Antiplasticization and local elastic constants in trehalose and glycerol mixtures

We have performed molecular dynamics simulations of glassy trehalose with various amounts of glycerol in order to explore the tendency for glycerol to antiplasticize the glass. We find that below a temperature of 300 K, the average density of the system containing 5%(wt) glycerol is larger than that of the pure trehalose system; the glass transition temperature is decreased, and the elastic constants are essentially unchanged. Taken together, these phenomena are indicative of mild antiplasticization, a type of behavior generally observed in polymeric systems. We have calculated the local elastic constants in our glassy materials and, consistent with previous simulations on a coarse-grained polymer, we find evidence of domains having negative elastic moduli. We have explored the ability of various measures of the Debye-Waller factor u(2) to predict the stiffness of our systems in terms of their elastic constants. We find that u(2) is indeed correlated with the behavior of the bulk elastic constants. On a local level, a correlation exists between the local moduli and u(2); however, that correlation is not strong enough to arrive at conclusive statements about the local elastic properties.     

Glass transition temperature of glucose, sucrose, and trehalose: an experimental and in silico study

Isothermal-isobaric molecular dynamics simulations are used to calculate the specific volume of models of different amorphous carbohydrates (glucose, sucrose, and trehalose) as a function of temperature. Plots of specific volume vs temperature exhibit a characteristic change in slope when the amorphous systems change from the glassy to the rubbery state. The intersection of the regression lines of data below (glassy state) and above (rubbery state) the change in slope provides the glass transition temperature (T(g)). These predicted glass transition temperatures are compared to experimental T(g) values as obtained from differential scanning calorimetry measurements. As expected, the predicted values are systematically higher than the experimental ones (about 12-34 K) as the cooling rates of the modeling methods are about a factor of 10(12) faster. Nevertheless, the calculated trend of T(g) values agrees exactly with the experimental trend: T(g)(glucose) < T(g)(sucrose) < T(g)(trehalose). Furthermore, the relative differences between the glass transition temperatures were also computed precisely, implying that atomistic molecular dynamics simulations can reproduce trends of T(g) values in amorphous carbohydrates with high quality.     

Dynamics of hemoglobin in human erythrocytes and in solution: influence of viscosity studied by ultrafast vibrational echo experiments

Ultrafast spectrally resolved stimulated vibrational echo experiments are used to measure the vibrational dephasing of the CO stretching mode of hemoglobin-CO (HbCO) inside living human erythrocytes (red blood cells), in liquid solutions, and in a glassy matrix. A method is presented to overcome the adverse impact on the vibrational echo signal from the strong light scattering caused by the cells. The results from the cytoplasmic HbCO are compared to experiments on aqueous HbCO samples prepared in different buffers, solutions containing low and high concentrations of glycerol, and in a solid trehalose matrix. Measurements are also presented that provide an accurate determination of the viscosity at the very high Hb concentration that is found inside the cells. It is demonstrated that the dynamics of the protein, as sensed by the CO ligand, are the same inside the erythrocytes and in aqueous solution and are independent of the viscosity. In solutions that are predominantly glycerol, the dynamics are modified somewhat but are still independent of viscosity. The experiments in trehalose give the dynamics at infinite viscosity and are used to separate the viscosity-dependent dynamics from the viscosity-independent dynamics. Although the HbCO dynamics are the same in the red blood cell and in the equivalent aqueous solutions, differences in the absorption spectra show that the distribution of a protein's equilibrium substates is sensitive to small pH differences.     

Restricted orientational motion of nitroxides in molecular glasses: direct estimation of the motional time scale basing on the comparative study of primary and stimulated electron spin echo decays

A comparative study of anisotropic relaxation in two-pulse primary and three-pulse stimulated electron spin echo decays provides a direct way to distinguish fast (correlation time tau(c)<10(-6) s) and slow (tau(c)>10(-6) s) motions. Anisotropic relaxation is detected as a difference of the decay rates for different resonance field positions in anisotropic electron paramagnetic resonance spectra. For fast motion anisotropic relaxation influences the primary echo decay and does not influence the stimulated echo decay. For slow motion it is seen in both two-pulse echo and three-pulse stimulated echo decays. For nitroxide spin probes dissolved in glassy glycerol only fast motion was found below 200 K. Increase of temperature above 200 K results in the appearance of slow motion. Its amplitude increases rapidly with temperature increase. While in glycerol glass slow motion appears above glass transition temperature T(g), in ethanol glass it is observable below T(g). The scenario of motional dynamics in glasses is proposed which involves the broadening of the correlation time distribution with increasing temperature.     

The influence of aqueous versus glassy solvents on protein dynamics: vibrational echo experiments and molecular dynamics simulations

Spectrally resolved infrared stimulated vibrational echo measurements are used to measure the vibrational dephasing of the CO stretching mode of carbonmonoxy-hemoglobin (HbCO), a myoglobin mutant (H64V), and a bacterial cytochrome c(552) mutant (Ht-M61A) in aqueous solution and trehalose glasses. The vibrational dephasing of the heme-bound CO is significantly slower for all three proteins embedded in trehalose glasses compared to that of aqueous protein solutions. All three proteins exhibit persistent but notably slower spectral diffusion when the protein surface is fixed by the glassy solvent. Frequency-frequency correlation functions (FFCFs) of the CO are extracted from the vibrational echo data to reveal that the structural dynamics, as sensed by the CO, of the three proteins in trehalose and aqueous solution are dominated by fast (tens of femtoseconds), motionally narrowed fluctuations. MD simulations of H64V in dynamic and "static" water are presented as models of the aqueous and glassy environments. FFCFs are calculated from the H64V simulations and qualitatively reproduce the important features of the experimentally extracted FFCFs. The suppression of long time scale (picoseconds to tens of picoseconds) frequency fluctuations (spectral diffusion) in the glassy solvent is the result of a damping of atomic displacements throughout the protein structure and is not limited to structural dynamics that occur only at the protein surface. The analysis provides evidence that some dynamics are coupled to the hydration shell of water, supporting the idea that the bioprotection offered by trehalose is due to its ability to immobilize the protein surface through a thin, constrained layer of water.     

Anisotropic pseudorotation of the photoexcited triplet state of fullerene C60 in molecular glasses studied by pulse EPR

Spin-polarized echo-detected electron paramagnetic resonance (EPR) spectra and the transversal relaxation rate T2(-1) of the photoexcited triplet state of fullerene C60 molecules were studied in o-terphenyl, 1-methylnaphthalene, and decalin glassy matrices. The model is composed of a fast (correlation time approximately 10(-12) s) pseudorotation of (3)C60 in a local anisotropic potential created by interaction of the fullerene molecule with the surrounding matrix molecules. In simulations, this potential is assumed to be axially symmetric around some axis of a preferable orientation in a matrix cage. The fitted value of the potential was found to depend on the type of glass and to decrease monotonically with a temperature increase. A sharp increase of the T2(-1) temperature dependence was found near 240 K in glassy o-terphenyl and near 100 K in glassy 1-methylnaphthalene and decalin. This increase probably is related to the influence on the pseudorotation of the onset of large-amplitude vibrational molecular motions (dynamical transition in glass) that are known for glasses from neutron scattering and molecular dynamics studies. The obtained results suggest that molecular and spin dynamics of the triplet fullerene are extremely sensitive to molecular motions in glassy materials.     

Confinement effects on the glass transition of hydrogen bonded liquids

The glass transition behavior of glycerol and propylene glycol confined in nanoporous glass is investigated using differential scanning calorimetry. Both silanized and unsilanized porous glasses are used to confine the liquids with nominal pore sizes ranging from 2.5 to 7.5 nm, and the glass transition temperature (T(g)) and the limiting fictive temperature (T(f )') are measured on cooling and heating, respectively. The effect of pore fullness is also examined. We find that differences in T(g), DeltaC(p), and the enthalpy overshoot behavior observed on heating are significant between partially and completely filled pores for the case of the unsilanized controlled pore glasses (CPGs) but that the effect of pore fullness is insignificant for the silanized CPGs. In general, the behavior in the silanized CPGs is similar to the behavior in the completely filled unsilanized pores. For glycerol, this includes a small depression in T(f )' on the order of 5 K at 2.5 nm. For propylene glycol, similar behavior is found except that an additional glass transition is observed in both silanized and unsilanized systems approximately 30 K higher than the bulk and a slightly smaller depression on the order of 3 K at 2.5 nm is observed in the completely filled unsilanized pores and in partially and completely filled silanized pores. The results are compared to those in the literature, and the confinement effects are discussed.     

Dynamics of glass-forming liquids. XV. Dynamical features of molecular liquids that form ultra-stable glasses by vapor deposition

The dielectric relaxation behavior of ethylbenzene (EBZ) in its viscous regime is measured, and the glass transition temperature (T(g) = 116 K) as well as fragility (m = 98) are determined. While the T(g) of EBZ from this work is consistent with earlier results, the fragility is found much higher than what has been assumed previously. Literature data is supplemented by the present results on EBZ to compile the dynamic behavior of those glass formers that are known to form ultra-stable glasses by vapor deposition. These dynamics are contrasted with those of ethylcyclohexane, a glass former for which a comparable vapor deposition failed to produce an equally stable glassy state. In a graph that linearizes Vogel-Fulcher-Tammann behavior, i.e., the derivative of -logτ with respect to T/T(g) raised to the power of -1/2 versus T/T(g), all ultra-stable glass formers fall onto one master curve in a wide temperature range, while ethylcyclohexane deviates for T ? T(g). This result suggests that ultra-stable glass formers share common behavior regarding the dynamics of their supercooled liquid state if scaled to their respective T(g) values, and that fragility and related features are linked to the ability to form ultra-stable materials.     

Study of dielectric relaxations of anhydrous trehalose and maltose glasses

We investigated the frequency dependent dielectric relaxation behaviors of anhydrous trehalose and maltose glasses in the temperature range which covers a supercooled and glassy states. In addition to the α-, Johari-Goldstein (JG) β-, and γ-relaxations in a typical glass forming system, we observed an extra relaxation process between JG β- and γ-relaxations in the dielectric loss spectra. We found that the unknown extra relaxation is a unique property of disaccharide which might originate from the intramolecular motion of flexible glycosidic bond. We also found that the temperature dependence of the JG β-relaxation time changes at 0.95T(g) and it might be universal.     

Molecular view of the isothermal transformation of a stable glass to a liquid

We have used neutron reflectivity to measure translational motion on the nanometer length scale in exceptionally stable glasses of tris(naphthylbenzene). These glasses are prepared by vapor deposition onto a substrate held somewhat below the glass transition temperature (T(g) = 342 K). When the most stable samples are annealed at 345 K, no translational motion is observed on the 12 nm length scale for over 10,000 s and full mixing requires more than 60,000 s. For comparison, the equilibrium supercooled liquid mixes in 1000 s at this temperature and on this length scale. These measurements provide insight into the mechanism by which a stable glass transforms into a liquid. "Melting" of the stable glass appears to occur by the growth of liquid regions into the surrounding glassy matrix, perhaps by a surface-initiated growth process. At 345 K, translational motion in the stable glass is at least 100 times slower than motion in the supercooled liquid.     

Bulk and interfacial glass transitions of water

Fast scanning calorimetry (FSC) was employed to investigate glass softening dynamics in bulk-like and ultrathin glassy water films. Bulk-like water samples were prepared by vapor-deposition on the surface of a tungsten filament near 140 K where vapor-deposition results in low enthalpy glassy water films. The vapor-deposition approach was also used to grow multiple nanoscale (approximately 50 nm thick) water films alternated with benzene and methanoic films of similar dimensions. When heated from cryogenic temperatures, the ultrathin water films underwent a well manifested glass softening transition at temperatures 20 K below the onset of crystallization. However, no such transition was observed in bulk-like samples prior to their crystallization. These results indicate that thin-film water demonstrates glass softening dynamics that are dramatically distinct from those of the bulk phase. We attribute these differences to water's interfacial glass transition, which occurs at temperatures tens of degrees lower than that in the bulk. Implications of these findings for past studies of glass softening dynamics in various glassy water samples are discussed.     

The trehalose coating effect on the internal protein dynamics

(15)N and (13)C NMR experiments were applied to conduct a comparative study of a cold shock protein (Csp) in two states-lyophilized powder and a protein embedded in a glassy trehalose matrix. Both samples were studied at various levels of rehydration. The experiments used (measuring relaxation rates R(1) and R(1ρ), motionally averaged dipolar couplings and solid state exchange method detecting reorientation of the chemical shift anisotropy tensor) allow obtaining abundant information on the protein structural features and internal motions in a range of correlation times from nanoseconds to seconds. The main results are: (a) the trehalose coating makes the protein structure more native in comparison with the dehydrated lyophilized powder, however, trehalose still cannot remove all non-native hydrogen bonds which are present in a dehydrated protein; (b) trehalose has an appreciable effect on the internal dynamics: the motion of the backbone N-H groups in the nanosecond and microsecond time scales becomes slower while the motional amplitude remains constant; (c) upon adding water to the Csp-trehalose mixture, water molecules accumulate around proteins forming a layer between the protein surface and the trehalose matrix. The protein dynamics become faster, however, not as fast as in the fully hydrated state; (d) the hydration response of dynamics of the NH and CH(CH(2)) groups in a protein is qualitatively different: upon increasing protein hydration, the correlation times of the N-H motions become shorter and the amplitude remains stable, and for CH(CH(2)) groups the motional amplitude increases and the correlation times do not change. This can be explained by a different ability of the NH and CH(CH(2)) groups to form hydrogen bonds.     

Effects of solvent viscosity on ligand interconversion dynamics in the primary docking site of heme proteins

Interconversion dynamics of the ligand in the primary docking site of myoglobin (Mb) and hemoglobin (Hb) in trehalose and glycerol/D2O mixtures at 283 K was investigated by probing time-resolved vibrational spectra of CO photolyzed from these proteins. The interconversion dynamics in viscous media are similar to those in aqueous solution, indicating that it is minimally coupled to the solvent-coupled large-scale protein motion. Interconversion rates in the heme pocket of Hb in water solution are slower than those of Mb in trehalose glass, suggesting that the interconversion barrier in Hb is intrinsically higher than that in Mb and is not modified by the solvent viscosity.     

Coupling between lysozyme and trehalose dynamics: microscopic insights from molecular-dynamics simulations

We have carried out molecular-dynamics simulations on fully flexible all-atom models of the protein lysozyme immersed in trehalose, an effective biopreservative, with the purpose of exploring the nature and extent of the dynamical coupling between them. Our study shows a strong coupling over a wide range of temperatures. We found that the onset of anharmonic behavior was dictated by changes in the dynamics and relaxation processes in the trehalose glass. The physical origin of protein-trehalose coupling was traced to the hydrogen bonds formed at the interface between the protein and the solvent. Moreover, protein-solvent hydrogen bonding was found to control the structural relaxation of the protein. The dynamics of the protein was found to be heterogeneous; the motions of surface and core atoms had different dependencies on temperature and, in addition, the surface atoms were more sensitive to the dynamics of the solvent than the core atoms. From the solvent perspective we found that the dynamics near the protein surface showed an unexpected enhanced mobility compared to the bulk. These results shed some light on the microscopic origins of the dynamical coupling in protein-solvent systems.     

Hiking down the energy landscape: progress toward the Kauzmann temperature via vapor deposition

Physical vapor deposition was employed to prepare amorphous samples of indomethacin and 1,3,5-(tris)naphthylbenzene. By depositing onto substrates held somewhat below the glass transition temperature and varying the deposition rate from 15 to 0.2 nm/s, glasses with low enthalpies and exceptional kinetic stability were prepared. Glasses with fictive temperatures that are as much as 40 K lower than those prepared by cooling the liquid can be made by vapor deposition. As compared to an ordinary glass, the most stable vapor-deposited samples moved about 40% toward the bottom of the potential energy landscape for amorphous materials. These results support the hypothesis that enhanced surface mobility allows stable glass formation by vapor deposition. A comparison of the enthalpy content of vapor-deposited glasses with aged glasses was used to evaluate the difference between bulk and surface dynamics for indomethacin; the dynamics in the top few nanometers of the glass are about 7 orders of magnitude faster than those in the bulk at Tg - 20 K.     

2H NMR studies of glycerol dynamics in protein matrices

We use (2)H NMR spectroscopy to investigate the rotational motion of glycerol molecules in matrices provided by the connective tissue proteins elastin and collagen. Analyzing spin-lattice relaxation, line-shape properties, and stimulated-echo decays, we determine the rates and geometries of the motion as a function of temperature and composition. It is found that embedding glycerol in an elastin matrix leads to a mild slowdown of glycerol reorientation at low temperatures and glycerol concentrations, while the effect vanishes at ambient temperatures or high solvent content. Furthermore, it is observed that the nonexponential character of the rotational correlation functions is much more prominent in the elastin matrix than in the bulk liquid. Results from spin-lattice relaxation and line shape measurements indicate that, in the mixed systems, the strong nonexponentiality is in large part due to the existence of distributions of correlation times, which are broader on the long-time flank and, hence, more symmetric than in the neat system. Stimulated-echo analysis of slow glycerol dynamics reveals that, when elastin is added, the mechanism for the reorientation crosses over from small-angle jump dynamics to large-angle jump dynamics and the geometry of the motion changes from isotropic to anisotropic. The results are discussed against the background of present and previous findings for glycerol and water dynamics in various protein matrices and compared with observations for other dynamically highly asymmetric mixtures so as to ascertain in which way the viscous freezing of a fast component in the matrix of a slow component differs from the glassy slowdown in neat supercooled liquids.     

Chiral studies in amorphous solids: the effect of the polymeric glassy state on the racemization kinetics of bridged paddled binaphthyls

Optical activity, used here for the first time to gain information about the amorphous solid state, allows previously unavailable insight into the dynamic properties of polymer glasses and their effect on a chemical process. This is accomplished by dispersing in polymer glasses atropisomeric bridged binaphthyls with appended oligophenyl paddles of varying sizes and studying the racemization kinetics as a function of temperature. The racemization occurs by a simple one-dimensional twisting motion and, without effect on the intrinsic mechanism, sweeps out a variable volume of the matrix as the paddle length is increased. The racemization is limited by the polymer matrix only for probes with a minimum paddle size and only when the time scale for racemization is comparable to the time scale for segmental motion of the polymer matrix. The high barrier for this racemization is unique in probe studies of glasses and causes these overlapping time scales to occur significantly below the glass transition temperature. These measurements yield a clear quantitative view of the role of segmental dynamics on the racemization kinetics of the binaphthyls and allow the important demonstration, via the transition from first-order to stretched exponential kinetics, that heterogeneous dynamics persist deep within the glassy state.     

A molecular view of vapor deposited glasses

Recently, novel organic glassy materials that exhibit remarkable stability have been prepared by vapor deposition. The thermophysical properties of these new "stable" glasses are equivalent to those that common glasses would exhibit after aging over periods lasting thousands of years. The origin of such enhanced stability has been elusive; in the absence of detailed models, past studies have discussed the formation of new polyamorphs or that of nanocrystals to explain the observed behavior. In this work, an atomistic molecular model of trehalose, a disaccharide of glucose, is used to examine the properties of vapor-deposited stable glasses. Consistent with experiment, the model predicts the formation of stable glasses having a higher density, a lower enthalpy, and higher onset temperatures than those of the corresponding "ordinary" glass formed by quenching the bulk liquid. Simulations reveal that newly formed layers of the growing vapor-deposited film exhibit greater mobility than the remainder of the material, thereby enabling a reorganization of the film as it is grown. They also reveal that "stable" glasses exhibit a distinct layered structure in the direction normal to the substrate that is responsible for their unusual properties.     

Trehalose glass-facilitated thermal reduction of metmyoglobin and methemoglobin

The reduction of ferric derivatives of hemeproteins in solution typically requires moderate to strong reducing agents. Reducing sugars are not adequate to reduce ferric myoglobins or hemoglobins under solution conditions favorable to protein stability. We find that embedding aquo-met derivatives of horse myoglobin and human adult hemoglobin in a glucose-doped glassy matrix derived from trehalose facilitates an efficient thermally initiated reduction that yields a five-coordinate high-spin ferrous heme. The trehalose glass plays a central role by stabilizing the reduction-prone bis-histidine heme (hemichrome) intermediate under the high-temperature conditions that favor the open reducing form of glucose. Due to glass-imposed limitations on conformational reorganization, this process has clear applications in biophysics where it can be used to generate nonequilibrium ferrous derivatives having the initial conformation of the aquo-met derivative. Since the glassy matrix can be redissolved to release the embedded protein, this technique is not only a basis for a relatively benign method of reducing hemoglobin-based blood substitutes that have undergone autoxidation during storage but may also be a way to reactivate stored proteins that have undergone oxidation.     

Determination of the Fe-CO bond energy in myoglobin using heterodyne-detected transient thermal phase grating spectroscopy

The bond energies at active sites of proteins are intimately coupled to the structure-function relationship in biological systems. Due to the unknown nature of the protein relaxation along a reaction coordinate, it has not been possible to directly determine bond energies relevant to protein function. By embedding proteins in trehalose glasses, it is possible to freeze out protein relaxation on short time scales and determine the bond energies of photolabile ligands using photothermal spectroscopies. As a prototypical example, the photodissociation dynamics and energetics of carboxy-myoglobin (MbCO) in a trehalose glass matrix at room temperature were studied by transient absorption (or pump-probe) and transient thermal phase grating spectroscopy to determine the CO recombination dynamics and associated energetics, respectively. Both the initial energetics of the bond breaking and the energy released upon bond reformation could be used, on a time scale faster than significant protein relaxation, to determine the Fe-CO bond energy as 34 +/- 4 kcal/mol. This bond energy is significantly larger than that typically cited (25 kcal/mol) on the basis of indirect measurements but is in good agreement with recent theoretical predictions (35 kcal/mol) (Rovira, C.; Parrinello, M. Int. J. Quantum Chem. 2000, 80, 1172). This result in combination with the theoretical study suggests that protein structure plays a significant role in the bond energies at active sites which in turn provides a tuning element of the effective barrier heights independent to the transition state region.