FTIR and SAXS study on MBCO-saccharide amorphous systems: protein-matrix reciprocal effects

Probing light-induced conformational transitions in bacterial photosynthetic reaction centers embedded in trehalose-water amorphous matrices.

Abstract The coupling between electron transfer and protein dynamics has been studied in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides by embedding the protein into room temperature solid trehalose–water matrices. Electron transfer kinetics from the primary quinone acceptor (Q A − ) to the photoxidized donor (P + ) were measured as a function of the duration of photoexcitation from 20 ns (laser flash) to more than 1 min. Decreasing the water content of the matrix down to ≈5×10 3 water molecules per RC causes a reversible four-times acceleration of P + Q A − recombination after the laser pulse. By comparing the broadly distributed kinetics observed under these conditions …

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…

Inhomogeneous Protein-Water-Sugar Structures of Single GFP-mut2 Proteins Caged in Trehalose-Water Matrixes

Bioprotection Can Be Tuned with Proper Protein/Saccharide Ratio: The Case of Solid Amorphous Matrices

Saccharides, and in particular trehalose, are well known for their high efficiency in protecting biostructures against adverse environmental conditions. The protein dynamics is known to be highly inhibited in a low-water trehalose host medium, the inhibition being markedly dependent on the amount of residual water. Besides hydration, the protein/sugar ratio is expected to affect the properties of saccharide amorphous matrices. In this work, we report an infrared spectroscopy study in dry amorphous matrices of various sugars (the disaccharides trehalose, maltose, sucrose, and lactose, and the trisaccharide raffinose) containing myoglobin, at different protein/sugar ratios. We analyze the str…

Single Molecule Study of GFP-mut2 Proteins Caged in Trehalose-Water Matrixes: Spatially Inhomogeneous Protein-Water-Sugar Structures

Interconversion among low tier substates in MbCO: an FTIR, Neutron Scattering and Molecular Dynamics simulation study

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.

Trehalose-hydroxyethylcellulose microspheres containing vancomycin for topical drug delivery.

Abstract A new formulation, in which vancomycin is entrapped into trehalose and hydroxyethylcellulose (Natrosol ® ) spherical matrices, is described. Microspheres were produced by the solvent evaporation method. The entrapped drug was fully recovered following microspheres dissolution. Differential scanning calorimetry analyses proved that Natrosol maintains trehalose in its amorphous form. The stabilizing effects of trehalose on vancomycin were evaluated even after long storage and heating of microspheres. Calorimetric data indicated no decomposition of the entrapped drug. In vitro drug release, already performed by using a general two-compartment linear time-invariant open model, suggests…

Internal dynamics and protein-matrix coupling in trehalose-coated proteins.

Abstract We review recent studies on the role played by non-liquid, water-containing matrices on the dynamics and structure of embedded proteins. Two proteins were studied, in water–trehalose matrices: a water-soluble protein (carboxy derivative of horse heart myoglobin) and a membrane protein (reaction centre from Rhodobacter sphaeroides ). Several experimental techniques were used: Mossbauer spectroscopy, elastic neutron scattering, FTIR spectroscopy, CO recombination after flash photolysis in carboxy-myoglobin, kinetic optical absorption spectroscopy following pulsed and continuous photoexcitation in Q B containing or Q B deprived reaction centre from R. sphaeroides . Experimental result…

Structure−Dynamics Coupling between Protein and External Matrix in Sucrose-Coated and in Trehalose-Coated MbCO:  An FTIR Study

We performed FTIR measurements on carboxy-myoglobin (MbCO) embedded in a sucrose−water matrix to study the degrees of freedom coupling between protein and external matrix in such a system. The work was undertaken on the light of recent results by Giuffrida et al. (J. Phys. Chem. B 2003, 107, 13211−13217), who evidenced, in trehalose-coated MbCO, a structured water−sugar environment of the protein, tightly coupled to the heme pocket structure. Such information was obtained through a suitable analysis of the temperature dependence of the CO stretching and of the water association bands in samples of different content of residual water. We applied here the same analysis to sucrose-coated MbCO.…

Internal Dynamics and Protein-Matrix Coupling in Saccharide Coated Proteins

The Role of Solvent on Protein-Matrix Coupling in MbCO Embedded in Water-Saccharide Systems

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…

STRUCTURE-DYNAMICS COUPLING BETWEEN PROTEIN AND EXTERNAL MATRIX IN MBCO EMBEDDED IN VARIOUS SACCHARIDE MATRICES: A FTIR STUDY

BIOPRESERVATION OF MODEL PROTEINS EMBEDDED IN MALTODEXTRINS- AND TREHALOSILDEXTRINS-WATER MATRICES

Elastic neutron scattering of dry and rehydrated trehalose coated carboxy-myoglobin

We report here a comparison between the hydrogen atoms mean square displacements measured by elastic neutron scattering on trehalose coated carboxy-myoglobin, at ILL on the backscattering spectrometers IN13 and IN16. An inconsistency is observed when comparing the mean square displacements measured on the two spectrometer, on samples of identical composition, since they resulted of larger amplitude on IN13 (either in condition of drought or after overnight rehydration under 75% D2O atmosphere), notwithstanding the lower time window accessible on this instrument with respect to IN16. Such inconsistency disappears when the data obtained on this last spectrometer are analyzed in two separate r…

Role of residual water hydrogen bonding in sugar/water/biomolecule systems: a possible explanation fortrehalose peculiarity

We report on the set of experimental and simulative evidences which enabled us to suggest how biological structures embedded in a non-liquid water–saccharide solvent are anchored to the surrounding matrix via a hydrogen bond network. Such a network, whose rigidity increases by decreasing the sample water content, couples the degrees of freedom of the biostructure to those of the matrix and gives place to protein–saccharide–water structures (protein–solvent conformational substates). In particular, the whole set of data evidences that, while the protein–sugar interaction is well described in terms of a water entrapment hypothesis, the water replacement hypothesis better describes the sugar–m…

Light-Induced Protein-Matrix Uncoupling and Protein Relaxation in Dry Samples of Trehalose-Coated MbCO at Room Temperature

In humid samples of trehalose-coated carboxy-myoglobin (MbCO), thermally driven conformational relaxation takes place after photodissociation of the carbon monoxide (CO) molecule at room temperature. In such samples, because of the extreme viscosity of the external matrix, photodissociated CO cannot diffuse out of the protein and explores the whole (proximal and distal side) heme pocket, experiencing averaged protein heme pocket structures, as a result of the presence of Brownian motions. At variance, in very dry samples, a lower portion of the photodissociated CO diffuses from the distal to the proximal heme pocket side probing in nonaveraged structures. We revisit here the flash photolysi…

Lipid Phase Transition in Saccharide-Coated Cholate-Containing Liposomes: Coupling to the Surrounding Matrix

The water association band as a marker of hydrogen bonds in trehalose amorphous matrices

The relevant role played by residual water in modulating the dynamics and structure of a protein, a matrix and their coupling has been thoroughly studied in bioprotective amorphous saccharide matrices via experiments and simulations. In order to better characterize this residual water and the hydrogen bond structures in which it is involved, in this work infrared spectroscopy experiments are conducted on trehalose-water systems. The properties of water are inferred from the study of a peculiar infrared band, the water association band, which we exploited as a marker of the hydrogen bonds in which water is involved. Our aim was the identification of populations of water molecules, which give…

Proteins in amorphous saccharide matrices: Structural and dynamical insights on bioprotection

Bioprotection by sugars, and in particular trehalose peculiarity, is a relevant topic due to the implications in several fields. The underlying mechanisms are not yet clearly elucidated, and remain the focus of current investigations. Here we revisit data obtained at our lab on binary sugar/water and ternary protein/sugar/water systems, in wide ranges of water content and temperature, in the light of the current literature. The data here discussed come from complementary techniques (Infrared Spectroscopy, Molecular Dynamics simulations, Small Angle X-ray Scattering and Calorimetry), which provided a consistent description of the bioprotection by sugars from the atomistic to the macroscopic …

Protein-Matrix Coupling/Uncoupling in “Dry” Systems of Photosynthetic Reaction Centre Embedded in Trehalose/Sucrose: The Origin of Trehalose Peculiarity

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 …

Lipid phase transition in saccharide-coated cholate-containing liposomes: coupling to the surrounding matrix.

We performed FTIR measurements on cholate-containing liposomes (CCL) embedded in saccharide (trehalose or sucrose) matrixes with different contents of residual water. We obtained information on the CCL phase transition following the thermal evolution (310-70 K) of the IR spectrum of the carbonyl moieties of phospholipids in the frequency range 4225-4550 cm(-1). Furthermore, we simultaneously followed the thermal evolution of the water association band, which gave information on the behavior of the surrounding water-saccharide matrix. The analysis revealed a small sub-band of the water association band present in CCL but not in cholate-free liposomes, the thermal evolution of which is tightl…

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

Protein-solvent coupling in carboxy-myoglobin/sugar/water systems by molecular dynamics simulations

Protein-solvent coupling in carboxy-myoglobin--sugar-water systems by Molecular Dynamics Simulation

Molecular Dynamics simulation of carboxy-myoglobin in trehalose and sucrose-water systems

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…

Light Induced Protein Matrix Decoupling and Protein Relaxation in Dry Samples of Trehalose Coated MbCO at Room Temperature

Dehydration and crystallization of trehalose and sucrose glasses containing carbonmonoxy-myoglobin

We report a study wherein we contemporarily measured 1) the dehydration process of trehalose or sucrose glasses embedding carbonmonoxy-myoglobin (MbCO) and 2) the evolution of the A substates in saccharide-coated MbCO. Our results indicate that microcrystallization processes, sizeably different in the two saccharides, take place during dehydration; moreover, the microcrystalline structure is maintained unless the dry samples are equilibrated with a humidity >/=75% (>/=60%) at 25 degrees C for the trehalose (sucrose) sample. The evolution of the parameters that characterize the A substates of MbCO indicates that 1) the effects of water withdrawal are analogous in samples dried in the presenc…

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

GFP-mut2 proteins in trehalose-water matrixes: spatially heterogeneous protein-water-sugar structure

A reduction of protein specific motions in co-ligated myoglobin embedded in a trehalose glass

Coupling between the Thermal Evolution of the Heme Pocket and the External Matrix Structure in Trehalose Coated Carboxymyoglobin

Proteins can assume a very large number of conformations (conformational substates), all concurring to its function. We present experimental evidence for the existence, in trehalose coated carboxymyoglobin, of a structured environment of the protein, tightly coupled to the heme pocket structure, as experienced by the bound CO molecule. This was evidenced by the strict correlation observed between the thermal evolution (300−20 K) of the CO stretching and of the water association bands in samples of carboxymyoglobin embedded in trehalose matrixes of different hydration. This observation put forward the coupling between the degrees of freedom of the matrix and those of the protein. In the drie…

FTIR AS A TOOL FOR STUDYING PROTEIN-SOLVENT COUPLING AND COMPLEMENT NEUTRON SCATTERING DATAA

Harmonic behavior of trehalose-coated carbon-monoxy-myoglobin at high temperature.

Abstract Embedding biostructures in saccharide glasses protects them against extreme dehydration and/or exposure to very high temperature. Among the saccharides, trehalose appears to be the most effective bioprotectant. In this paper we report on the low-frequency dynamics of carbon monoxy myoglobin in an extremely dry trehalose glass measured by neutron spectroscopy. Under these conditions, the mean square displacements and the density of state function are those of a harmonic solid, up to room temperature, in contrast to D 2 O-hydrated myoglobin, in which a dynamical transition to a nonharmonic regime has been observed at ∼180K (Doster et al., 1989. Nature. 337:754–756). The protective ef…

MOLECULAR DYNAMICS SIMULATIONS COMPLEMENT EXPERIMENTAL DATA ON PROTEIN EMBEDDED IN SUGAR-WATER SYSTEMS

Atomic mean square displacements in proteins by Molecular Dynamics: a case for analysis of variance

Structure-dynamics-function of proteins: Molecular Dynamics simulations of myoglobin in water and complex solvents

Thermal evolution of heme pocket structure in trehalose coated carboxy-myoglobin probed by FTIR measurements

BSA Aggregation in Trehalose-Water Systems

Introduction Recent studies [1] on ternary protein-trehalose-water samples have shown that the protein thermal denaturation temperature is linearly correlated with the glass transition temperature of the system, despite the quite large temperature difference between the two processes. In such studies it is stated that the collective, long range properties of the matrix that regulate the glass transition are strictly correlated with local features on which the denaturation of the protein depends. In order to ascertain whether an analogous correlation exists between the effects of trehalose on the protein’s aggregation process and the glass transition temperature of the system, we performed L…

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

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

Single molecule investigation of Trehalose coated proteins

Spatially heterogeneous protein-solvent coupling in non-liquid, water trehalose systems

Silk-Water: a dynamic duo

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

MbCo in Saccharide Solid Amorphous Systems: A Combined FTIR and SAXS Study

Saccharides, and in particular trehalose, are known for their efficiency in protecting biostructures against environmental stress [1], although the preservation mechanism is still debated. Experiments and simulations [2,3] on carboxy-myoglobin (MbCO) showed that the protein dynamics is highly inhibited in dry trehalose matrices, the inhibition being dependent on the water content. In these conditions, a mutual protein-matrix structural and dynamic influence is observed.Here we report a combined FTIR and SAXS study on MbCO embedded in dry amorphous matrices of trehalose and sucrose. FTIR measurements were performed at different protein/sugar ratios, focussing on the stretching band of the bo…

Water effects on trehalose matrices studied through Molecular Dynamics

MAPPING THE FREE ENERGY LANDSCAPE OF CO DIFFUSION IN MYOGLOBIN

Myoglobin embedded in saccharide amorphous matrices: water-dependent domains evidenced by small angle X-ray scattering

We report Small Angle X-ray Scattering (SAXS) measurements performed on samples of carboxy-myoglobin (MbCO) embedded in low-water trehalose glasses. Results showed that, in such samples, "low-protein" trehalose-water domains are present, surrounded by a protein-trehalose-water background; such finding is supported by Infrared Spectroscopy (FTIR) measurements. These domains, which do not appear in the absence of the protein and in analogous sucrose systems, preferentially incorporate the incoming water at the onset of rehydration, and disappear following large hydration. This observation suggests that, in organisms under anhydrobiosis, analogous domains could play a buffering role against th…

MbCO↔matrix reciprocal effects in low hydration amorphous saccharide systems: a FTIR study

Role of Solvent on Protein-Matrix Coupling in MbCO Embedded in Water-Saccharide Systems: A Fourier Transform Infrared Spectroscopy Study

AbstractEmbedding protein in sugar systems of low water content enables one to investigate the protein dynamic-structure function in matrixes whose rigidity is modulated by varying the content of residual water. Accordingly, studying the dynamics and structure thermal evolution of a protein in sugar systems of different hydration constitutes a tool for disentangling solvent rigidity from temperature effects. Furthermore, studies performed using different sugars may give information on how the detailed composition of the surrounding solvent affects the internal protein dynamics and structural evolution. In this work, we compare Fourier transform infrared spectroscopy measurements (300–20K) o…

Molecular Dynamics simulation of carboxy-myoglobin in trehalose and sucrose-water systems

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…

Water association band : anuseful tool for studying the water structure in samples of low water content

Sugar-induced stabilization of the monoolein Pn3m bicontinuous cubic phase during dehydration

To explore the molecular mechanism of the protective function of sugars on cubic lipidic systems, the mesomorphic properties of the monoolein-water system, dehydrated in the presence of a series of sugars, have been studied by osmotic stress experiments. Two bicontinuous inverse cubic structures $(Pn3m$ and $\mathrm{Ia}3d)$ and a lamellar ${L}_{\ensuremath{\alpha}}$ phase form under dehydration in pure water. In sugar solutions, the $\mathrm{Pn}3m$ phase shows an extraordinary stability: as a function of sugar concentration, the lattice parameter decreases to very low values, but no phase transitions occur. Instead, the $\mathrm{Pn}3m$ to $\mathrm{Ia}3d$ phase transition is obtained by equi…

Protein-solvent coupling in myoglobin-sugar-water systems: a Molecular Dynamics study

Water Association Band as marker of the matrix structure in amorphous saccharide and saccharide–protein samples

Effects of Disaccarides On Thermal Aggregation of Bovine Serum Albumin.

Thermal Behavior of dry and hydrated MbCO crowded systems

Protein-Matrix Coupling in MbCO embedded in saccharide matrices

IN13 vs. IN16 measurements on protein-trehalose samples: A puzzle.

Trehalose-induced local distorsions in iron proteins

Biopreservation of model proteins embedded in maltodextrins- and trehalosildextrins–water matrices

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 coupling between electron transfer and protein dynamics in the bacterial photosynthetic reaction center: trapping of conformational substates in room temperature amorphous matrices

The photosynthetic reaction center (RC) from purple bacteria is becoming a prototype in exploring the coupling between internal protein motions and long-range electron transfer (ET). This interplay has been extensively investigated by hampering RC substate interconversion and relaxations at low temperatures (McMahon et al., 1998, Biophys. J. 74, 2567). As a complementary approach for limiting RC dynamics at room temperature we embedded the protein within a dehydrated trehalose matrix. Spectroscopic studies and molecular dynamics simulations performed on myoglobin/trehalose/water systems have shown that the nonharmonic contributions to the protein motions (attributed to thermal fluctuations …

Protein-solvent coupling in carboxymyoglobin/sugar/water systems by molecular dynamics simulation

Thermal aggregation of BSA in trehalose or saccharose solutions

Protein-solvent coupling in carboxy-myoglobin-sugar-water systems Molecular Dynamics Simulations and Experiments

Hydrogen bonding properties of amorphous water-saccharide matrixes and bioprotection

Disaccarides effects on thermal aggregation of Bovine Serum Albumin

Structure and Dynamics in Glassy and Plasticized Amorphous Disaccharide-Water Samples: A FTIR Study

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…

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 …

Thermal evolution of the CO stretching band in carboxy-myoglobin in the light of neutron scattering and molecular dynamics simulations

Abstract As it is well known, the thermal behaviour of the CO stretching band in MbCO reflects the interconversion among protein’s taxonomic and lower tier substates. We compare here FTIR data on the thermal behaviour of the CO stretching band in MbCO embedded in non-liquid, water–trehalose matrixes, and neutron scattering data on dry and hydrated proteins and nucleic acids. The comparison, also in the light of simulative data, gives relevant information on the relationship between the mean square displacements of hydrogen atoms and the heme pocket thermal rearrangements in MbCO, as experienced by the bound CO, in the temperature region 100–200 K, and at higher temperature when large scale …

Hydrogen bonding properties of amorphous low-water protein-saccharide matrixes and bioprotection

Ocular gelling microspheres: in vitro precorneal retention time and drug permeation through reconstituted corneal epithelium.

Purpose: The model drug norfloxacin (NOR)was encapsulated into trehalose (TRH) and hydroxyethylcellulose(NAT) microspheres to obtain a novel gelling ophthalmic delivery system for prolonged release on corneal tissue. Methods: We assessed NOR release from microspheres, prepared by the emulsion-solvent evaporation method. A new in vitro tear turnover model, including inserts containing reconstituted human corneal epithelium (RHC), was designed to evaluate the TRH/NAT microspheres’ precorneal retention time. Bioadhesive properties of TRH/NAT microspheres were validated by using drug-loaded microspheres prepared with gelatine (GLT) commonly used as reference material in adhesion studies. Result…

SAXS and FTIR study on MbCO-Saccharide amorphous systems

A FTIR study on low hydration saccharide amorphous matrices: Thermal behaviour of the Water Association Band

Abstract We report a study on the thermal behaviour of the infrared Water Association Band (WAB) in dry binary saccharide–water systems (containing trehalose, sucrose, maltose, and raffinose). This is a follow-up of preceding studies on analogous carboxymyoglobin-saccharide–water ternary systems, which pointed out a mutual protein-matrix influence (coupling). A comparison between binary and ternary systems, for all the saccharides studied, evidences a reduction in the residual water content in the latter and, except for trehalose, a sizable modification in the thermal behaviour, which is discussed in terms of structure and hydrogen bonding properties of the sugars. The study allowed us also…

Stabilization of the monoolein Pn 3 m cubic structure on trehalose glasses

Trehalose is known to protect some organisms from various stresses due to drought and high temperature. To explore the molecular mechanism of the protective function, the mesomorphic properties of the monoolein-water system, dried in the presence of trehalose, were studied by X-ray diffraction. While, in pure water, two bicontinuous inverse cubic structures (the Pn3m and Ia3d phases) and a lamellar Lα phase exist as a function of concentration, only the Pn3m cubic phase has been detected in concentrated trehalose solutions or in trehalose glasses, even under extremely dry conditions. Depending on the sugar concentration, or after glass dehydration, the Pn3m cubic unit cell decreases to very…

GFP-mut2 Proteins in Trehalose-Water Matrixes: Spatially Heterogeneous Protein-Water-Sugar Structures

We report investigations on the properties of nanoenvironments around single-GFP-mut2 proteins in trehalose-water matrixes. Single-GFPmut2 molecules embedded in thin trehalose-water films were characterized in terms of their fluorescence brightness, bleaching dynamics, excited state lifetime, and fluorescence polarization. For each property, sets of approximately 100-150 single molecules have been investigated as a function of trehalose content and hydration. Three distinct and interconverting families of proteins have been found which differ widely in terms of bleaching dynamics, brightness, and fluorescence polarization, whose relative populations sizably depend on sample hydration. The r…

Cytochrome c in a Dry Trehalose Matrix: Structural and Dynamical Effects Probed by X-Ray Absorption Spectroscopy

AbstractWe report on the structure and dynamics of the Fe ligand cluster of reduced horse heart cytochrome c in solution, in a dried polyvinyl alcohol (PVA) film, and in two trehalose matrices characterized by different contents of residual water. The effect of the solvent/matrix environment was studied at room temperature using Fe K-edge x-ray absorption fine structure (XAFS) spectroscopy. XAFS data were analyzed by combining ab initio simulations and multi-parameter fitting in an attempt to disentangle structural from disorder parameters. Essentially the same structural and disorder parameters account adequately for the XAFS spectra measured in solution, both in the absence and in the pre…

Molecular dynamics simulation of carboxy-myoglobin embedded in a trehalose-water matrix

AbstractWe report on a molecular dynamics (MD) simulation of carboxy-myoglobin (MbCO) embedded in a water-trehalose system. The mean square fluctuations of protein atoms, calculated at different temperatures in the 100–300K range, are compared with those from a previous MD simulation on an H2O-solvated MbCO and with experimental data from Mössbauer spectroscopy and incoherent elastic neutron scattering on trehalose-coated MbCO. The results show that, for almost all the atomic classes, the amplitude of the nonharmonic motions stemming from the interconversion among the protein’s conformational substates is reduced with respect to the H2O-solvated system, and their onset is shifted toward hig…

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

Molecular dynamics simulation of sucrose- and trehalose-coated carboxy-myoglobin

We performed a room temperature molecular dynamics (MD) simulation on a system containing 1 carboxy-myoglobin (MbCO) molecule in a sucrose–water matrix of identical composition (89% [sucrose/(sucrose + water)] w/w) as for a previous trehalose–water–MbCO simulation (Cottone et al., Biophys J 2001;80:931–938). Results show that, as for trehalose, the amplitude of protein atomic mean-square fluctuations, on the nanosecond timescale, is reduced with respect to aqueous solutions also in sucrose. A detailed comparison as a function of residue number evidences mobility differences along the protein backbone, which can be related to a different efficacy in bioprotection. Different heme pocket struc…

FTIR Study on Reciprocal Protein↔Matrix effects in dry amorphous saccharide systems

Role of Solvent on Protein-Matrix Coupling in MbCO Embedded in Water-Saccharide Systems: an FTIR study.

Embedding protein in sugar systems of low water content enables one to investigate the protein dynamicstructure function in matrixes whose rigidity is modulated by varying the content of residual water. Accordingly, studying the dynamics and structure thermal evolution of a protein in sugar systems of different hydration constitutes a tool for disentangling solvent rigidity from temperature effects. Furthermore, studies performed using different sugars may give information on how the detailed composition of the surrounding solvent affects the internal protein dynamics and structural evolution. In this work, we compare Fourier transform infrared spectroscopy measurements (300–20 K) on MbCO e…

Water association band as a marker of the matrix structure in amorphous saccharide and saccharide-protein samples

Different degrees in protein thermal aggregation processes in presence of trealose

MbCO embedded in trehalosyldextrin matrices: thermal effects and protein-matrix coupling

Saccharide-based biopreservation is widely studied because of its scientific importance and possible technological outcomes for food and pharmaceutical industries. Ternary protein/saccharide/water systems have been extensively exploited to model the characteristics of the in vivo biopreservation process. A tight, water dependent, protein–matrix coupling has been shown to occur in various simple saccharide amorphous matrices, which is stronger in trehalose. The efficiency as bioprotectant of trehalose has been ascribed to this tight coupling, since the appearance of damages on biological structures will more involve structural variations of the surrounding matrix. Here we present, as an appl…

Local dynamics of DNA probed with optical absorption spectroscopy of bound ethidium bromide

We have studied the local dynamics of calf thymus double-helical DNA by means of an "optical labeling" technique. The study has been performed by measuring the visible absorption band of the cationic dye ethidium bromide, both free in solution and bound to DNA, in the temperature interval 360-30 K and in two different solvent conditions. The temperature dependence of the absorption line shape has been analyzed within the framework of the vibronic coupling theory, to extract information on the dynamic properties of the system; comparison of the thermal behavior of the absorption band of free and DNA-bound ethidium bromide gave information on the local dynamics of the double helix in the prox…

Thermal Aggregation of Bovine Serum Albumin in Trehalose and Sucrose Aqueous Solutions

We report results of static and dynamic light scattering measurements performed on bovine serum albumin (BSA) in saccharide (trehalose and sucrose) solutions. Our aim is to study the effects of the two disaccharides on the first steps of thermal aggregation of BSA in aqueous solutions at two protein concentrations (1 and 30 mg/mL) at increasing sugar/water ratio. Results show that sugars modify early stages of aggregation mainly by perturbing the thermodynamic behavior of the solvent (i.e., general solvent effects) without involving direct, specific sugar-protein interactions. This agrees with current hypotheses on sugar action in protein solutions. (1-3) The linear correlation detected bet…

Atomic Mean-Square Displacements in Proteins by Molecular Dynamics: A Case for Analysis of Variance

AbstractInformation on protein internal motions is usually obtained through the analysis of atomic mean-square displacements, which are a measure of variability of the atomic positions distribution functions. We report a statistical approach to analyze molecular dynamics data on these displacements that is based on probability distribution functions. Using a technique inspired by the analysis of variance, we compute unbiased, reliable mean-square displacements of the atoms and analyze them statistically. We applied this procedure to characterize protein thermostability by comparing the results for a thermophilic enzyme and a mesophilic homolog. In agreement with previous experimental observ…