A broad glass transition in hydrated proteins

We performed Raman and Brillouin scattering measurements to estimate glass transition temperature, T_g, of hydrated protein. The measurements reveal very broad glass transition in hydrated lysozyme with approximate T_g ∼ 180 ± 15 K. This result agrees with a broad range of T_g ∼ 160–200 K reported in literature for hydrated globular proteins and stresses the difference between behavior of hydrated biomolecules and simple glass-forming systems. Moreover, the main structural relaxation of the hydrated protein system that freezes at T_g ∼ 180 K remains unknown. We emphasize the difference between the “dynamic transition”, known as a sharp rise in mean-squared atomic displacement <r²> at temperatures around T_D ∼ 200–230 K, and the glass transition. They have different physical origin and should not be confused.     

H and C NMR studies on the temperature-dependent water and protein dynamics in hydrated elastin,myoglobin and collagen

²H NMR spin-lattice relaxation and line-shape analyses are performed to study the temperature-dependent dynamics of water in the hydration shells of myoglobin, elastin, and collagen. The results show that the dynamical behaviors of the hydration waters are similar for these proteins when using comparable hydration levels of h = 0.25–0.43. Since water dynamics is characterized by strongly nonexponential correlation functions, we use a Cole–Cole spectral density for spin-lattice relaxation analysis, leading to correlation times, which are in nice agreement with results for the main dielectric relaxation process observed for various proteins in the literature. The temperature dependence can roughly be described by an Arrhenius law, with the possibility of a weak crossover in the vicinity of 220 K. Near ambient temperatures, the results substantially depend on the exact shape of the spectral density so that deviations from an Arrhenius behavior cannot be excluded in the high-temperature regime. However, for the studied proteins, the data give no evidence for the existence of a sharp fragile-to-strong transition reported for lysozyme at about 220 K. Line-shape analysis reveals that the mechanism for the rotational motion of hydration waters changes in the vicinity of 220 K. For myoglobin, we observe an isotropic motion at high temperatures and an anisotropic large-amplitude motion at low temperatures. Both mechanisms coexist in the vicinity of 220 K. ¹³C CP MAS spectra show that hydration results in enhanced elastin dynamics at ambient temperatures, where the enhancement varies among different amino acids. Upon cooling, the enhanced mobility decreases. Comparison of ²H and ¹³C NMR data reveals that the observed protein dynamics is slower than the water dynamics.     

Electron spin relaxation of the electron paramagnetic resonance spectra of cytochrome c

The progressive power saturation of the electron paramagnetic resonance (EPR) spectrum of ferricytochrome c has been investigated in order to determine the spin-lattice relaxation time of the center. We have generalized the usual saturation treatments to include the effects of extended sample size and anisotropic g values as well as derivative spectra. We find that the results are consistent with a T7 power law in the temperature range 6--25 K. At temperatures above 25 K the relaxation time is too short for successful power saturation. Observation of the linewidth shows that the relaxation behavior continues as a first-order Raman process to 50 K.     

Protein Brownian Rotation at the Glass Transition Temperature of a Freeze-Concentrated Buffer Probed by Superparamagnetic Nanoparticles

For applications from food science to the freeze-thawing of proteins it is important to understand the often complex freezing behavior of solutions of biomolecules. Here we use a magnetic method to monitor the Brownian rotation of a quasi-spherical cage-shaped protein, apoferritin, approaching the glass transition T_g in a freeze-concentrated buffer (Tris-HCl). The protein incorporates a synthetic magnetic nanoparticle (Co-doped Fe₃O₄ (magnetite)). We use the magnetic signal from the nanoparticles to monitor the protein orientation. As T decreases toward T_g of the buffer solution the protein’s rotational relaxation time increases exponentially, taking values in the range from a few seconds up to thousands of seconds, i.e., orders of magnitude greater than usually accessed, e.g., by NMR. The longest relaxation times measured correspond to estimated viscosities >2 MPa s. As well as being a means to study low-temperature, high-viscosity environments, our method provides evidence that, for the cooling protocol used, the following applies: 1), the concentration of the freeze-concentrated buffer at T_g is independent of its initial concentration; 2), little protein adsorption takes place at the interface between ice and buffer; and 3), the protein is free to rotate even at temperatures as low as 207 K.     

Protein Brownian Rotation at the Glass Transition Temperature of a Freeze-Concentrated Buffer Probed by Superparamagnetic Nanoparticles

For applications from food science to the freeze-thawing of proteins it is important to understand the often complex freezing behavior of solutions of biomolecules. Here we use a magnetic method to monitor the Brownian rotation of a quasi-spherical cage-shaped protein, apoferritin, approaching the glass transition T_g in a freeze-concentrated buffer (Tris-HCl). The protein incorporates a synthetic magnetic nanoparticle (Co-doped Fe₃O₄ (magnetite)). We use the magnetic signal from the nanoparticles to monitor the protein orientation. As T decreases toward T_g of the buffer solution the protein’s rotational relaxation time increases exponentially, taking values in the range from a few seconds up to thousands of seconds, i.e., orders of magnitude greater than usually accessed, e.g., by NMR. The longest relaxation times measured correspond to estimated viscosities >2 MPa s. As well as being a means to study low-temperature, high-viscosity environments, our method provides evidence that, for the cooling protocol used, the following applies: 1), the concentration of the freeze-concentrated buffer at T_g is independent of its initial concentration; 2), little protein adsorption takes place at the interface between ice and buffer; and 3), the protein is free to rotate even at temperatures as low as 207 K.     

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (T_m) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer T_m between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the T_m for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.     

Post-yield relaxation behavior of bovine cancellous bone

Relaxation studies were conducted on specimens of bovine cancellous bone at post-yield strains. Stress and strain were measured for 1000 s and the relaxation modulus was determined. Fifteen cylindrical, cancellous bone specimens were removed from one bovine femur in the anterior–posterior direction. The relaxation modulus was found to be a function of strain. Therefore cancellous bone is non-linearly viscoelastic/viscoplastic in the plastic region. A power law regression was fit to the relaxation modulus data. The multiplicative constant was found to be statistically related through a power law relationship to both strain (p<0.0005) and apparent density (p<0.0005) while the power coefficient was found to be related through a power law relationship, E(t, ε)=A(ε)t^?n(ε), to strain (p<0.0005), but not apparent density.     

Effect of water activity on the mechanical glass transition and dynamical transition of bacteria

The purpose of this study was to clarify the glass-transition behavior of bacteria (Cronobacter sakazakii) as a function of water activity (a_w). From the water sorption isotherm (298 K) for C. sakazakii, monolayer water content and monolayer a_w were determined to be 0.0724 g/g-dry matter and 0.252, respectively. Mechanical relaxation was investigated at 298 K. In a higher a_w range of over 0.529, the degree of mechanical relaxation increased with an increase in a_w. From the effect of a_w on the degree of mechanical relaxation, the mechanical a_wc (a_w at which mechanical glass transition occurs at 298 K) was determined to be 0.667. Mean-square displacement of atoms in the bacteria was investigated by incoherent elastic neutron scattering. The mean-square displacement increased gradually with an increase in temperature depending on the a_w of samples. From the linear fitting, two or three dynamical transition temperatures (low, middle, and high T_ds) were determined at each a_w. The low-T_d values (142–158 K) were almost independent from a_w. There was a minor effect of a_w on the middle T_d (214–234 K) except for the anhydrous sample (261 K). The high T_d (252–322 K) largely increased with the decrease in a_w. From the a_w dependence of the high T_d, the dynamical a_wc was determined to be 0.675, which was almost equivalent to the mechanical a_wc. The high T_d was assumed to be the glass-transition temperature (T_g), and anhydrous T_g was estimated to be 409 K. In addition, molecular relaxation time (τ) of the bacteria was calculated as a function of a_w. From the result, it is suggested that the progress of metabolism in the bacterial system requires a lower τ than approximately 6 × 10^?5 s.     

Effects of temperature and field strength on the NMR relaxation times of 23Na in frog striated muscle

Pulsed NMR techniques have been applied to the study of the relaxation parameters characterizing ²³Na within frog striated muscle. Experiments were performed at 3°C, 22–24°C and 39°C at a Larmor frequency of 15.7 MHz; at 22–24°C, measurements were obtained both at 15.7 MHz and at 7.85 MHz.As previously reported, only a single spine-lattice relaxation time (T₁) was observed, but both slow (T₂)_I and fast (T₂)_II components of the spin-spin relaxation time were measured. The effect of temperature (θ) upon (1/T₁) was qualitatively similar to that reported for ²³Na in free solution; (θ) did not significantly affect (1/T₂) over the range of temperatures studied. (1/T₂)_I, and to a lesser degreee, (1/T₁) exhibited a modest inverse dependence of doubtful significance on the Larmor frequency.The data are examined within the framework of a simple specific model; a conservative values in assumed for the quadrupolar coupling constant characterizing immobilized intracellular Na⁺. Within this framework, the results suggest that the fraction of bound ions whose molecular tumbling is severely restricted does not exceed some few percent of the total sodium population.     

pH-Dependent Raman spectra and thermal melting profiles for polycytidylic acid

The Raman spectrum of polycytidylic acid was investigated in the pH range of 6.6–4.1. The thermal melting temperatures and the nature of the thermal melting profiles change in this range as monitored by the three Raman band envelopes, which include the 780-, 805-cm^?1 bands, the 1190-, 1285-cm^?1 bands, and the 1527-cm^?1 band. By coupling these data with the theory of Raman scattering intensity and quantitative pH profiles for cytidine, it is shown that the band envelopes studied exhibit specific, yet different information regarding the thermal melting process. The band envelopes at 1170–1310 and 1527 cm^?1, which are a sensitive function of both the extent of protonation and base stacking (hypochromic), reveal T_m values which agree with values derived from uv melting profiles. The 760–830-cm^?1 envelope, which is not directly sensitive to cytosine residue protonation, but includes information associated with base stacking (the 780-cm^?1 band) and the nature of the phosphodiester backbone (the frequency-dependent 805-cm^?1 component), exhibits T_m values which deviate from the values obtained from the other bands. The observed differences are pH-dependent and correlate well with the extent of deprotonation that takes place in the denaturation process. Details of the spectrum of neutral and protonated poly(C) from pH 7 to 4.1 are discussed and related to the nature of the thermal denaturation process.     

Phospholipid Reorientation at the Lipid/Water Interface Measured by High Resolution P Field Cycling NMR Spectroscopy

The magnetic field dependence of the ³¹P spin-lattice relaxation rate, R₁, of phospholipids can be used to differentiate motions for these molecules in a variety of unilamellar vesicles. In particular, internal motion with a 5- to 10-ns correlation time has been attributed to diffusion-in-a-cone of the phosphodiester region, analogous to motion of a cylinder in a liquid hydrocarbon. We use the temperature dependence of ³¹P R₁ at low field (0.03-0.08 T), which reflects this correlation time, to explore the energy barriers associated with this motion. Most phospholipids exhibit a similar energy barrier of 13.2 ± 1.9 kJ/mol at temperatures above that associated with their gel-to-liquid-crystalline transition (T_m); at temperatures below T_m, this barrier increases dramatically to 68.5 ± 7.3 kJ/mol. This temperature dependence is broadly interpreted as arising from diffusive motion of the lipid axis in a spatially rough potential energy landscape. The inclusion of cholesterol in these vesicles has only moderate effects for phospholipids at temperatures above their T_m, but significantly reduces the energy barrier (to 17 ± 4 kJ/mol) at temperatures below the T_m of the pure lipid. Very-low-field R₁ data indicate that cholesterol inclusion alters the averaged disposition of the phosphorus-to-glycerol-proton vector (both its average length and its average angle with respect to the membrane normal) that determines the ³¹P relaxation.     

Intracellular water in Artemia cysts (brine shrimp): Investigations by deuterium and oxygen-17 nuclear magnetic resonance

The dormant cysts of Artemia undergo cycles of hydration-dehydration without losing viability. Therefore, Artemia cysts serve as an excellent intact cellular system for studying the dynamics of water-protein interactions as a function of hydration. Deuterium spin-lattice (T₁) and spin-spin (T₂) relaxation times of water in cysts hydrated with D₂O have been measured for hydrations between 1.5 and 0.1 g of D₂O per gram of dry solids. When the relaxation rates (I/T₁, I/T₂) of ²H and ¹⁷O are plotted as a function of the reciprocal of hydration (1/H), an abrupt change in slope is observed near 0.6 g of D₂O (or H₂ ¹⁷O)/gram of dry solids, the hydration at which conventional metabolism is activated in this system. The results have been discussed in terms of the two-site and multisite exchange models for the water-protein interaction as well as protein dynamics models. The ²H and ¹⁷O relaxation rates as a function of hydration show striking similarities to those observed for anisotropic motion of water molecules in protein crystals.

It is suggested here that although the simple two-site exchange model or n-site exchange model could be used to explain our data at high hydration levels, such models are not adequate at low hydration levels (<0.6 g H₂O/g) where several complex interactions between water and proteins play a predominant role in the relaxation of water nuclei. We further suggest that the abrupt change in the slope of I/T₁ as a function of hydration in the vicinity of 0.6 g H₂O/g is due to a change in water-protein interactions resulting from a variation in the dynamics of protein motion.

     

Power law dispersion in animal horn

The dielectric response of sheep horn has been measured in the frequency range from 10^–3–10⁵ Hz and over temperatures in the range 304–500 K. The dynamic behaviour of the conductance and capacitance in sheep horn has been observed to follow fractional power law dependences on frequency. It is shown that the over all dielectric response of these dead cells correspond to a dispersive imperfect bulk in series with a dispersive barrier region. It is further shown that the increase in temperature influences the reponse by eliminating the room temperature dc conductance and affecting the magnitude of the dispersion in capacitance. The magnitudes of activation energies are found as 0.33±0.02 eV for conductance, 0.40±0.02 eV for relaxation and 0.33±0.02 eV for the frequency shift.     

Quantitative analysis of backbone motion in proteins using MAS solid-state NMR spectroscopy

We present a comprehensive analysis of protein dynamics for a micro-crystallin protein in the solid-state. Experimental data include ¹⁵N T ₁ relaxation times measured at two different magnetic fields as well as ¹H–¹⁵N dipole, ¹⁵N CSA cross correlated relaxation rates which are sensitive to the spectral density function J(0) and are thus a measure of T ₂ in the solid-state. In addition, global order parameters are included from a ¹H,¹⁵N dipolar recoupling experiment. The data are analyzed within the framework of the extended model-free Clore–Lipari–Szabo theory. We find slow motional correlation times in the range of 5 and 150 ns. Assuming a wobbling in a cone motion, the amplitude of motion of the respective amide moiety is on the order of 10° for the half-opening angle of the cone in most of the cases. The experiments are demonstrated using a perdeuterated sample of the chicken α-spectrin SH3 domain.     

Hemoglobin-water interactions in normal and sickle erythrocytes by proton magnetic resonance T1ϱ measurements

The dependence of the water proton magnetic resonance spin-lattice relaxation rate (T_1?^?1) in the rotating frame on the strength of the spin-locking (H₁) field has been investigated for packed oxy and deoxy normal and sickle erythrocytes at temperatures from 9 to 40 °C. The T_1?^?1 of oxy or deoxy normal erythrocytes shows no dependence on H₁ up to ~7 G at any temperature studied. On the other hand, T_1?^?1 decreases from about 40 s^?1 to 15 s^?1 (H₁ from 0 to ~7 G) for deoxygenated packed sickle cells at 40 °C. The magnitude of this variation of T_1?^?1 with H₁ decreases with decreasing temperature. Oxy packed sickle cells also show a dependence of T_1?^?1 on H₁ but the magnitude is <10% of that of the deoxygenated samples. These results suggest that water proton T_1?^?1 measurements are a sensitive probe of hemoglobin S polymerization and provide a novel technique for the study of slow water motions in these systems. The T_1?^?1 results are compared with low frequency T₁^?1 results of other investigators on hemoglobin S solutions. Analysis of the data suggests that water proton motions with correlation times of the order of 10^?5 s are present in the deoxygenated sickle cell samples at temperatures above 10 °C.     

Nuclear magnetic resonance investigation of human erythrocytes in the presence of manganese ions. Evidence for a thermal transition

Water proton transverse relaxation was investigated in whole blood and washed erythrocytes samples, respectively, at various temperatures and manganese concentrations. Water diffusional exchange controls proton relaxation in whole blood samples at higher Mn²⁺ concentrations (20–30 mM) or in washed erythrocyte samples at low Mn²⁺ content (1–5 mM). Mn²⁺ uptake is significant in washed normal erythrocyte samples when its concentrations is about 18 mM or higher in the medium, at temperatures below about 26°C. The thermal transition as revealed by the NMR doping method represents a switch from a water exchange process, mainly seen in the higher temperature range, to a paramagnetic ion controlled water proton relaxation in the lower temperature range.     

Characterization of heat injury in grapes using h nuclear magnetic resonance methods : changes in transverse relaxation times

Transverse relaxation times (T₂) of tissue water (¹H) in leaves and suspension cultured cells of grape hybrids (Vitis spp. cv `Venus' and `Veeblanc') were measured by nuclear magnetic resonance at various temperatures. The tissue water was characterized by two T₂ time constants. A sharp decrease in T₂ for the major fraction of tissue water was observed in association with heat injury, as measured by electrolyte leakage and triphenyltetrazolium chloride reduction in both leaves and suspension cultured cells. The changes in T₂ as a result of heat injury were irreversible, as indicated by a temperature dependent hysteresis of T₂. Studies using a paramagnetic probe (Mn⁺²) indicated that the plasma membrane was irreversibly damaged at the killing temperature, resulting in a loss of cell compartmentalization. Tissue water in heat-killed samples was characterized by only a single T₂.     

Electron-electron distances in spin-labeled low-spin metmyoglobin variants by relaxation enhancement

Thirteen single-cysteine variants of myoglobin were prepared by overexpression of apoprotein, spin labeling, and reconstitution with hemin. This procedure resulted in a protein with fewer hemichrome impurities than was obtained by an overexpression of holo-protein followed by spin labeling. Coordination of cyanide to the met heme formed low-spin complexes. Iron-nitroxyl interspin distances in the range of 17-30 Å were determined by saturation recovery measurements of the enhancement of the nitroxyl spin lattice relaxation rates between ∼30-140 K, and by spin-echo measurements of the enhancement of spin-spin relaxation rates at 10-30 K. Interspin distances were also calculated, using the molecular modeling program Insight II (Accelrys, San Diego, CA). For most variants, distances determined from the temperature dependence of spin-echo intensities at a pulse spacing of 200 ns agree with distances measured by saturation recovery and calculated with Insight II within about an angstrom, which is within experimental uncertainties. Measurements of interspin distances via spin-spin relaxation enhancement have the advantages that maximum effects are observed for slower metal relaxation rates than are required for spin-lattice relaxation enhancement, and the impact diminishes as r⁻³ instead of r⁻⁶, as with spin-lattice relaxation enhancement, which permits measurements at longer distances.     

Low temperature study of myoglobin-ligand rebinding kinetics with Mössbauer spectroscopy

We have studied the recombination kinetics of carboxymyoglobin (after photodissociation of the CO ligand) by Mössbauer spectroscopy for temperatures in the range 4.2 – 60 K. The observed kinetics display non-exponential behaviour which was monitored over periods of a few days. It is shown that the time dependence of the kinetics can be reduced to a single universal function of the temperature-dependent variable (t/τ _1/2(T)) ^β(T) . The half-decay time τ _1/2(T) and the scaling parameter β(T) are analysed for the presence of tunneling effects. The non-Arrhenius temperature dependence of the half-decay time below 60 K is interpreted as activated tunneling in models with an Eckart barrier or a fluctuating barrier.