyme at physiological pH. The time evolution of the aggregation extent and the conformational changes of the protein were followed by dynamic light scattering (DLS) and FTIR spectroscopy, respectively, whereas the morphology of the aggregates was observed by Atomic Force Microscopy (AFM). The conformational changes of the secondary and tertiary structures were simultaneous and distinct in time with respect to the formation of aggregates. Oligomer formation occurred through at least two different aggregation processes: a nucleation process and a homogeneous non-nucleative diffusion-controlled process. FTIR measuremen…