0000000000043057
AUTHOR
Giorgio Schirò
showing 27 related works from this author
Quaternary transition pathway in sol–gel encapsulated haemoglobin tracked by NIR and UV spectral relaxations
2008
→T structural transition of haemoglobin (hb), the protein responsible for oxygen (o) transport in the red blood cells of vertebrates, is the hall mark example. This transition, which regu lates o2 uptake in the lungs and o2 release in the tissues, is a switch in the quaternary structure of the protein from a low-affinity state (T) to a high-affinity state (R), two well-characterised structures. The struc tural pathway connecting the end states of this transition remains unclear, however, although recently several experimental 1 or
Quaternary relaxations in sol-gel encapsulated hemoglobin studied via NIR and UV spectroscopy.
2007
In this work, we study the kinetics of the R --T transition in hemoglobin using a combination of near-infrared and near-ultraviolet spectroscopy. We use a sol-gel encapsulation protocol to decelerate the conformational transitions and to avoid spectral perturbations arising from ligand migration and recombination. We monitor two spectroscopic markers: band III in the near-IR, which is a fine probe of the heme pocket conformation, and the tryptophan band in the near-UV, which probes the formation of the Trpbeta37-Aspalpha94 hydrogen bond, characteristic of the T structure, at the critical alpha1beta2 subunit interface. The time evolution of these two bands is monitored after deoxygenation of…
The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin
2012
International audience; The acknowledged success of the Monod-Wyman-Changeux (MWC) allosteric model stems from its efficacy in accounting for the functional behavior of many complex proteins starting with hemoglobin (the paradigmatic case) and extending to channels and receptors. The kinetic aspects of the allosteric model, however, have been often neglected, with the exception of hemoglobin and a few other proteins where conformational relaxations can be triggered by a short and intense laser pulse, and monitored by time-resolved optical spectroscopy. Only recently the application of time-resolved wide-angle X-ray scattering (TR-WAXS), a direct structurally sensitive technique, unveiled th…
Hemoglobin loaded polymeric nanoparticles: preparation and characterizations.
2011
In the present work polymeric nanoparticles based on Poly (maleic anhydride-alt-butyl vinyl ether) 5% grafted with m-PEG (2000) and 95% grafted with 2-methoxyethanol (VAM41-PEG) were loaded with human hemoglobin (Hb) and characterized from a physicochemical point of view. The assessment of structural and functional features of the loaded Hb was performed and the effect of the introduction of different reducing agents as aimed at minimizing Hb oxidation during the nanoparticles formulation process, was also investigated. Nanoparticles possessing an average diameter of 138 ± 10 nm and physicochemical features suitable for this kind of application were successfully obtained. Although the oxida…
Protein/Hydration Water Dynamics in Hard Confinement: Dielectric Relaxations and Picoseconds Hydrogen Fluctuations
2011
In this review we report on some experimental studies on the dynamics of Myoglobin in a confined geometry, obtained by encapsulation in a porous silica matrix, at low hydration levels. After formation through the solgel method, the samples were left aging/drying in order to reach a condition where only one or two water layers surround the proteins. In order to put in evidence the specific effect of confinement in the silica host, we compared this system with another one (i.e. hydrated powder) where proteins are confined by other proteins. Using elastic neutron scattering we investigate the temperature dependence of the mean square displacements of non-exchangeable hydrogen atoms of sol-gel …
The boson peak of deeply cooled confined water reveals the existence of a low-temperature liquid-liquid crossover.
2014
International audience; The Boson peak of deeply cooled water confined in the pores of a silica xerogel is studied by inelastic neutron scattering at different hydration levels to separate the contributions from matrix, water on the pore surfaces and "internal" water. Our results reveal that at high hydration level, where the contribution from internal water is dominant, the temperature dependence of the Boson peak intensity shows an inflection point at about 225 K. The complementary use of differential scanning calorimetry to describe the thermodynamics of the system allows identifying the inflection point as the signature of a water liquid-liquid crossover.
The protein dynamical transition does not require the protein polypeptide chain
2011
We give experimental evidence that the main features of protein dynamics revealed by neutron scattering, i.e., the “protein dynamical transition” and the “boson peak”, do not need the protein polypeptide chain. We show that a rapid increase of hydrogen atoms fluctuations at about 220 K, analogous to the one observed in hydrated myoglobin powders, is also observed in a hydrated amino acids mixture with the chemical composition of myoglobin but lacking the polypeptide chain; in agreement with the protein behavior, the transition is abolished in the dry mixture. Further, an excess of low-frequency vibrational modes around 3 meV, typically observed in protein powders, is also observed in our mi…
Dynamic properties of solvent confined in silica gels studied by broadband dielectric spectroscopy
2007
Abstract We report the results of a broadband (10−2–107 Hz) dielectric spectroscopy study on a solvent system (glycerol–water solution) confined in a porous silica matrix. The dielectric relaxation of the system is studied as a function of both temperature (120–280 K) and solvent composition (0–36 glycerol molar percentage), at constant matrix composition. Our data show that glycerol–water systems confined inside silica gel are characterized by a very complex dynamics quite different from that observed in solution, thus indicating that confinement may deeply modify solvent dynamics. Indeed in addition to the relaxation processes similar to those occurring in bulk samples, new dielectric rel…
Impulsive solvent heating probed by picosecond x-ray diffraction
2006
The time-resolved diffraction signal from a laser-excited solution has three principal components: the solute-only term, the solute-solvent cross term, and the solvent-only term. The last term is very sensitive to the thermodynamic state of the bulk solvent, which may change during a chemical reaction due to energy transfer from light-absorbing solute molecules to the surrounding solvent molecules and the following relaxation to equilibrium with the environment around the scattering volume. The volume expansion coefficient alpha for a liquid is typically approximately 1 x 10(-3) K(-1), which is about 1000 times greater than for a solid. Hence solvent scattering is a very sensitive on-line t…
IN13 Backscattering Spectrometer at ILL: Looking for Motions in Biological Macromolecules and Organisms
2008
In 1998, three partner groups (the French institutions Institut de Biologie Structurale and the Leon Brillouin Laboratory and the Italian Istituto Nazionale per la Fisica della Materia, now merged with the Consiglio Nazionale delle Ricerche, INFM-CNR) applied to operate the thermal backscattering spectrometer IN13, at the Institut Laue Langevin, as a French-Italian Collaborative Research Group (CRG). The plan was to have access to a dedicated spectrometer in order to explore how far neutron scattering could contribute to the understanding of dynamics in biological macromolecules: how “flexible” must be a biological object to perform its function?
Physical Origin of Anharmonic Dynamics in Proteins: New Insights From Resolution-Dependent Neutron Scattering on Homomeric Polypeptides
2012
Neutron scattering reveals a complex dynamics in polypeptide chains, with two main onsets of anharmonicity whose physical origin and biological role are still debated. In this study the dynamics of strategically selected homomeric polypeptides is investigated with elastic neutron scattering using different energy resolutions and compared with that of a real protein. Our data spotlight the dependence of anharmonic transition temperatures and fluctuation amplitudes on energy resolution, which we quantitatively explain in terms of a two-site model for the protein-hydration water energy landscape. Experimental data strongly suggest that the protein dynamical transition is not a mere resolution …
Decoding vibrational states of Concanavalin A amyloid fibrils.
2015
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…
Hydration dependent dynamics in sol-gel encapsulated myoglobin.
2008
In this work we study the effect of hydration on the dynamics of a protein in confined geometry, i.e. encapsulated in a porous silica matrix. Using elastic neutron scattering we investigate the temperature dependence of the mean square displacements of non-exchangeable hydrogen atoms of sol-gel encapsulated met-myoglobin. The study is extended to samples at 0.2, 0.3 and 0.5 g water/g protein fractions and comparison is made with met-myoglobin powders at the same average hydration and with a dry powder sample. Elastic data are analysed using a model of dynamical heterogeneity to take into account deviations of elastic intensity from gaussian behaviour in a large momentum transfer range and r…
Direct Evidence of the Amino Acid Side Chain and Backbone Contributions to Protein Anharmonicity
2010
Elastic incoherent neutron scattering has been used to study the temperature dependence of the mean-square displacements of nonexchangeable hydrogen atoms in powders of a series of homomeric polypeptides (polyglycine, polyalanine, polyphenylalanine and polyisoleucine) in comparison with myoglobin at the same hydration level (h = 0.2). The aim of the work was to measure the dynamic behavior of different amino acid residues separately and assess the contribution of each type of side chain to the anharmonic dynamics of proteins. The results provide direct experimental evidence that the first anharmonic activation, at approximately 150 K, is largely due to methyl group rotations entering the ti…
Dynamics of myoglobin in confinement: An elastic and quasi-elastic neutron scattering study
2008
In order to clarify the role of hard confinement on protein dynamics, elastic and quasi-elastic neutron scattering experiments have been performed on ferric horse myoglobin in two different systems: the protein embedded in a porous silica matrix, and the corresponding hydrated protein powder. Elastic data have been analysed using two different models (dynamical heterogeneity and anharmonic double-well potential) that take into account deviations of elastic intensity from Gaussian behaviour. The profile of quasi-elastic spectra has been approximated by a combination of Lorentzian and Gaussian components. Comparison between the data relative to the two different samples indicates that geometr…
Communication: Protein dynamical transition vs. liquid-liquid phase transition in protein hydration water
2013
In this work, we compare experimental data on myoglobin hydrated powders from elastic neutron scattering, broadband dielectric spectroscopy, and differential scanning calorimetry. Our aim is to obtain new insights on the connection between the protein dynamical transition, a fundamental phenomenon observed in proteins whose physical origin is highly debated, and the liquid-liquid phase transition (LLPT) possibly occurring in protein hydration water and related to the existence of a low temperature critical point in supercooled water. Our results provide a consistent thermodynamic/dynamic description which gives experimental support to the LLPT hypothesis and further reveals how fundamental …
Spectroscopic markers of the T-R quaternary transition in human hemoglobin
2004
n questo lavoro, usiamo un protocollo sol-gel per intrappolare e confrontare gli stati quaternari R e T di entrambi i deossigenati (deossiHb) ederivati di ossido di carbonio (HbCO) dell'emoglobina umana. La banda di assorbimento ottico del vicino infrarosso III e lo stretching di CO a infrarossibanda sono utilizzati per rilevare l'effetto della struttura quaternaria sulle proprietà spettrali di deoxyHb e HbCO; confronto con mioglobinaconsente una valutazione dei contributi terziari e quaternari ai turni di banda misurati. La RXLa transizione T è indicata per causare un bluspostamento della banda III di ~ 35 cm?1per deoxyHb e uno spostamento rosso della banda di allungamento CO di soli ~ 0…
Probing in cell protein structural changes with time-resolved X-ray scattering
2012
International audience; Investigating protein structural changes inside the cell is a major goal in molecular biology. Here we show that time-resolved wide-angle X-ray scattering is a valuable tool for this purpose. Hemoglobin has been chosen as a model system and its tertiary and quaternary conformational changes following laser flash-photolysis have been tracked in intact red blood cells with nanosecond time resolution.
Neutron Scattering Reveals Enhanced Protein Dynamics in Concanavalin A Amyloid Fibrils
2012
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…
Molecular origin and hydration dependence of protein anharmonicity: an elastic neutron scattering study.
2010
Two main onsets of anharmonicity are present in protein dynamics. Neutron scattering on protein hydrated powders revealed a first onset at about 150 K and a second one at about 230 K (the so called dynamical transition). In order to assess the molecular origin of protein anharmonicity, we study different homomeric polypeptides by incoherent elastic neutron scattering, thus disentangling the contribution of different molecular groups in proteins. We show that methyl group rotations are the main contributors to the low temperature onset. Concerning the dynamical transition, we show that it also occurs in absence of side chains; however, the presence and mobility of side chains substantially i…
High Fluorescence of Thioflavin T Confined in Mesoporous Silica Xerogels
2013
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…
Experimental evidence for a liquid-liquid crossover in deeply cooled confined water.
2014
International audience; In this work we investigate, by means of elastic neutron scattering, the pressure dependence of mean square displacements (MSD) of hydrogen atoms of deeply cooled water confined in the pores of a three-dimensional disordered SiO 2 xerogel; experiments have been performed at 250 and 210 K from atmospheric pressure to 1200 bar. The " pressure anomaly " of supercooled water (i.e., a mean square displacement increase with increasing pressure) is observed in our sample at both temperatures; however, contrary to previous simulation results and to the experimental trend observed in bulk water, the pressure effect is smaller at lower (210 K) than at higher (250 K) temperatur…
Observing heme doming in myoglobin with femtosecond X-ray absorption spectroscopy.
2015
International audience; We report time-resolved X-ray absorption measurements after photolysis of carbonmonoxy myoglobin performed at the LCLS X-ray free electron laser with nearly 100 fs (FWHM) time resolution. Data at the Fe K-edge reveal that the photoinduced structural changes at the heme occur in two steps, with a faster (∼70 fs) relaxation preceding a slower (∼400 fs) one. We tentatively attribute the first relaxation to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and the second relaxation to a residual Fe motion out of the heme plane that is coupled to the displacement of myoglobin F-helix
The Boson Peak of Amyloid Fibrils: Probing the Softness of Protein Aggregates by Inelastic Neutron Scattering
2014
Proteins and polypeptides are characterized by low-frequency vibrations in the terahertz regime responsible for the so-called "boson peak". The shape and position of this peak are related to the mechanical properties of peptide chains. Amyloid fibrils are ordered macromolecular assemblies, spontaneously formed in nature, characterized by unique biological and nanomechanical properties. In this work, we investigate the effects of the amyloid state and its polymorphism on the boson peak. We used inelastic neutron scattering to probe low-frequency vibrations of the glucagon polypeptide in the native state and in two different amyloid morphologies in both dry and hydrated sample states. The dat…
Dielectric Relaxations in Confined Hydrated Myoglobin
2009
In this work we report the results of a broadband dielectric spectroscopy study on the dynamics of a globular protein, myoglobin, in confined geometry, i.e. encapsulated in a porous silica matrix, at low hydration levels, where about only one or two water layers surround the proteins. In order to highlight the specific effect of confinement in the silica host, we compared this system with hydrated myoglobin powders at the same hydration levels. The comparison between the data relative to the two different systems indicates that geometrical confinement within the silica matrix plays a crucial role in protein-water dielectric relaxations, the effect of sol-gel encapsulation being essentially …
Hydration dependence of myoglobin dynamics studied with elastic neutron scattering, differential scanning calorimetry and broadband dielectric spectr…
2014
In this work we present a thorough investigation of the hydration dependence of myoglobin dynamics. The study is performed on D2O-hydrated protein powders in the hydration range 0<h<0.5 (h≡gr[D2O]/gr[protein]) and in the temperature range 20-300K. The protein equilibrium fluctuations are investigated with Elastic Neutron Scattering using the spectrometer IN13 at ILL (Grenoble), while the relaxations of the protein + hydration water system are investigated with Broadband Dielectric Spectroscopy; finally, Differential Scanning Calorimetry is used to obtain a thermodynamic description of the system. The effect of increasing hydration is to speed up the relaxations of the myoglobin + hydration …
Anharmonic activations in proteins and peptide model systems and their connection with supercooled water thermodynamics
2016
International audience; — Proteins, the nano-machines of living systems, are highly dynamic molecules. The timescale of functionally relevant motions spans over a very broad range, from femtoseconds to several seconds. In particular, the pico-to nanoseconds region is characterized by side-chain and backbone anharmonic fluctuations that are responsible for many biological tasks like ligand binding, substrate recognition and enzymatic activity. Neutron scattering on hydrated protein powders reveals two main activations of anharmonic dynamics, characterized by different onset temperature and amplitude. Here we review our work on synthetic polypeptides, native proteins, and single amino acids t…