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ABSTRACT: Biopreservation by saccharides is a widely studied issue due to its scientific and technological importance; in particular, ternary amorphous protein-saccharide-water systems are extensively exploited to model the characteristics of the in vivo biopreservation process. We present here a differential scanning calorimetry (DSC) study on amorphous trehalose-water systems with embedded different proteins (myoglobin, lysozyme, BSA, hemoglobin), which differ for charge, surface, and volume properties. In our study, the protein/trehalose molar ratio is kept constant at 1/40, while the water/sugar molar ratio is varied between 2 and 300; results are compared with those obtained for binary trehalose-water systems. DSC upscans offer the possibility of investigating, in the same measurement, the thermodynamic properties of the matrix (glass transition, T(g)) and the functional properties of the encapsulated protein (thermal denaturation, T(den)). At high-to-intermediate hydration, the presence of the proteins increases the glass transition temperature of the encapsulating matrix. The effect mainly depends on size properties, and it can be ascribed to confinement exerted by the protein on the trehalose-water solvent. Conversely, at low hydration, lower T(g) values are measured in the presence of proteins: the lack of water promotes sugar-protein interactions, thus weakening the confinement effect and softening the matrix with respect to the binary system. A parallel T(den) increase is also observed; remarkably, this stabilization can reach ∼70 K at low hydration, a finding potentially of high biotechnological relevance. A linear relationship between T(g) and T(den) is also observed, in line with previous results; this finding suggests that collective water-trehalose interactions, responsible for the glass transition, also influence the protein denaturation.
The Journal of Physical Chemistry B 05/2011; 115(19):6340-6. · 3.70 Impact Factor
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ABSTRACT: 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 the daily variations of the atmospheric moisture. The reported results are rationalized by assuming sizably different protein-matrix coupling in trehalose with respect to sucrose, analogous to the one suggested for the photosynthetic reaction centre from Rhodobacter sphaeroides (F. Francia et al., J. Am. Chem. Soc., 2008, 130, 10240-10246).
Physical Chemistry Chemical Physics 07/2010; 12(25):6852-8. · 3.57 Impact Factor
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ABSTRACT: 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 varies from very low to very large values, depending on the water content. At a high water/saccharide mole ratio, homogeneous glass formation does not occur; regions of glass form, whose T(g) does not vary by varying the saccharide content, and the disaccharide barely affects the myoglobin denaturation temperature. At a suitably low water/saccharide mole ratio, by lowering the temperature, the systems undergo transition to the glassy state whose T(g) is determined by the water content; the Gordon-Taylor relationship between T(g) and the water/disaccharide mole ratio is obeyed; and T(den) increases by decreasing the hydration regardless of the disaccharide, such effect being entropy-driven. The presence of the protein was found to lower the T(g). Furthermore, for nonreducing disaccharides, plots of T(den) vs T(g) give linear correlations, whereas for reducing disaccharides, data exhibit an erratic behavior below a critical water/disaccharide ratio. We ascribe this behavior to the likelihood that in the latter samples, proteins have undergone Maillard reaction before thermal denaturation.
The Journal of Physical Chemistry B 09/2009; 113(33):11543-9. · 3.70 Impact Factor
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ABSTRACT: Trehalose is a nonreducing disaccharide of glucose found in organisms, which can survive adverse conditions such as extreme drought and high temperatures. Furthermore, isolated structures, as enzymes or liposomes, embedded in trehalose are preserved against stressing conditions [see, e.g., Crowe, L. M. Comp. Biochem. Physiol. A 2002, 131, 505-513]. Among other hypotheses, such protective effect has been suggested to stem, in the case of proteins, from the formation of a water-mediated, hydrogen bond network, which anchors the protein surface to the water-sugar matrix, thus coupling the internal degrees of freedom of the biomolecule to those of the surroundings [Giuffrida, S.; et al. J. Phys. Chem. B 2003, 107, 13211-13217]. Analogous protective effect is also accomplished by other saccharides, although with a lower efficiency. Here, we studied the recombination kinetics of the primary, light-induced charge separated state (P(+)Q(A)(-)) and the thermal stability of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides in trehalose-water and in sucrose-water matrixes of decreasing water content. Our data show that, in sucrose, at variance with trehalose, the system undergoes a "nanophase separation" when the water/sugar mole fraction is lower than the threshold level approximately 0.8. We rationalize this result assuming that the hydrogen bond network, which anchors the RC surface to its surrounding, is formed in trehalose but not in sucrose. We suggest that both the couplings, in the case of trehalose, and the nanophase separation, in the case of sucrose, start at low water content when the components of the system enter in competition for the residual water.
Journal of the American Chemical Society 08/2008; 130(31):10240-6. · 9.91 Impact Factor
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ABSTRACT: 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.
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.
In vitro drug release showed a typical trend of swelling systems. Precorneal retention tests showed that TRH/NAT microspheres maintained fluorescence in tear fluid for 81.7 min, whereas TRH/GLT microspheres and water solution maintained fluorescence for 51.8 and 22.3 min, respectively. NOR released from microspheres permeated throughout RHC slower (J(s) = 23.08 microg/cm(2)h) than NOR from commercial eye drops (J(s) = 42.77 microg/cm(2)h) used as the control.
Adequate drug concentrations in aqueous humor could be prolonged after the administration of TRH/NAT/NOR microspheres. Good bioadhesive properties of the system and slow drug release on corneal surface might increase ocular NOR bioavailability.
Journal of Ocular Pharmacology and Therapeutics 05/2008; 24(2):186-96. · 1.51 Impact Factor
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ABSTRACT: 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 reported results evidence the simultaneous presence of different protein-trehalose-water nanostructures whose rigidity increases by lowering the sample hydration. Such spatial inhomogeneity is in line with the well-known heterogeneous dynamics in supercooled fluids and in nonsolid carbohydrate glasses and gives a pictorial representation of the sharp, sudden reorganization of the above structures after uptake <==>release of water molecules.
Biophysical Journal 07/2007; 93(1):284-93. · 3.65 Impact Factor
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ABSTRACT: We 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 presence of glycerol, and in the PVA film, showing that this polymer interacts weakly with the embedded protein. Instead, incorporation in trehalose leads to severe structural changes, more prominent in the more dried matrix, consisting of 1), an increase up to 0.2 A of the distance between Fe and the imidazole N atom of the coordinating histidine residue and 2), an elongation up to 0.16 A of the distance between Fe and the fourth-shell C atoms of the heme pyrrolic units. These structural distortions are accompanied by a substantial decrease of the relative mean-square displacements of the first ligands. In the extensively dried trehalose matrix, extremely low values of the Debye Waller factors are obtained for the pyrrolic and for the imidazole N atoms. This finding is interpreted as reflecting a drastic hindering in the relative motions of the Fe ligand cluster atoms and an impressive decrease in the static disorder of the local Fe structure. It appears, therefore, that the dried trehalose matrix dramatically perturbs the energy landscape of cytochrome c, giving rise, at the level of local structure, to well-resolved structural distortions and restricting the ensemble of accessible conformational substates.
Biophysical Journal 03/2007; 92(4):1350-60. · 3.65 Impact Factor
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ABSTRACT: Embedding 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 (300-20 K) on MbCO embedded in trehalose, sucrose, maltose, raffinose, and glucose matrixes of different water content. At all the water contents investigated, the protein-solvent coupling was tighter in trehalose than in the other sugars, thus suggesting a molecular basis for the trehalose peculiarity. These results are in line with the observation that protein-matrix phase separation takes place in lysozyme-lactose, whereas it is absent in lysozyme-trehalose systems; indeed, these behaviors may respectively be due to the lack or presence of suitable water-mediated hydrogen-bond networks, which match the protein surface to the surroundings. The above processes might be at the basis of pattern recognition in crowded living systems; indeed, hydration shells structural and dynamic matching is first needed for successful come together of interacting biomolecules.
Biophysical Journal 09/2006; 91(3):968-80. · 3.65 Impact Factor
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ABSTRACT: 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 supporting the applicability of the short-times approximation to the analysis of hemeprotein spectra; in particular, they clearly indicate the presence of spectral heterogeneity in the Soret band of carbon-monoxy myoglobin. The analyses also show that a temperature dependence of the Gaussian width parameter steeper than the hyperbolic cotangent law predicted by the Einstein harmonic oscillator and/or a temperature dependence of inhomogeneous broadening are not sufficient to obtain quantitative information on the magnitude of an-harmonic contributions to the iron-heme plane motion. However, the dependence of the previous two quantities may be used to obtain semiquantitative information on the overall coupling of the Soret transition to the low-frequency modes and therefore on the dynamic properties of the heme pocket in different states of the protein.
European Biophysics Journal 11/2005; 34(7):881-9. · 2.14 Impact Factor
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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 results, together with the outcome of molecular dynamics simulations, concurred to give a picture of how water-containing matrices control the internal dynamics of the embedded proteins. This occurs, in particular, via the formation of hydrogen bond networks that anchor the protein surface to the surrounding matrix, whose stiffness increases by lowering the sample water content. In the conclusion section, we also briefly speculate on how the protein-matrix interactions observed in our samples may shed light on the protein-solvent coupling also in liquid aqueous solutions.
Biochimica et Biophysica Acta 07/2005; 1749(2):252-81. · 4.66 Impact Factor
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ABSTRACT: 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 tightly coupled to that of the spectrum of the carbonyl moieties of phospholipids. We suggest that this band arises from water molecules, which are inserted within the lipidic structure, in the region located at the border between the hydrophilic and the hydrophobic moieties of phospholipids in the presence of cholic acid. Such water molecules could be responsible for the peculiar flexibility and hydrophilicity of CCL. Following Giuffrida et al. (J. Phys. Chem. B 2003, 107, 13211-13217), we also performed a Spectra Distance analysis, which enabled us to detect an overall liposomes-matrix structural coupling.
Langmuir 05/2005; 21(9):4108-16. · 4.19 Impact Factor
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ABSTRACT: 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 photolysis data by Librizzi et al. (2002) and report on new, room temperature experiments in MbCO-trehalose samples, shortly illuminated prior the laser pulse. In dry samples, pre-illumination increased the diffusion of CO from the distal to the proximal heme pocket side, which resulted in less structure than in non-pre-illuminated samples. Such an effect, which is absent in humid samples, stems from a decoupling of the protein internal degrees of freedom from those of the external water-sugar matrix. We suggest that such a decoupling can be brought about by the continuous attempts performed by the protein during pre-illumination to undergo relaxation toward the photodissociated deoxy state. This, in turn, causes a collapse in the hydrogen bond network, which connects the protein surface to the water-sugar matrix, as reported by Cottone et al. (2002) and Giuffrida et al. (2003). In the conclusion section, we discuss the possible involvement of the processes invoked to rationalize the present data, in the function of macromolecules and interactions in living cells.
Cell Biochemistry and Biophysics 02/2005; 43(3):431-7. · 3.74 Impact Factor
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ABSTRACT: 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. Comparison between the results obtained in the two saccharide systems points out the different free-energy landscape experienced by the protein and matrix atoms. This is put forward by the differences in heme pocket structure (shape of the CO stretching band) and in protein environment (shape of the water association band). Furthermore, our data evidence a tighter protein−matrix coupling in trehalose than in sucrose. We suggest this difference to be at the basis for the better efficiency, as biopreservant, of trehalose with respect to sucrose, since the appearance of damages on biological structures will more involve structural variations of the surrounding matrix in the former sugar.
08/2004;
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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 approximately 5x10(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 with the ones measured in glycerol-water mixtures at cryogenic temperatures, we conclude that RC relaxation from the dark-adapted to the light-adapted state and thermal fluctuations among conformational substates are hindered in the room temperature matrix over the time scale of tens of milliseconds. When the duration of photoexcitation is increased from a few milliseconds to the second time scale, recombination kinetics of P(+)Q(A)(-) slows down progressively and becomes less distributed, indicating that even in the driest matrices, during continuous illumination, the RC is gaining a limited conformational freedom that results in partial stabilization of P(+)Q(A)(-). This behavior is consistent with a tight structural and dynamical coupling between the protein surface and the trehalose-water matrix.
Biochimica et Biophysica Acta 08/2004; 1658(1-2):50-7. · 4.66 Impact Factor
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ABSTRACT: Information 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 observations, our analysis suggests that the proteins surface regions can play a role in the different thermal behavior.
Biophysical Journal 06/2004; 86(5):2765-72. · 3.65 Impact Factor
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ABSTRACT: The role of protein dynamics in the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), was studied at room temperature in isolated reaction centers (RC) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in trehalose water systems of different trehalose/water ratios. The effects of dehydration on the reaction kinetics were examined by analyzing charge recombination after different regimes of RC photoexcitation (single laser pulse, double flash, and continuous light) as well as by monitoring flash-induced electrochromic effects in the near infrared spectral region. Independent approaches show that dehydration of RC-containing matrices causes reversible, inhomogeneous inhibition of Q(A)(-)-to-Q(B) electron transfer, involving two subpopulations of RCs. In one of these populations (i.e., active), the electron transfer to Q(B) is slowed but still successfully competing with P(+)Q(A)(-) recombination, even in the driest samples; in the other (i.e., inactive), electron transfer to Q(B) after a laser pulse is hindered, inasmuch as only recombination of the P(+)Q(A)(-) state is observed. Small residual water variations ( approximately 7 wt %) modulate fully the relative fraction of the two populations, with the active one decreasing to zero in the driest samples. Analysis of charge recombination after continuous illumination indicates that, in the inactive subpopulation, the conformational changes that rate-limit electron transfer can be slowed by >4 orders of magnitude. The reported effects are consistent with conformational gating of the reaction and demonstrate that the conformational dynamics controlling electron transfer to Q(B) is strongly enslaved to the structure and dynamics of the surrounding medium. Comparing the effects of dehydration on P(+)Q(A)(-)-->PQ(A) recombination and Q(A)(-)Q(B)-->Q(A)Q(B)(-) electron transfer suggests that conformational changes gating the latter process are distinct from those stabilizing the primary charge-separated state.
Biophysical Journal 11/2003; 85(4):2760-75. · 3.65 Impact Factor
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ABSTRACT: 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 driest sample, in which only tightly bound, nonremovable water molecules were present, temperature induced structural variations of both the heme pocket and the external matrix were small, even at room temperature. At variance, such variations were larger in two water richer samples in which their onset was already at 50 K. Further, the thermal evolution of the CO stretching and of the water association bands showed a single linear correlation for the drier samples in the whole temperature range investigated. The same correlation was observed for the water richest sample up to 180 K, that is, the temperature at which a dynamic transition for the protein motions has been recurrently observed by experimental and computational means, in water containing systems. The data presented enable us to suggest the existence of a rigid water dipole network, which extends throughout the sample, impeding structural heme pocket rearrangements, which imply charge displacement. This in turn brings about the lack of thermally induced variations of the stretching band of the bound CO, which reflects the distribution of taxonomic (A) and lower tier conformational substates. Accordingly, in agreement with previous suggestions, we speculate that, in solution, slaving of the protein internal dynamics to the dynamics of the external solvent is brought about by the “attempts” of the protein structure to match the rapidly evolving structure of the water dipole network.
10/2003;
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ABSTRACT: Some organisms can survive complete dehydration and/or high temperature
in a state of suspended animation called anydrobiosis, in which all
metabolic processes are ``switched off'' however, upon rehydration,
their normal life cycle is restored, without formation of irreversible
damages. A common feature of these organisms, when in anhydrobiosis, is
the presence of large amounts of sugar, particularly trehalose, which
has been found to protect most effectively biomaterials. Several studies
have attempted to understand how trehalose interacts with biomolecules.
To address this problem, we performed molecular dynamics simulations of
carboxy-myoglobin embedded in a trehalose aqueous solution and in a
trehalose-water plasticized amorphous matrix. The results show that, in
an aqueous solution, trehalose is excluded from the protein domain. This
behavior extends also to the trehalose-water plasticized amorphous
matrix, where we find sugar-water-protein structures with more water
molecules that those derived from system concentration, and only few
trehalose molecules bound to the protein, mainly through single hydrogen
bonds.
Chemical Physics 11/2002; 117:9862-9866. · 1.90 Impact Factor
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ABSTRACT: We report on room temperature electron transfer in the reaction center (RC) complex purified from Rhodobacter sphaeroides. The protein was embedded in trehalose-water systems of different trehalose/water ratios. This enabled us to get new insights on the relationship between RC conformational dynamics and long-range electron transfer. In particular, we measured the kinetics of electron transfer from the primary reduced quinone acceptor (Q(A)(-)) to the primary photo oxidized donor (P(+)), by time-resolved absorption spectroscopy, as a function of the matrix composition. The composition was evaluated either by weighing (liquid samples) or by near infrared spectroscopy (highly viscous or solid glasses). Deconvolution of the observed, nonexponential kinetics required a continuous spectrum of rate constants. The average rate constant ( = 8.7 s(-1) in a 28% (w/w) trehalose solution) increases smoothly by increasing the trehalose/water ratio. In solid glasses, at trehalose/water ratios > or = 97%, an abrupt increase is observed ( = 26.6 s(-1) in the driest solid sample). A dramatic broadening of the rate distribution function parallels the above sudden increase. Both effects fully revert upon rehydration of the glass. We compared the kinetics observed at room temperature in extensively dried water-trehalose matrices with the ones measured in glycerol-water mixtures at cryogenic temperatures and conclude that, in solid trehalose-water glasses, the thermal fluctuations among conformational substates are inhibited. This was inferred from the large broadening of the rate constant distribution for electron transfer obtained in solid glasses, which was due to the free energy distribution barriers having become quasi static. Accordingly, the RC relaxation from dark-adapted to light-adapted conformation, which follows primary charge separation at room temperature, is progressively hindered over the time scale of P(+)Q(A)(-) charge recombination, upon decreasing the water content. In solid trehalose-water glasses the electron transfer process resulted much more affected than in RC dried in the absence of sugar. This indicated a larger hindering of the internal dynamics in trehalose-coated RC, notwithstanding the larger amount of residual water present in comparison with samples dried in the absence of sugar.
Biophysical Journal 02/2002; 82(2):558-68. · 3.65 Impact Factor
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ABSTRACT: We report on dynamic properties of carbon monoxy myoglobin (MbCO) embedded in trehalose matrices of different water content, studied by FTIR spectroscopy and CO rebinding after flash-photolysis. FTIR spectroscopy was used to study the thermal behavior of the bound CO stretching and of the adjacent bands arising from trehalose and residual water, as a function of the sample water content. These measurements enabled us to get information on the relation between the interconversion among A substates (as evidenced by the thermal behavior of the CO stretching band) and the dynamics of the trehalose-water matrix. Under condition of drought, the protein internal dynamics is tightly coupled to the dynamics of the external matrix and is modulated by traces of residual water. Under such condition, substates interconversion is hindered due to extreme increase of energy barriers. At variance sizeable substates interconversion takes place following a small water uptake, obtained by exposure of the dry sample to a nondry surrounding atmosphere. FTIR results were in full agreement with flash-photolysis data. In particular, the heme pocket dynamics, which regulates the migration of the flashed off CO molecule within the protein matrix, can be well described on the basis of the information obtained by FTIR measurements. Under condition of drought, in which vanishing temperature dependence of the CO stretching band is observed, the rebinding kinetics is governed by quasi-static rate constant distributions. At variance, stretched exponentials describe geminate rebinding following slight water uptake by the sample, which, as shown by FTIR, brings about interconversion among conformational A substates. © 2002 American Institute of Physics.
The Journal of Chemical Physics. 01/2002; 116(3):1193-1200.
