Effect of conformational states on protein dynamical transition

In order to examine the properties specific to the folded protein, the effect of the conformational states on protein dynamical transition was studied by incoherent elastic neutron scattering for both wild type and a deletion mutant of staphylococcal nuclease. The deletion mutant of SNase which lacks C-terminal 13 residues takes a compact denatured structure, and can be regarded as a model of intrinsic unstructured protein. Incoherent elastic neutron scattering experiments were carried out at various temperature between 10 K and 300 K on IN10 and IN13 installed at ILL. Temperature dependence of mean-square displacements was obtained by the q-dependence of elastic scattering intensity. The measurements were performed on dried and hydrated powder samples. No significant differences were observed between wild type and the mutant for the hydrated samples, while significant differences were observed for the dried samples. A dynamical transition at ∼ 140 K observed for both dried and hydrated samples. The slopes of the temperature dependence of MSD before transition and after transition are different between wild type and the mutant, indicating the folding induces hardening. The hydration water activates a further transition at ∼ 240 K. The behavior of the temperature dependence of MSD is indistinguishable for wild type and the mutant, indicating that hydration water dynamics dominate the dynamical properties.     

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.     

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T_g′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T_g′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T_g′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T_g′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T_g′ of freeze-drying buffer and temperature during the drying phase.     

The protein-solvent glass transition

The protein dynamical transition and its connection with the liquid-glass transition (GT) of hydration water and aqueous solvents are reviewed. The protein solvation shell exhibits a regular glass transition, characterized by steps in the specific heat and the thermal expansion coefficient at the calorimetric glass temperature T_G ≈ 170 K. It implies that the time scale of the structural α-relaxation has reached the experimental time window of 1–100 s. The protein dynamical transition, identified from elastic neutron scattering experiments by enhanced amplitudes of molecular motions exceeding the vibrational level [1], probes the α-process on a shorter time scale. The corresponding liquid-glass transition occurs at higher temperatures, typically 240 K. The GT is generally associated with diverging viscosities, the freezing of long-range translational diffusion in the supercooled liquid. Due to mutual hydrogen bonding, both, protein- and solvent relaxational degrees of freedom slow down in paralled near the GT. However, the freezing of protein motions, where surface-coupled rotational and librational degrees of freedom are arrested, is better characterized as a rubber-glass transition. In contrast, internal protein modes such as the rotation of side chains are not affected. Moreover, ligand binding experiments with myoglobin in various glass-forming solvents show, that only ligand entry and exit rates depend on the local viscosity near the protein surface, but protein-internal ligand migration is not coupled to the solvent. The GT leads to structural arrest on a macroscopic scale due to the microscopic cage effect on the scale of the intermolecular distance. Mode coupling theory provides a theoretical framework to understand the microcopic nature of the GT even in complex systems. The role of the α- and β-process in the dynamics of protein hydration water is evaluated. The protein-solvent GT is triggered by hydrogen bond fluctuations, which give rise to fast β-processes. High-frequency neutron scattering spectra indicate increasing hydrogen bond braking above T_G.     

Molecular mobility studies on the amorphous state of disaccharides. I—thermally stimulated currents and differential scanning calorimetry

The relaxational processes in amorphous solid gentiobiose and cellobiose are studied by thermally stimulated depolarization currents (TSDC) in the temperature region from 108 K up to 423 K. The slow molecular mobility was characterized in the crystal and in the glassy state. The features of different motional components of the secondary relaxation have been monitored as a function of time as the glass structurally relaxes on aging. It is concluded that some modes of motion of this mobility are aging independent, while others are affected by aging. The value of the steepness index or fragility (T_g-normalized temperature dependence of the relaxation time) was obtained by differential scanning calorimetry (DSC) from the analysis of the scanning rate dependency of T_g.     

Predict the glass transition temperature of glycerol-water binary cryoprotectant by molecular dynamic simulation

Vitrification is proposed to be the best way for the cryopreservation of organs. The glass transition temperature (T_g) of vitrification solutions is a critical parameter of fundamental importance for cryopreservation by vitrification. The instruments that can detect the thermodynamic, mechanical and dielectric changes of a substance may be used to determine the glass transition temperature. T_g is usually measured by using differential scanning calorimetry (DSC). In this study, the T_g of the glycerol-aqueous solution (60%, wt/%) was determined by isothermal-isobaric molecular dynamic simulation (NPT-MD). The software package Discover in Material Studio with the Polymer Consortium Force Field (PCFF) was used for the simulation. The state parameters of heat capacity at constant pressure (C_p), density (ρ), amorphous cell volume (V_cell) and specific volume (V_specific) and radial distribution function (rdf) were obtained by NPT-MD in the temperature range of 90–270 K. These parameters showed a discontinuity at a specific temperature in the plot of state parameter versus temperature. The temperature at the discontinuity is taken as the simulated T_g value for glycerol–water binary solution. The T_g values determined by simulation method were compared with the values in the literatures. The simulation values of T_g (160.06–167.51 K) agree well with the DSC results (163.60–167.10 K) and the DMA results (159.00 K). We drew the conclusion that molecular dynamic simulation (MDS) is a potential method for investigating the glass transition temperature (T_g) of glycerol–water binary cryoprotectants and may be used for other vitrification solutions.     

Slow molecular mobility in the crystalline and amorphous solid states of glucose as studied by Thermally Stimulated Depolarization Currents (TSDC)

Thermally Stimulated Depolarization Currents (TSDC) measurements on α-d-glucose have been carried out in the temperature region from −165 °C (108 K) to 120 °C (393 K). The slow molecular mobility was characterized in the crystalline and in the glassy states, as well as in the glass transition region. The influence of aging on the measured TSDC peaks of the secondary relaxation has been discussed and it was concluded that there are motional modes that are aging independent while others are affected by aging. Important discrepancies were reported in the value of the steepness index or fragility (T_g—normalized temperature dependence of the relaxation time) obtained by different, and well-established, experimental techniques. A careful discussion of the possible origins of these discrepancies is presented.     

Temperature and pressure effects on structural and conformational properties of POPC/SM/cholesterol model raft mixtures—a FT-IR, SAXS, DSC, PPC and Laurdan fluorescence spectroscopy study

We report on the effects of temperature and pressure on the structure, conformation and phase behavior of aqueous dispersions of the model lipid “raft” mixture palmitoyloleoylphosphatidylcholine (POPC)/bovine brain sphingomyelin (SM)/cholesterol (Chol) (1:1:1). We investigated interchain interactions, hydrogen bonding, conformational and structural properties as well as phase transformations of this system using Fourier transform-infrared (FT-IR) spectroscopy, small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), and Laurdan fluorescence spectroscopy. The IR spectral parameters in combination with the scattering patterns from the SAXS measurements were used to detect structural and conformational transformations upon changes of pressure up to 7-9 kbar and temperature in the range from 1 to about 80 °C. The generalized polarization function (GP) values, obtained from the Laurdan fluorescence spectroscopy studies also reveal temperature and pressure dependent phase changes. DSC and PPC were used to detect thermodynamic properties accompanying the temperature-dependent phase changes. In combination with literature fluorescence spectroscopy and microscopy data, a tentative p,T stability diagram of the mixture has been established. The data reveal a broad liquid-order/solid-ordered (l_o + s_o) two-phase coexistence region below 8 ± 2 °C at ambient pressure. With increasing temperature, a l_o + l_d + s_o three-phase region is formed, which extends up to ∼27 °C, where a liquid-ordered/liquid-disordered (l_o + l_d) immiscibility region is formed. Finally, above 48 ± 2 °C, the POPC/SM/Chol (1:1:1) mixture becomes completely fluid-like (liquid-disordered, l_d). With increasing pressure, all phase transition lines shift to higher temperatures. Notably, the l_o + l_d (+s_o) phase coexistence region, mimicking raft-like lateral phase separation in natural membranes, extends over a rather wide temperature range of about 40 °C, and a pressure range, which extends up to about 2 kbar for T = 37 °C. Interestingly, in this pressure range, ceasing of membrane protein function in natural membrane environments has been observed for a variety of systems.     

Glass transition behavior of the vitrification solutions containing propanediol, dimethyl sulfoxide and polyvinyl alcohol

Knowledge of the glass transition behavior of vitrification solutions is important for research and planning of the cryopreservation of biological materials by vitrification. This brief communication shows the analysis for the glass transition and glass stability of the multi-component vitrification solutions containing propanediol (PE), dimethyl sulfoxide (Me₂SO) and polyvinyl alcohol (PVA) by using differential scanning calorimetry (DSC) during the cooling and subsequent warming between 25 and −150 °C. The glass formation of the solutions was enhanced by introduction of PVA. Partial glass formed during cooling and the fractions of free water in the partial glass matrix increased with the increasing of PVA concentration, which caused slight decline of glass transition temperature, T_g. Exothermic peaks of devitrification were delayed and broadened, which may result from the inhibition of ice nucleation or recrystallization of PVA.     

The use of small subcutaneous transponders for quantifying thermal biology and torpor in small mammals

Remote measurements of body temperature (T_b) in animals require implantation of relatively large temperature-sensitive radio-transmitters or data loggers, whereas rectal temperature (T_rec) measurements require handling and therefore may bias the results. We investigated whether ∼0.1 g temperature-sensitive subcutaneously implanted transponders can be reliably used to quantify thermal biology and torpor use in small mammals. We examined (i) the precision of transponder readings as a function of temperature and (ii) whether subcutaneous transponders can be used to remotely record subcutaneous temperature (T_sub). Five adult male dunnarts (Sminthopsis macroura, body mass 24 g) were implanted with subcutaneous transponders to determine T_sub as a function of time and ambient temperature (T_a), and in comparison to thermocouple readings of T_rec. Transponder temperature was highly correlated with water bath temperature (r²=0.96–0.99) over a range of approximately 10.0–40.0 °C. Transponders provided reliable data (±0.6 °C) over the T_sub of 21.4–36.9 °C and could be read from a distance of up to 5 cm. Below 21.4 °C, accuracy was reduced to ±2.8 °C, but individual transponder accuracy varied. Consequently, small subcutaneous transponders are useful to remotely quantify thermal physiology and torpor patterns without having to disturb the animal and disrupt torpor. Even at T_sub<21.4 °C where the accuracy of the temperature readings was reduced, transponders do provide reliable data on whether and when torpor is used.     

Dielectric properties of two diastereoisomers of the arabinose and their equimolar mixture

Dielectric relaxation measurements were performed on two enantiomers, d- and l-arabinose and their equimolar mixture, and compared to dielectric data obtained for d-ribose. d-Arabinose differs from d-ribose by having the opposite configuration at C2. This study reveals that both d- and l- of arabinose exhibit α-relaxation peaks with the same shape for the same α-relaxation time τ_α, and the same steepness index for the T_g-scale T-dependence of τ_α. However, the two isomers have slightly different glass transition temperatures T_g’s, and their secondary γ-relaxation times also differ slightly from the previously observed γ-relaxation in d-ribose at the same temperature. However, when samples of both investigated monosaccharides are annealed at higher temperatures, their glass transition temperatures become nearly identical. This is an effect of the mutarotation process, which leads to the formation of pairs of the enantiomers and accordingly they should have the same physical properties. The width of the α-relaxation of d- and l-arabinose is broader than that of d-ribose, as reflected by the smaller stretch exponent in the Kohlrausch-Williams-Watts function used to fit the data of the former (β_KWW = 0.46 ± 0.01) than the latter (β_KWW = 0.55 ± 0.01). The width of the α-relaxation of racemic mixture of the d- and l-arabinose is slightly broader than that of the pure isomers. While the dielectric loss data of d-ribose in the glassy state at ambient and elevated pressures show an inflexion indicating the presence of the JG β-relaxation, the data of d- and l-arabinose show no such feature for identification of the supposedly universal JG β-relaxation. Nevertheless, on comparing the loss spectra of d-arabinose with that of d-ribose, the presence of the JG β-relaxation in d-arabinose has been rationalized.     

Application of the Kwei equation to model the Tg behavior of binary blends of sugars and salts

Vitrification of sugar-based solutions plays an important role in cryopreservation, lyophilization, and the emerging field of anhydrous preservation. An understanding of the glass transition characteristics of such formulations is essential for determining an appropriate storage temperature to ensure an extended shelf life of vitrified products. To better understand the effect of salts on the glass transition temperature (T_g) of glass-forming sugars, we investigated several data-fitting models (Fox, Gordon–Taylor and Kwei) for sugar–salt formulations using data from the literature, as well as new data generated on blends of trehalose and choline dihydrogen phosphate (CDHP). CDHP has recently been shown to have promise as a stabilizing agent for proteins and DNA. The Kwei equation, which has a specific parameter characterizing intermolecular interactions, provides good fits to the T_g data for sugar–salt blends, and complements other commonly used models that are frequently used to model T_g data.     

Molecular dynamics simulations of a cyclic-DP-240 amylose fragment in a periodic cell: Glass transition temperature and water diffusion

Molecular dynamics simulations using AMB06C, an in-house carbohydrate force field, (NPT ensembles, 1 atm) were carried out on a periodic cell that contained a cyclic 240 glucose residue amylose fragment (c-DP-240) and TIP3P water molecules. Molecular conformation and movement of the amylose fragment and water molecules at different temperatures were examined. The periodic cell volume, density, and potential energy were determined at temperatures above and below the glass transition temperature (T_g) in 25 K increments_. The amorphous cell is constructed through successive dynamic equilibration steps at temperatures above the assumed T_g value and the temperature successively lowered until several temperature points were obtained below T_g. Molecular dynamics simulations were continued for at least 500 ps or until the volume drift stopped and remained constant for several hundred picoseconds. The T_g values were found by noting the discontinuity in slope of the volume (V), potential energy (PE), or density (ρ) versus 1/T. The changes in flexibility and motion of the amylose chain as well as differences in self diffusion coefficients of water molecules are described. The final average T_g value found (316 K) is in agreement with experimental values, i.e. 320 K.     

Near-Infrared Analysis of Hydrogen-Bonding in Glass- and Rubber-State Amorphous Saccharide Solids

Near-infrared (NIR) spectroscopic analysis of noncrystalline polyols and saccharides (e.g., glycerol, sorbitol, maltitol, glucose, sucrose, maltose) was performed at different temperatures (30–80°C) to elucidate the effect of glass transition on molecular interaction. Transmission NIR spectra (4,000–12,000 cm⁻¹) of the liquids and cooled-melt amorphous solids showed broad absorption bands that indicate random configuration of molecules. Heating of the samples decreased an intermolecular hydrogen-bonding OH vibration band intensity (6,200–6,500 cm⁻¹) with a concomitant increase in a free and intramolecular hydrogen-bonding OH group band (6,600–7,100 cm⁻¹). Large reduction of the intermolecular hydrogen-bonding band intensity at temperatures above the glass transition (T _g) of the individual solids should explain the higher molecular mobility and lower viscosity in the rubber state. Mixing of the polyols with a high T _g saccharide (maltose) or an inorganic salt (sodium tetraborate) shifted both the glass transition and the inflection point of the hydrogen-bonding band intensity to higher temperatures. The implications of these results for pharmaceutical formulation design and process monitoring (PAT) are discussed.     

Evidence of α fluctuations in myoglobin's denaturation in the high temperature region: Average relaxation time from an Adam–Gibbs perspective

In this work, we derive an analytical expression for the relaxation time τ as a function of temperature T for myoglobin protein (Mb, PDB:1MBN) in the high temperature limit (T > T_g = 200 K). The method is based on a modified version of the Adam–Gibbs theory (AG theory) for the glass transition in supercooled liquids and an implementation of differential geometry techniques. This modified version of the AG theory takes into account that the entropic component in protein's denaturation has two major sources: a configurational contribution ΔS_c due to the unfolding of the highly ordered native state N and a hydration contribution ΔS^hyd arising from the exposure of non-polar residues to direct contact with solvent polar molecules. Our results show that the configurational contribution ΔS_c is temperature-independent and one order of magnitude smaller than its hydration counterpart ΔS^hyd in the temperature range considered. The profile obtained for log τ(T) from T = 200 K to T = 300 K exhibits a non-Arrhenius behavior characteristic of α relaxation mechanisms in hydrated proteins and glassy systems. This result is in agreement with recent dielectric spectroscopy data obtained for hydrated myoglobin, where at least two fast relaxation processes in the high temperature limit have been observed. The connection between the relaxation process calculated here and the experimental results is outlined.     

Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium–HCl,urea, and pH

The stability of bovine lactoperoxidase to denaturation by guanidinium–HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near T_m is controlled by kinetics. The values for the thermal transition, T_m, at slow heating scan rate were 66.8, 61.1, and 47.2 °C in the presence of 0.5, 1, and 2 M guanidinium–HCl, respectively. The extrapolated value for T_m in the absence of guanidinium–HCl is 73.7 °C, compared with 70.2 °C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, C_p, at T_m and E_a for the thermal transition decrease under conditions where T_m is lowered. At a given concentration, urea is less effective than guanidinium–HCl in reducing T_m, but urea reduces C_p relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 °C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164–1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change.     

Photosynthetic electron transport activity in heat-treated barley leaves: The role of internal alternative electron donors to photosystem II

Electron transport processes were investigated in barley leaves in which the oxygen-evolution was fully inhibited by a heat pulse (48 °C, 40 s). Under these circumstances, the K peak (∼ F_400 μs) appears in the chl a fluorescence (OJIP) transient reflecting partial Q_A reduction, which is due to a stable charge separation resulting from the donation of one electron by tyrozine Z. Following the K peak additional fluorescence increase (indicating Q_A⁻ accumulation) occurs in the 0.2-2 s time range. Using simultaneous chl a fluorescence and 820 nm transmission measurements it is demonstrated that this Q_A⁻ accumulation is due to naturally occurring alternative electron sources that donate electrons to the donor side of photosystem II. Chl a fluorescence data obtained with 5-ms light pulses (double flashes spaced 2.3-500 ms apart, and trains of several hundred flashes spaced by 100 or 200 ms) show that the electron donation occurs from a large pool with t_1/2 ∼ 30 ms. This alternative electron donor is most probably ascorbate.     

Synthesis and characterization of two stable paramagnetic octahedral chromium(IV) complexes with dianionic tridentate SNO donor ligands and of a chromium(III) complex with a ONO donor ligand

Two novel paramagnetic octahedral chromium(IV) complexes with dianionic tridentate SNO donor ligands containing extended π-system have been synthesized while only a paramagnetic octahedral chromium(III) complex is obtained when a related dianionic tridentate ONO donor ligand is used under similar conditions. These bischelate complexes [Cr(abtsal)₂] (1) (abtsalH₂ is the Schiff base of o-aminobenzenethiol and salicylaldehyde), [Cr(4-PhTSCsal)₂] · H₂O (2) (4-PhTSCsalH₂ is the Schiff base of 4-phenylthiosemicarbazide and salicylaldehyde) and K[Cr(sap)₂] · H₂O (3) (sapH₂ is the tridentate Schiff base of salicylaldehyde and o-aminophenol) are characterized by elemental analyses, magnetic moment measurements, IR, UV-Vis and EPR spectroscopic studies. Compound 3 has been structurally characterized by X-ray crystallography. Measured room temperature (RT) magnetic moment values are 2.98 BM for 1 and 2.83 BM for 2 indicating a d² system with a triplet ground state in both the cases. On the other hand, the magnetic moment value for 3 is found to be 3.74 BM at RT and is consistent with the presence of three unpaired electrons for a d³ Cr(III) ion. The magnetic moment values rule out the large spin-orbit coupling which is substantiated by the presence of RT EPR signals. Compounds 1 and 2 exhibit very similar powder EPR spectra at RT and LNT, which show the allowed transition ΔM_s = ±1 (g = 2.004 for both 1 and 2) as well as the “forbidden” half-field transition (ΔM_s = ±2) at g = 4.105 for 1 and g = 4.318 for 2, respectively. The X-band LNT frozen glass EPR spectrum of 1 in DMF shows the presence of zero-field split rhombic symmetry character, and results in the parameters g ≅ 2.0, D = 740 G, and E = 260 G. It suggests that the intensity of ΔM_s = ±2 forbidden transition is large due to the large D value. The X-band frozen glass EPR spectrum of compound 3 in DMF is found to be very similar to that reported for trans-[Cr(py)₄F₂]⁺ in DMF-H₂O-MeOH glass. The large difference (∼700 mV) in the reduction potential for the two octahedral complexes 1 (−1.40 V) and 3 (−0.70 V) is attributed to the difference in their metal ion oxidation states.     

Anti-plasticization of cassava starch by complexing fatty acids

The effect of adding 1–8% amylose complexing fatty acids (CFA), such as linoleic and oleic acids, on the glass transition temperature (T_g) of cassava starch (CS) with moisture content varying from 5 to 35% (dry basis) was studied. The main relaxation temperature (T_α), associated with the glass transition temperature of the samples (T_g), was determined by dynamic-mechanical-thermal analysis. The plasticizing behavior of water in the blends was evidenced by a decrease of T_α values with moisture content. The effect of CFA on CS was found to be a function of moisture content. At low moisture (<11%) it caused an anti-plasticization effect, while at higher moisture contents it produced plasticization. The anti-plasticizing effect of CFA on CS was attributed to amylose–lipid complex formation.