Functionally relevant electric-field induced perturbations of the prosthetic group of yeast ferrocytochrome c mutants obtained from a vibronic analysis of low-temperature absorption spectra.

We have measured the low temperature (T = 20 K) absorption spectra of the N52A, N52V, N52I, Y67F, and N52AY67F mutants of ferrous Saccharomyces cerevisiae (baker's yeast) cytochrome c. All the bands in the Q0- and Q(v)-band region are split, and the intensity distributions among the split bands are highly asymmetric. The spectra were analyzed by a decomposition into Voigtian profiles. The spectral parameters thus obtained were further analyzed in terms of the vibronic coupling model of Schweitzer-Stenner and Bigman (Schweitzer-Stenner, R.; Bigman, D. J. Phys. Chem. B 2001, 7064-7073) to identify parameters related to electronic and vibronic perturbations of the heme macrocycle. We report th…

Quaternary transition pathway in sol–gel encapsulated haemoglobin tracked by NIR and UV spectral relaxations

→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

The importance of vibronic perturbations in ferrocytochrome c spectra: a reevaluation of spectral properties based on low-temperature optical absorption, resonance Raman, and molecular-dynamics simulations.

We have measured and analyzed the low-temperature (T=10 K) absorption spectrum of reduced horse heart and yeast cytochrome c. Both spectra show split and asymmetric Q(0) and Q(upsilon) bands. The spectra were first decomposed into the individual split vibronic sidebands assignable to B(1g) (nu15) and A(2g) (nu19, nu21, and nu22) Herzberg-Teller active modes due to their strong intensity in resonance Raman spectra acquired with Q(0) and Q(upsilon) excitations. The measured band splittings and asymmetries cannot be rationalized solely in terms of electronic perturbations of the heme macrocycle. On the contrary, they clearly point to the importance of considering not only electronic perturbati…

Quaternary relaxations in sol-gel encapsulated hemoglobin studied via NIR and UV spectroscopy.

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…

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…

Low temperature optical spectroscopy of low-spin ferric hemeproteins

We report the Soret absorption spectra (500-350 nm) of the cyanomet derivatives of human hemoglobin and horse myoglobin, in the temperature range 300-20 K and in two different solvents (65% v/v glycerol-water or 65% v/v ethylene glycol-water). In order to obtain information on stereodynamic properties of active site of the two hemeproteins, we perform an analysis of the band profiles within the framework of electron-vibrations coupling. This approach enables us to single out the various contributions to the spectral bandwidth, such as those arising from non-radiative decay of the excited electronic state (homogeneous broadening) and from the coupling of the electronic transition i) with hig…

More than a Confinement: “Soft” and “Hard” Enzyme Entrapment Modulates Biological Catalyst Function

Catalysis makes chemical and biochemical reactions kinetically accessible. From a technological point of view, organic, inorganic, and biochemical catalysis is relevant for several applications, from industrial synthesis to biomedical, material, and food sciences. A heterogeneous catalyst, i.e., a catalyst confined in a different phase with respect to the reagents’ phase, requires either its physical confinement in an immobilization matrix or its physical adsorption on a surface. In this review, we will focus on the immobilization of biological catalysts, i.e., enzymes, by comparing hard and soft immobilization matrices and their effect on the modulation of the catalysts’ function. Indeed, …

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

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…

Dynamic properties of solvent confined in silica hydrogels studied by broadband dielectric spectroscopy

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 mon…

Calorimetric study of myoglobin embedded in trehalose-water matrixes

It has been suggested that in ‘dry’ protein–trehalose–water systems, water–mediated hydrogen bond network, whose strength increases by drying, anchors the protein to its surroundings. To further characterize this effect, we performed a DSC study on low-water myoglobin–trehalose systems. The denaturation temperature resulted to increase by decreasing hydration, and linearly correlated to the glass transition temperature of both the ternary protein–water–trehalose and the binary water–trehalose systems. Further measurements are being performed to investigate eventual differences among different saccharides.

Dynamics of ligand binding to hemoglobin: quaternary structure dependence

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 oxida…

Supercooled Water Confined in a Silica Xerogel: Temperature and Pressure Dependence of Boson Peak and of Mean Square Displacements

A silica xerogel can be obtained from an alcoxide precursor (TMOS, tetramethylortosilcate) via the sol-gel method: TMOS hydrolysis and subsequent polycondensation yields a solid, disordered, porous SiO2 matrix (average pore dimensions ~20Å). Inside the pores water is trapped and the hydration level h=gr[H2O]/gr[SiO2] can be easily controlled. The presence and temperature dependence of the boson peak (BP) in xerogel confined supercooled water was studied with inelastic neutron scattering (spectrometer IN6 at ILL, Grenoble) in xerogel samples having h=0.4 and h=0.2. After careful subtraction of the contributions arising from the matrix and from quasi-elastic scattering, the BP contribution wa…

Protein/Hydration Water Dynamics in Hard Confinement: Dielectric Relaxations and Picoseconds Hydrogen Fluctuations

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 …

Dynamics of water confined in silica hydrogels studied with elastic and quasi-elastic neutron scattering

Conformational relaxations in hemoglobin studied by time-resolved absorption spectroscopy

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…

Application of Broadband Dielectric Spectroscopy to Cultural Heritage: characterization and preservation of ancient paper artwork

Within the cultural heritage the characterization and conservation of artworks based on paper represents a significant issue for both restorers and scientists. The paper deterioration is affected by the degree of hydrolytic and oxidative reactions which occur upon aging. Moreover, the durability of cellulose fibers depends on the intrinsic composition/structure of the paper as well as on the conservation conditions, such as temperature and humidity. The structural and dynamical characterization of the cellulose matrix and of the water confined within its pores is therefore of central interest. Our working hypothesis is that WATER DYNAMICS is one of the main determinants of paper degradation…

Dynamic properties of myoglobin embedded in silica hydrogels studied through elastic and quasi-elastic neutron scattering

Dynamics of myoglobin confined in silica hydrogel in comparison with hydrated powder: an elastic and quasi-elastic neutron scattering study

Dynamic properties of myoglobin embedded in silica hydrogels studied through elastic neutron scattering

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 …

Low-temperature optical spectroscopy of cobalt in Cu,Co superoxide dismutase: a structural dynamics study of the solvent-unaccessible metal site.

The temperature dependence (300 to 10 K) of the electronic absorption spectra of the cobalt chromophore in bovine superoxide dismutase (SOD) having the native Zn(II) ion selectivity replaced by Co(II) has been investigated in four different derivatives: Cu(II),Co(II) SOD, N3(-)-Cu(II), Co(II) SOD, Cu(I),Co(II) SOD, and E,Co(II) SOD in which the copper ion has been selectively removed. In the Cu(II),Co(II) SOD, the cobalt spectrum is characterized at room temperature by three bands centered at 18,472, 17,670, and 16,793 cm-1; the low-frequency band is split, at low temperatures, into two components, indicating a lower symmetry contribution to a predominantly tetrahedral crystal field. Additi…

A calorimetric study of ternary protein-trehalose-water systems: matrix glass transition and protein thermal denaturation

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…

Effects of confinement on the dynamics of a protein-solvent system studied by elastic and quasi-elastic neutron scattering

Tracking protein motions in solution

Spectral broadening of the Soret band in myoglobin: an interpretation by the full spectrum of low-frequency modes from a normal modes analysis.

In this work the temperature dependence of the Soret band line shape in carbon-monoxy myoglobin is re-analyzed by using both the full correlator approach in the time domain and the frequency domain approach. The new analyses exploit the full density of vibrational states of carbon-monoxy myoglobin available from normal modes analysis, and avoid the artificial division of the entire set of vibrational modes coupled to the Soret transition into "high-frequency" and "low-frequency" subsets; the frequency domain analysis, however, makes use of the so-called short-times approximation, while the time domain one avoids it. Time domain and frequency domain analyses give very similar results, thus s…

Different relaxations in myoglobin after photolysis

To clarify the interplay of kinetic hole-burning (KHB), structural relaxation, and ligand migration in myoglobin (Mb), we measured time-resolved absorption spectra in the Soret region after photolysis of carbon monoxide Mb (MbCO) in the temperature interval 120-260 K and in the time window 350 ns to 200 ms. The spectral contributions of both photolyzed (Mb * ) and liganded Mb (MbCO) have been analyzed by taking into account homogeneous bandwidth, coupling to vibrational modes, and static conformational heterogeneity. We succeeded in separating the “time-dependent” spectral changes, and this work provides possibilities to identify the events in the process of ligand rebinding. KHB is domina…

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…

Solvation of a probe molecule by fluid supercooled water in a hydrogel at 200 K

By combining electron paramagnetic resonance (EPR) measurements on a nitroxide probe and differential scanning calorimetry (DSC), we demonstrate existence of liquid supercooled water in a silica hydrogel with high hydration level down to temperatures of at least 198 K. Besides the major fraction of liquid supercooled water, a minor fraction crystallizes at about 236 K during cooling and melts at 246 K during heating. The liquid domains are of sufficient size to solvate the nearly spherical paramagnetic probe molecule TEMPO with a diameter of about 6 angstrom. Analysis of EPR spectra provides the rotational correlation time of the probe that is further used to compare the viscosity of the su…

Observing myoglobin proteinquake with an X-ray free-electron laser

The events following the photodissociation of the bond be- tween myoglobin and its ligand have been extensively studied with a variety of experimental, theoretical and computational methods [1]. The results of these investigations have been rationalized in terms of a model that implies a protein quake- like motion [2], i.e. the propagation of the strain released upon photoexcitation through the protein similar to the prop- agation of acoustic waves during an earthquake. The exper- imental investigations performed so far have been based on spectroscopic measurements or did not have sufficient time- resolution to measure the timescale of such “proteinquake”. We have obtained direct experiment…

Unveiling the timescale of the R-T transition in human hemoglobin.

Time-resolved wide-angle X-ray scattering, a recently developed technique allowing to probe global structural changes of proteins in solution, was used to investigate the kinetics of R-T quaternary transition in human hemoglobin and to systematically compare it to that obtained with time-resolved optical spectroscopy under nearly identical experimental conditions. Our data reveal that the main structural rearrangement associated with the R-T transition takes place approximately 2 mus after the photolysis of hemoglobin at room temperature and neutral pH. This finding suggests that the 20-mus step observed with time-resolved optical spectroscopy corresponds to a small and localized structural…

Setting the basis for the study of intermediate conformers of human neuroserpin by Double Electron Electron Resonance

The Double Electron Electron Resonance (DEER) is an innovative technique that allows to measure the distance distribution between two spin labels within a range of 2-8 nm. This technique does not require crystalline samples, thus it is possible to determine the position of two different spin labelled domains of intrinsically flexible macromolecular systems. Our idea is to determinate the structural details of the intermediate conformers of human neuroserpin (hNS) by DEER. hNS is a protein that in the native state is folded in a metastable conformation. In particular conditions, hNS can either adopt a more stable conformation with the reactive centre loop (RCL) inserted into a β-sheet (laten…

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

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

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

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…

On the molecular structure of human neuroserpin polymers. Coagulation, fragmentation and latentization control serpin aggregation

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 structure…

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 …

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

Abstract 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…

Effect of protein net charge and steric hindrance on the glass transition of Protein-Trehalose-Water systems and on protein thermal denaturation

Time-resolved X-ray scattering as a tool to probe heme proteins structural dynamics in solution

Time-resolved wide-angle X-ray scattering (TR-WAXS) is a recently developed technique allowing to probe global structural changes of proteins in solution. We have used TR-WAXS to investigate large conformational changes of heme proteins (wild-type and mutant hemoglobin, neuroglobin, etc.) that cannot take place when these macromolecules are in a crystalline environment. Our data revealed detailed information on kinetic and thermodynamic properties of the investigated proteins and demonstrate the potentiality of the TR-WAXS technique.

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering (vol 5, pg 881, 2008)

Ferricytochrome c encapsulated in silica nanoparticles: structural stability and functional properties.

Using a modified sol-gel technique, we have succeeded in encapsulating ferric cytochrome c in silica nanoparticles obtained from hydrolysis and polycondensation of tetramethylorthosilicate. Particles dimensions have been determined with dynamic light scattering; this technique yields an hydrodynamic radius of about 100 nm, each nanoparticle containing about 10(2)-10(3) proteins. If stored in the cold at low ionic strength, nanoparticles are stable for more than one week, even if a slow radius increase with time is observed. CD measurements show that encapsulated proteins exhibit substantially increased stability against guanidinium hydrochloride induced denaturation. Reduction kinetics of e…

Effects of confinement on protein dynamics: a neutron scattering study on myoglobin confined in silica hydrogel at different average hydration levels

Immobilization of proteins in silica gel: Biochemical and biophysical properties

The development of silica-based sol-gel techniques compatible with the retention of protein structure and function started more than 20 years ago, mainly for the design of biotechnological devices or biomedical applications. Silica gels are optically transparent, exhibit good mechanical stability, are manufactured with different geometries, and are easily separated from the reaction media. Biomolecules encapsulated in silica gel normally retain their structural and functional properties, are stabilized with respect to chemical and physical insults, and can sometimes exhibit enhanced activity in comparison to the soluble form. This review briefly describes the chemistry of protein encapsulat…

Reply to “Comment to ‘Dynamics of supercooled confined water measured by deep inelastic neutron scattering’ by Y. Finkelstein and R. Moreh”

We reply to the comment [Front. Phys. 14(5), 53605 (2019)] by Y. Finkelstein and R. Moreh on our article Front. Phys. 13(1), 138205 (2018). We agree with some of their criticisms about our calculation of the temperature effect on the kinetic energy of hydrogen atoms of supercooled confined water; we also agree with their statement that, in view of the current sensitivity of the technique, possible effects of the liquid-liquid water transition are hardly detected with deep inelastic neutron scattering (DINS). However, we disagree with their use of the translational mass ratio of a single water molecule and, in general, with their underestimation of collective effects.

Impulsive solvent heating probed by picosecond x-ray diffraction

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…

Dielectric properties of myoglobin at 10 GHz by microwave cavity perturbation measurements

We report on the temperature dependence, at microwave (mw) frequency, of the imaginary part of the dielectric constant (En) in myoglobin powder samples with different hydration levels (h). The measurements have been performed by the cavity perturbation technique, in the range of temperature 80-345 K. The sample is located inside a glass capillary along the axis of a cylindrical copper cavity, resonating in the TE011 mode at 9.6 GHz, where the mw electric field has a node. By measuring the variation of the quality factor of the resonant cavity, one can extract the imaginary part of the dielectric constant. At temperatures higher than 230 K we observe an evident increase of the dielectric los…

Ferricytochrome c encapsulated in silica hydrogels: correlation between active site dynamics and solvent structure.

Ferricytochrome c encapsulated in silica hydrogels has been prepared by the sol-gel technique following, with some modifications, the procedure originally developed by Ellerby et al. (Science 255 1113 (1992)). A suitable preparation of hydrogels enables having both 'wet' and 'dry' samples. Wet samples have a high water content: as the temperature is lowered below approximately 260 K, water freezes and the samples crack. On the contrary, dry samples have a low water content (hydration h approximately equal 0.35): in these conditions water does not freeze even at cryogenic temperatures and the samples remain transparent and non-cracking. The dynamics of ferricytochrome c and its dependence on…

Quaternary structure specific dynamics and reactivity in human hemoglobin under cryogenic conditions

IN13 Backscattering Spectrometer at ILL: Looking for Motions in Biological Macromolecules and Organisms

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?

On the physical origin of the protein dynamical transition

Low-Temperature Optical Spectroscopy of Native and Azide-Reacted Bovine Cu,Zn Superoxide Dismutase. A Structural Dynamics Study

The optical absorption spectra of native and N(3-)-reacted Cu,Zn superoxide dismutase (SOD) has been studied in the temperature range 300-10 K. The broad d-d bands observed in the room temperature spectrum, centered at 14,700 cm-1 (native enzyme) and at 15,550 cm-1 (N(3-)-reacted enzyme), are clearly split at low temperature into two bands each, centered at 12,835 and 14,844 cm-1 and at 14,418 and 16,300 cm-1, respectively. The thermal behavior of the 23,720 cm-1 band present in the spectrum of the native enzyme indicates that this band belongs to the His61-->Cu(II) ligand to metal charge transfer transition. Analysis of the zeroth, first, and second moments of the various bands as a functi…

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 res…

Is the polypeptide chain necessary for the dynamical transition in proteins?

Quaternary structure control of geminate ligand rebinding after photolysis of carbonmonoxy hemoglobin

Protein dynamics: conformational disorder, vibrational coupling and anharmonicity in deoxy-hemoglobin and myoglobin.

In this work we study the temperature dependence of the Soret band lineshape of deoxymyoglobin and deoxyhemoglobin, in the range 300-20 K. To fit the measured spectra we use an approach originally proposed by Champion and coworkers (Srajer et al. 1986; Srajer and Champion 1991). The band profile is modelled as a Voigt function that accounts for the coupling with low frequency vibrational modes, whereas the coupling with high frequency modes is responsible for the vibronic structure of the spectra. Moreover, owing to the position of the iron atom out of the mean heme plane, inhomogeneous broadening brings about a non-Gaussian distribution of 0-0 electronic transition frequencies. The reporte…

Dynamic properties of deoxyhemoglobin in T and R quaternary conformation probed by the temperature dependence of NIR band III

Heme symmetry, vibronic structure, and dynamics in heme proteins: ferrous nicotinate horse myoglobin and soybean leghemoglobin.

We report the visible and Soret absorption bands, down to cryogenic temperatures, of the ferrous nicotinate adducts of native and deuteroheme reconstituted horse heart myoglobin in comparison with soybean leghemoglobin-a. The band profile in the visible region is analyzed in terms of vibronic coupling of the heme normal modes to the electronic transition in the framework of the Herzberg–Teller approximation. This theoretical approach makes use of the crude Born–Oppenheimer states and therefore neglects the mixing between electronic and vibrational coordinates; however, it takes into account the vibronic nature of the visible absorption bands and allows an estimate of the vibronic side bands…

Conformational substates of the Fe2+-His F8 linkage in deoxymyoglobin and hemoglobin probed in parallel by the Raman band of the Fe-His stretching vibration and the near-infrared absorption band III

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 …

Relationship between the Glass Transition of Myoglobin-Water-Disaccharide systems and Protein Thermal Denaturation

Near-Infrared Spectra of Water Confined in Silica Hydrogels in the Temperature Interval 365−5 K

We have used a sol−gel technique to obtain optically transparent hydrogels in which water is trapped within a tridimensional disordered silica matrix. A suitable aging of these hydrogels enables to have transparent noncracking samples down to cryogenic temperatures. We report the optical absorption spectra, in the near-infrared region, of water trapped in our silica hydrogels, measured in the temperature range 365−5 K, and we compare them with the same spectra of liquid water, measured in the temperature range 365−263 K. The data show that it is possible to have noncrystallizing water even at 5 K:  indeed, the overtone bands at ∼1.41 μm and at ∼1.155 μmtypical of “weakly bonded” water molec…

Conformational relaxations and kinetic hole burning in hemoglobin

Time-resolved small-angle scattering study of the R to T transition in hemoglobin

The R-T quaternary transition in deoxyhemoglobin studied by NIR spectral relaxation

Hemoglobin dynamics probed by picosecond wide-angle x-ray scattering

Interfacial water structure controls protein conformation.

A phenomenological theory of salt-induced Hofmeister phenomena is presented, based on a relation between protein solubility in salt solutions and protein-water interfacial tension. As a generalization of previous treatments, it implies that both kosmotropic salting out and chaotropic salting in are manifested via salt-induced changes of the hydrophobic/hydrophilic properties of protein-water interfaces. The theory is applied to describe the salt-dependent free energy profiles of proteins as a function of their water-exposed surface area. On this basis, three classes of protein conformations have been distinguished, and their existence experimentally demonstrated using the examples of bacter…

Dynamics of myoglobin in confinement: an incoherent elastic and quasi-elastic neutron scattering study

Matrix glass transition and embedded protein denaturation: effect of trehalose on different proteins

Anharmonic activations in proteins and peptide model systems and their connection with supercooled water thermodynamics

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…

Hydration dependent dynamics in sol-gel encapsulated myoglobin.

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…

Dynamics of protein–solvent systems in hard confinement studied by Broadband Dielectric Spectroscopy and Neutron Scattering

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…

Structure and dynamics of water confined in silica hydrogels: X-ray scattering and dielectric spectroscopy studies.

We have used a sol-gel technique to obtain optically transparent hydrogels in which water is confined within a 3D silica matrix. In this work we report X-ray scattering and dielectric spectroscopy measurements on samples having different aging times and compare them with previously obtained results with near-infrared (NIR) absorption spectroscopy. X-ray scattering at room temperature enables to characterize the structure and size of the matrix pores and the non-uniform distribution of water inside the hydrogel. Broad band dielectric spectroscopy in the temperature range 130-280 K enables to study water dynamics. In aged hydrogels two relaxations are clearly evident and show characteristic t…

Direct Evidence of the Amino Acid Side Chain and Backbone Contributions to Protein Anharmonicity

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…

Incoherent elastic and quasi-elastic neutron scattering investigation of hemoglobin dynamics.

In this work we investigate the dynamic properties of hemoglobin in glycerolD(8)/D(2)O solution using incoherent elastic (ENS) and quasi-elastic (QENS) neutron scattering. Taking advantage of complementary energy resolutions of backscattering spectrometers at ILL (Grenoble), we explore motions in a large space-time window, up to 1 ns and 14 A; moreover, in order to cover the harmonic and anharmonic protein dynamics regimes, the elastic experiments have been performed over the wide temperature interval of 20-300 K. To study the dependence of the measured dynamics upon the protein quaternary structure, both deoxyhemoglobin (in T quaternary conformation) and carbonmonoxyhemoglobin (in R quater…

“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,…

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…

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

Dynamics of myoglobin in confinement: An elastic and quasi-elastic neutron scattering study

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…

Probing protein structural dynamics in a human cell

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 …

Spectroscopic markers of the T-R quaternary transition in human hemoglobin

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…

Dynamics of homomeric polypeptides studied with neutron scattering, dielectric spectroscopy and calorimetry

Structural and Dynamic Properties of the Homodimeric Hemoglobin from Scapharca inaequivalvis Thr-72→Ile Mutant: Molecular Dynamics Simulation, Low Temperature Visible Absorption Spectroscopy, and Resonance Raman Spectroscopy Studies

AbstractMolecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72→Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy …

The contribution of methyl groups dynamics to the dynamical transition of proteins

Probing in cell protein structural changes with time-resolved X-ray scattering

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.

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.

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…

Structure-dynamics-function relationships in Asian elephant (Elephas maximus) myoglobin. An optical spectroscopy and flash photolysis study on functionally important motions

In this work we report the thermal behavior (10–300 K) of the Soret band lineshape of deoxy and carbonmonoxy derivatives of Asian elephant (Elephas maximus) and horse myoglobins together with their carbon monoxide recombination kinetics after flash photolysis; the results are compared to analogous data relative to sperm whale myoglobin. The Soret band profile is modeled as a Voigt function that accounts for the coupling with high and low frequency vibrational modes, while inhomogeneous broadening is taken into account with suitable distributions of purely electronic transition frequencies. This analysis makes it possible to isolate the various contributions to the overall lineshape that; in…

The necessary chances of a thermodynamically metastable protein: inactivation and polymeritzation of human neuroserpin

Serpins are a wide class of proteins with high structural similarity, characterized by a unique substrate-like inhibitory mechanism that resembles a "molecular mousetrap". The active serpin is characterized by a main 5-stranded β-sheet and an exposed Reactive Centre Loop, which acts as a bait for the target protease. The cleavage of the loop by the protease triggers the insertion of the loop into the β-sheet as a strand and the disruptive translocation of the protease. This peculiar conformational mobility is achievable since serpins fold into a metastable native conformation. This feature gives a selective advantage to the serpin family to develop inhibitory activities, but leaves these pr…

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…

Quaternary structure dependence of kinetic hole burning and conformational substates interconversion in hemoglobin.

Using a sol-gel encapsulation technique, we have prepared samples of CO saturated human adult hemoglobin locked in the R or T quaternary conformation. We report time-resolved spectra of these samples in the Soret region following flash photolysis, in the time interval ranging from 250 ns to 200 ms and in the temperature interval of 100-170 K. A suitable analysis of the measured difference spectra enables us to obtain the spectral contribution of deoxyHb and HbCO molecules as a function of time and/or of the fraction N(t) of deoxyHb molecules. In our experimental time window geminate CO rebinding to hemoglobin in the T quaternary conformation is about 2 orders of magnitude slower than to hem…

Sub-diffusive dynamics in hydrated myoglobin powder?

Structural factors controlling ligand binding to myoglobin: a kinetic hole-burning study.

Using temperature-derivative spectroscopy in the temperature range below 100 K, we have studied the dependence of the Soret band on the recombination barrier in sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation at 12 K. The spectra were separated into contributions from the photodissociated species, Mb*CO, and CO-bound myoglobin. The line shapes of the Soret bands of both photolyzed and liganded myoglobin were analyzed with a model that takes into account the homogeneous bandwidth, coupling of the electronic transition to vibrational modes, and static conformational heterogeneity. The analysis yields correlations between the activation enthalpy for rebinding and the model p…

Probing the role of water in protein conformation and function

Life began in a bath of water and has never escaped it. Cellular function has forced the evolution of many mechanisms ensuring that cellular water concentration has never changed significantly. To free oneself of any conceptual distinction among all small molecules, solutes and solvents, means that experiments to probe water's specific role in molecular function can be designed like any classical chemical reaction. Such an ‘osmotic stress’ strategy will be described in general and for an enzyme, hexokinase. Water behaves like a reactant that competes with glucose in binding to hexokinase, and modulates its conformational change and activity. This ‘osmotic stress’ strategy, now applied to ma…

Water and proteins confined in silica hydrogels and silica nanoparticles: structural, dynamic and functional studies.

Functional and dysfunctional isoforms of human neuroserpin

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 NS and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric conformer. Using 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 par- ticular, emission spectra of aromatic residues yi…

Dynamics of supercooled confined water measured by deep inelastic neutron scattering

In this paper, we present the results of deep inelastic neutron scattering (DINS) measurements on supercooled water confined within the pores (average pore diameter ~ 20 Å) of a disordered hydrophilic silica matrix obtained through hydrolysis and polycondensation of the alkoxide precursor Tetra-Methyl-Ortho-Silicate via the sol-gel method. Experiments were performed at two temperatures (250 K and 210 K, i.e., before and after the putative liquid–liquid transition of supercooled confined water) on a “wet” sample with hydration h ~ 40% w/w, which is high enough to have water-filled pores but low enough to avoid water crystallization. A virtually “dry” sample at h ~ 7% was also inve…

Anomalous water dynamics in brain: a combined diffusion magnetic resonance imaging and neutron scattering investigation

International audience; Water diffusion is an optimal tool for investigating the architecture of brain tissue on which modern medical diagnostic imaging techniques rely. However, intrinsic tissue heterogeneity causes systematic deviations from pure free-water diffusion behaviour. To date, numerous theoretical and empirical approaches have been proposed to explain the non-Gaussian profile of this process. The aim of this work is to shed light on the physics piloting water diffusion in brain tissue at the micrometre-to-atomic scale. Combined diffusion magnetic resonance imaging and first pioneering neutron scattering experiments on bovine brain tissue have been performed in order to probe dif…

Dynamical properties of water in living cells

With the aim of studying the effect of water dynamics on the properties of biological systems, in this paper, we present a quasi-elastic neutron scattering study on three different types of living cells, differing both in their morphological and tumor properties. The measured scattering signal, which essentially originates from hydrogen atoms present in the investigated systems, has been analyzed using a global fitting strategy using an optimized theoretical model that considers various classes of hydrogen atoms and allows disentangling diffusive and rotational motions. The approach has been carefully validated by checking the reliability of the calculation of parameters and their 99% confi…

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…

Kinetic energy and radial momentum distribution of hydrogen and oxygen atoms of water confined in silica hydrogel in the temperature interval 170–325 K

Water is an ubiquitous liquid and it is necessary for life;. Studies on water are therefore of obvious scientific and .... technological relevance. In view of its peculiar physicalproperties (the so-called water anomalies, particularly relevant at low temperatures [1]), studies on water structureand dynamics in ample temperature intervals, covering also the supercooling region, have attracted much interest in recent years. In particular, studies focused on the supercooled phase are important in order to test theories and hypotheses[2,3], including the liquid-liquid phase transition hypothesis [4-6] and the related fragile-to-strong crossover observed inwater confined in silica matrices and …

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 underg…

Molecular origin and hydration dependence of protein anharmonicity: an elastic neutron scattering study.

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

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…

Conformational Properties of Nickel(II) Octaethylporphyrin in Solution. 2. A Low-Temperature Optical Absorption Spectroscopy Study

We have measured the absorption spectrum of Ni(II) octaethylporphyrin in CH2Cl2 and in a 50% v/v isopentane/ethyl ether mixture as a function of temperature between 150 and 300 K and 40 and 300 K, respectively. The Soret band can be decomposed into two subbands whose frequencies differ by 220 cm-1. By analogy with resonance Raman results (Jentzen et al. J. Phys. Chem. 1996, 100, 14184−14191 (preceding paper)), we attribute the low-frequency subband to a conformer with a nonplanar macrocycle structure, whereas the high-frequency subband is interpreted as resulting from a planar conformer. The subbands' intensity ratios exhibit a solvent-dependent van't Hoff behavior between 300 and 160 K. Cr…

Dynamical properties of myoglobin in an ultraviscous water-glycerol solvent investigated with elastic neutron scattering and FTIR spectroscopy

Abstract Proteins have distinctive dynamical properties, characterized by the fluctuations of protein molecules among the different minima of their energy landscape. These fluctuations, progressively activated for temperature values larger than ~180 K, lead to a steep increase in the temperature dependence of all measurable dynamical properties. This phenomenon is known as Protein Dynamical Transition and, in spite of the intense studies due to its importance in protein function and to the relation with the fascinating fundamental thermodynamics of complex systems, many aspects of it are not yet clearly understood. Among these, the relationship with the properties of the external solvent an…

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 res…

Thermal denaturation of myoglobin in water--disaccharide matrixes: relation with the glass transition of the system.

Proteins embedded in glassy saccharide systems are protected against adverse environmental conditions [Crowe et al. Annu. Rev. Physiol. 1998, 60, 73-103]. To further characterize this process, we studied the relationship between the glass transition temperature of the protein-containing saccharide system (T(g)) and the temperature of thermal denaturation of the embedded protein (T(den)). To this end, we studied by differential scanning calorimetry the thermal denaturation of ferric myoglobin in water/disaccharide mixtures containing nonreducing (trehalose, sucrose) or reducing (maltose, lactose) disaccharides. All the samples studied are, at room temperature, liquid systems whose viscosity …

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

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…

Quaternary structure dependence of hemoglobin ligand rebinding kinetics under cryogenic conditions

The effects of pressure on the energy landscape of proteins

AbstractProtein 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 Dynam…

Observing heme doming in myoglobin with femtosecond X-ray absorption spectroscopy.

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

Conformational substates of ferricytochrome c revealed by combined optical absorption and electronic circular dichroism spectroscopy at cryogenic temperature.

We have investigated the heterogeneity of the Fe(III)–Met80 linkage of horse heart ferricytochrome c by probing the 695 nm charge transfer band with absorption and electronic circular dichroism (ECD) spectroscopy. In order to verify the connection between conformational substates of the Fe(III)–Met80 linkage and the 695 nm band spectral heterogeneity, we have performed experiments as a function of pH (neutral and acidic) and temperature (room and 20 K). At room temperature, the ECD spectrum is blue shifted with respect to the absorption one; the shift is more pronounced at acidic pH and is compatible with the presence of sub-bands. ECD measurements at 20 K highlighted the heterogeneous natu…

Dynamics of nanoparticles in a supercooled liquid

The dynamic properties of nanoparticles suspended in a supercooled glass forming liquid are studied by x-ray photon correlation spectroscopy. While at high temperatures the particles undergo Brownian motion the measurements closer to the glass transition indicate hyperdiffusive behavior. In this state the dynamics is independent of the local structural arrangement of nanoparticles, suggesting a cooperative behavior governed by the near-vitreous solvent.

The Boson Peak of Amyloid Fibrils: Probing the Softness of Protein Aggregates by Inelastic Neutron Scattering

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…

Thermal Denaturation of Myoglobin in Water–Sugar Matrices and Relationship with the Glass Transition of the System

Dielectric Relaxations in Confined Hydrated Myoglobin

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 …

Specific Heat and Transport Functions of Water

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 value…

The effect of water on protein dynamics

Neutron diffraction and spectroscopy were applied to describe the hydration and dynamics of a soluble protein and a natural membrane from extreme halophilic Archaea. The quantitative dependence of protein motions on water activity was clearly illustrated, and it was established that a minimum hydration shell is required for the systems to access their functional resilience, i.e. a dynamics state that allows biological activity.

Anharmonic onsets in polypeptides revealed by neutron scattering: experimental ecidences and quantitative description of energy resolution dependence

Tracking protein intra-molecular motions in solution with time-resolved Wide Angle X-ray Scattering

Exploring Cell Biodiversity

Brain tissue is a really complex system composed of different cell types that change in shape and size. A single neuron itself has a cell body, dendrites and an axon. About 80% of cerebral tissue consists of water molecules that are confined (intra and extra cellular) in its disordered biologic networks. Using neutron scattering on IN13 we are able to explore hydrogens (H) dynamics in time scale at about 40 ps and in size scale at about 1 Å. Such characteristic make it suitable to investigate brain tissue heterogeneity exploiting hydrogens as a probe since major constituent of macromolecules and water. Elastic neutron scattering (ENS) gives information about means square displacement (MSDs)…

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<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 …

Hydration dependence of mean square displacements, relaxation times and calorimetric glass transition in myoglobin hydrated powders

Erratum to: Dynamical properties of water in living cells (Front. Phys, (2018) 13, 1, 138301, 10.1007/s11467-017-0731-5)

In the original publication of the article, the label Q2(A-2) in Fig. 4 should be replaced with Q(A-1). Below is the correct Fig. 4.[Figure not available: see fulltext.]. © 2018, Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature.

Exploring cell biodiversity - Neutron scattering investigation of water diffusion in complex system

Scientists from biophysics, biology and medicine fields are interested in exploring and characterizing topologically cerebral tissue in order to diagnostic different diseases which affect brain in many patients [1-3]. One of the most diffuse diagnostic techniques is dMRI (diffusion magnetic resonance imaging) which extracts information about heterogeneity and asymmetries in brain tissue studying water diffusion dynamics (~80% mass constituent of tissues). The experimental limit of this technique is related to the acquisition time, TA, of the order of milliseconds. Water molecules diffuse within micrometre distance using TA as diffuse time (Eistein equation D~2TA). Cells have micrometric siz…

The dynamic transition of myoglobin encapsulated in silica hydrogel is solvent induced?