"Water Association" Band in Saccharide Amorphous Matrices: Role of Residual Water on Bioprotection.

Saccharides protect biostructures against adverse environmental conditions mainly by preventing large scale motions leading to unfolding. The efficiency of this molecular mechanism, which is higher in trehalose with respect to other sugars, strongly depends on hydration and sugar/protein ratio. Here we report an Infrared Spectroscopy study on dry amorphous matrices of the disaccharides trehalose, maltose, sucrose and lactose, and the trisaccharide raffinose. Samples with and without embedded protein (Myoglobin) are investigated at different sugar/protein ratios, and compared. To inspect matrix properties we analyse the Water Association Band (WAB), and carefully decompose it into sub-bands, since their relative population has been shown to effectively probe water structure and dynamics in different matrices. In this work the analysis is extended to investigate the structure of protein-sugar-water samples, for the first time. Results show that several classes of water molecules can be identified in the protein and sugar environment and that their relative population is dependent on the type of sugar and, most important, on the sugar/protein ratio. This gives relevant information on how the molecular interplay between residual waters, sugar and protein molecules affect the biopreserving properties of saccharides matrices.

Specific Heat and Transport Functions ofWater.

Numerous water characteristics are essentially ascribed to its peculiarity to form stronghydrogen bonds that become progressively more stable on decreasing the temperature. However, thestructural and dynamical implications of the molecular rearrangement are still subject of debate andintense studies. In this work, we observe that the thermodynamic characteristics of liquid water arestrictly connected to its dynamic characteristics. In particular, we compare the thermal behaviourof the isobaric specific heat of water, measured in different confinement conditions at atmosphericpressure (and evaluated by means of theoretical studies) with its configurational contribution obtainedfrom the values of the measured self-diffusion coefficient through the use of the Adam-Gibbsapproach. Our results confirm the existence of a maximum in the specific heat of water at about 225K and indicate that especially at low temperature the configurational contributions to the entropy aredominant.

Hysteresis in the temperature dependence of the IR bending vibration of deeply cooled confined water.

Using Fourier Transform Infrared (FTIR) spectroscopy, we investigate the temperature dependence of the bending vibrations of water confined in the pores of a silica hydrogel in the temperature interval of 270-180 K. We also investigate the presence of thermal hysteresis by cooling and reheating temperature scans. The results clearly show the presence, at about 230 K, of a crossover in the temperature dependence of the IR spectra; moreover, the presence of hysteresis is clearly demonstrated. By comparing FTIR data with neutron diffraction data and previous calorimetric data on the same samples, we conclude that the crossover and the hysteretical behavior do not involve a water glass transition or crystallization but are related to a first-order-like liquid-liquid transition.

Palmitoylation is a post-translational modification of Alix regulating the membrane organization of exosome-like small extracellular vesicles.

BACKGROUND: Virtually all cell types have the capacity to secrete nanometer-sized extracellular vesicles, which have emerged in recent years as potent signal transducers and cell-cell communicators. The multifunctional protein Alix is a bona fide exosomal regulator and skeletal muscle cells can release Alix-positive nano-sized extracellular vesicles, offering a new paradigm for understanding how myofibers communicate within skeletal muscle and with other organs. S-palmitoylation is a reversible lipid post-translational modification, involved in different biological processes, such as the trafficking of membrane proteins, achievement of stable protein conformations, and stabilization of protein interactions. METHODS: Here, we have used an integrated biochemical-biophysical approach to determine whether S-palmitoylation contributes to the regulation of extracellular vesicle production in skeletal muscle cells. RESULTS: We ascertained that Alix is S-palmitoylated and that this post-translational modification influences its protein-protein interaction with CD9, a member of the tetraspanin protein family. Furthermore, we showed that the structural organization of the lipid bilayer of the small (nano-sized) extracellular vesicle membrane with altered palmitoylation is qualitatively different compared to mock control vesicles. CONCLUSIONS: We propose that S-palmitoylation regulates the function of Alix in facilitating the interactions among extracellular vesicle-specific regulators and maintains the proper structural organization of exosome-like extracellular vesicle membranes. GENERAL SIGNIFICANCE: Beyond its biological relevance, our study also provides the means for a comprehensive structural characterization of EVs.

The effects of pressure on the energy landscape of proteins.

Protein dynamics is characterized by fluctuations among different conformational substates, i.e. the different minima of their energy landscape. At temperatures above ~200 K, these fluctuations lead to a steep increase in the thermal dependence of all dynamical properties, phenomenon known as Protein Dynamical Transition. In spite of the intense studies, little is known about the effects of pressure on these processes, investigated mostly near room temperature. We studied by neutron scattering the dynamics of myoglobin in a wide temperature and pressure range. Our results show that high pressure reduces protein motions, but does not affect the onset temperature for the Protein Dynamical Transition, indicating that the energy differences and barriers among conformational substates do not change with pressure. Instead, high pressure values strongly reduce the average structural differences between the accessible conformational substates, thus increasing the roughness of the free energy landscape of the system.

Biopreservation of Myoglobin in Crowded Environment: A Comparison between Gelatin and Trehalose Matrixes.

Biopreservation by sugar and/or polymeric matrixes is a thoroughly studied research topic with wide technological relevance. Ternary amorphous systems containing both saccharides and proteins are extensively exploited to model the in vivo biopreservation process. With the aim of disentangling the effect of saccharides and polypeptidic crowders (such as gelatin) on the preservation of a model protein, we present here a combined differential scanning calorimetry and UV-vis spectrophotometry study on samples of myoglobin embedded in amorphous gelatin and trehalose + gelatin matrixes at different hydrations, and compare them with amorphous myoglobin-only and myoglobin-trehalose samples. The results point out the different effects of gelatin, which acts mainly as a crowding agent, and trehalose, which acts mainly by direct interaction. Gelatin is able to improve effectively the protein thermal stability at very low hydration; however, it has small effects at medium to high hydration. Consistently, gelatin appears to be more effective than trehalose against massive denaturation in the long time range, while the mixed trehalose + collagen matrix is most effective in preserving protein functionality, outdoing both gelatin-only and trehalose-only matrixes.

Ultrafast myoglobin structural dynamics observed with an X-ray free-electron laser.

Light absorption can trigger biologically relevant protein conformational changes. The light-induced structural rearrangement at the level of a photoexcited chromophore is known to occur in the femtosecond timescale and is expected to propagate through the protein as a quake-like intramolecular motion. Here we report direct experimental evidence of such 'proteinquake' observed in myoglobin through femtosecond X-ray solution scattering measurements performed at the Linac Coherent Light Source X-ray free-electron laser. An ultrafast increase of myoglobin radius of gyration occurs within 1 picosecond and is followed by a delayed protein expansion. As the system approaches equilibrium it undergoes damped oscillations with a ~3.6-picosecond time period. Our results unambiguously show how initially localized chemical changes can propagate at the level of the global protein conformation in the picosecond timescale.

Functional and dysfunctional conformers of human neuroserpin characterized by optical spectroscopies and Molecular Dynamics.

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latent serpins, we propose a structural model for latent NS, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatial distribution of intramolecular salt-bridges and hydrogen bonds.

Experimental evidence for a liquid-liquid crossover in deeply cooled confined water.

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 SiO2 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) temperature. Elastic neutron scattering results are complemented by differential scanning calorimetry data that put in evidence, besides the glass transition at about 170 K, a first-order-like endothermic transition occurring at about 230 K that, in view of the neutron scattering results, can be attributed to a liquid-liquid crossover. Our results give experimental evidence for the presence, in deeply cooled confined water, of a crossover occurring at about 230 K (at ambient pressure) from a liquid phase predominant at 210 K to another liquid phase predominant at 250 K; therefore, they are fully consistent with the liquid-liquid transition hypothesis.

The boson peak of deeply cooled confined water reveals the existence of a low-temperature liquid-liquid crossover.

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.

Hydration dependence of myoglobin dynamics studied with elastic neutron scattering, differential scanning calorimetry and broadband dielectric spectroscopy.

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

Communication: Protein dynamical transition vs. liquid-liquid phase transition in protein hydration water.

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 properties of water and proteins are tightly related.

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 producing a highly fluorescent material with a ThT quantum yield 1500 times greater than that of the free molecule. The study of spectroscopical properties of this system and the comparison with fluorescence of an uncharged analogue of ThT give indications about the mechanism responsible for the fluorescence switching-on of ThT molecules during their uptaking into the glass. Diffusion and nanocapillarity are responsible for ThT absorption, whereas electrostatic interaction between positive ThT molecules and negative dangling ≡SiO groups covering the pore surfaces causes the immobilization of ThT molecules inside the pores and the enhancement of its fluorescence, in line with the molecular rotor model proposed for this dye. We also show that entrapment efficiency and kinetics can be tuned by varying the electrostatic properties of the dye and/or the matrix.

Physical origin of anharmonic dynamics in proteins: new insights from resolution-dependent neutron scattering on homomeric polypeptides.

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 effect but is due to a real physical effect. Activation barriers and free energy values obtained for the protein dynamical transition allow us to make a connection with the two-well interaction potential of supercooled-confined water proposed to explain a low-density→high-density liquid-liquid transition.

The Monod-Wyman-Changeux allosteric model accounts for the quaternary transition dynamics in wild type and a recombinant mutant human hemoglobin.

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 the time scale of hemoglobin quaternary structural transition. In order to test the generality of the MWC kinetic model, we carried out a TR-WAXS investigation in parallel on adult human hemoglobin and on a recombinant protein (HbYQ) carrying two mutations at the active site [Leu(B10)Tyr and His(E7)Gln]. HbYQ seemed an ideal test because, although exhibiting allosteric properties, its kinetic and structural properties are different from adult human hemoglobin. The structural dynamics of HbYQ unveiled by TR-WAXS can be quantitatively accounted for by the MWC kinetic model. Interestingly, the main structural change associated with the R-T allosteric transition (i.e., the relative rotation and translation of the dimers) is approximately 10-fold slower in HbYQ, and the drop in the allosteric transition rate with ligand saturation is steeper. Our results extend the general validity of the MWC kinetic model and reveal peculiar thermodynamic properties of HbYQ. A possible structural interpretation of the characteristic kinetic behavior of HbYQ is also discussed.

The tempered polymerization of human neuroserpin.

Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimer's disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate monomeric conformer, which then associates with a native monomer to yield a dimeric species. After the formation of small polymers, the aggregation proceeds via monomer addition as well as polymer-polymer association. No further secondary mechanism takes place up to very high temperatures, thus resulting in the formation of neuroserpin linear polymeric chains. Most interesting, the overall aggregation is tuned by the co-occurrence of monomer inactivation (i.e. the formation of latent neuroserpin) and by a mechanism of fragmentation. The polymerization kinetics exhibit a unique modulation of the average mass and size of polymers, which might suggest synchronization among the different processes involved. Thus, fragmentation would control and temper the aggregation process, instead of enhancing it, as typically observed (e.g.) for amyloid fibrillation.

On the molecular structure of human neuroserpin polymers.

The polymerization of serpins is at the root of a large class of diseases; the molecular structure of serpin polymers has been recently debated. In this work, we study the polymerization kinetics of human neuroserpin by Fourier Transform Infra Red spectroscopy and by time-lapse Size Exclusion Chromatography. First, we show that two distinct neuroserpin polymers, formed at 45 and 85°C, display the same isosbestic points in the Amide I' band, and therefore share common secondary structure features. We also find a concentration independent polymerization rate at 45°C suggesting that the polymerization rate-limiting step is the formation of an activated monomeric species. The polymer structures are consistent with a model that predicts the bare insertion of portions of the reactive center loop into the A ß-sheet of neighboring serpin molecule, although with different extents at 45 and 85°C.

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 indicate that the protein is "softer" in the fibril state with respect to the native and amorphous aggregate states. We discuss this finding in terms of a structural interpretation and suggest that the paradigm ordered structure ↔ lower flexibility can be questioned when considering the local fast side-chain protein dynamics.

Protein thermal denaturation and matrix glass transition in different protein-trehalose-water systems.

Biopreservation by saccharides is a widely studied issue due to its scientific and technological importance; in particular, ternary amorphous protein-saccharide-water systems are extensively exploited to model the characteristics of the in vivo biopreservation process. We present here a differential scanning calorimetry (DSC) study on amorphous trehalose-water systems with embedded different proteins (myoglobin, lysozyme, BSA, hemoglobin), which differ for charge, surface, and volume properties. In our study, the protein/trehalose molar ratio is kept constant at 1/40, while the water/sugar molar ratio is varied between 2 and 300; results are compared with those obtained for binary trehalose-water systems. DSC upscans offer the possibility of investigating, in the same measurement, the thermodynamic properties of the matrix (glass transition, T(g)) and the functional properties of the encapsulated protein (thermal denaturation, T(den)). At high-to-intermediate hydration, the presence of the proteins increases the glass transition temperature of the encapsulating matrix. The effect mainly depends on size properties, and it can be ascribed to confinement exerted by the protein on the trehalose-water solvent. Conversely, at low hydration, lower T(g) values are measured in the presence of proteins: the lack of water promotes sugar-protein interactions, thus weakening the confinement effect and softening the matrix with respect to the binary system. A parallel T(den) increase is also observed; remarkably, this stabilization can reach ∼70 K at low hydration, a finding potentially of high biotechnological relevance. A linear relationship between T(g) and T(den) is also observed, in line with previous results; this finding suggests that collective water-trehalose interactions, responsible for the glass transition, also influence the protein denaturation.

Hemoglobin loaded polymeric nanoparticles: preparation and characterizations.

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 oxidation of the protein was not avoided during its loading into nanoparticles, the presence of acidic moieties in the polymeric structure is proposed to be directly involved in the protein inactivation mechanism.