Dielectric properties of hydrated collagen

Dielectric measurements have been carried out on partially hydrated collagen in the frequency ranges 100 kHz–5 MHz, 100 MHz–1 GHz, and 8–23 GHz. In the low-frequency range, a dispersion was observed around 100 kHz which results from inhomogeneous conductivity of the samples. A dielectric relaxation was observed aroud 0.3 GHz using time-domain-spectroscopy techniques. This relaxation can be considered to originate from mobile side chains. Microwave measurements indicate that the water relaxation may extend into the 10-GHz region. An apparent discrepancy between the main water relaxation time and the average rotational correlation time of water as measured by nmr line widths was resolved by the assumption that a fraction of the water molecules is bound to the collagen with residence times on the order of 10^?6 sec, whereas the remainder of the water is only weakly bound and exhibits rotational rates on the order of 10^?10 sec.     

A pulsed N.M.R study of D2O bound to 1,2 dipalmitoyl phosphatidylcholine

Spin lattice relaxation times in both the lab and rotating frame, have been measured for deuterons (²H) in a number of unsonicated dispersions of 1,2 dipalmitoyl phosphatidylcholine in D₂O over a range of resonant frequencies from 13 MHz to 1 MHz for temperatures from ?20°C to 65°C.The proton (¹H) spin lattice relaxation time for the lecithin was measured for resonant frequencies of 8.5 MHz, and 40 MHz over a similar range of temperatures.The results agree with broadline measurements by Salsbury et al. [1], and for the liquid crystal phase are consistent with an anisotropic tumbling model of the water molecules bound to the lecithin headgroup. This tumbling occurs with correlation times of ≤10^?10 sec and ≈ 10^?6 sec about axes parallel to and perpendicular to the bisector of the D-O-D angle within a D₂O molecule, hydrogen bonded to the negatively charged phosphate headgroup.     

Thermodynamic analysis of hydration in human serum heme-albumin

Ferric human serum heme-albumin (heme-HSA) shows a peculiar nuclear magnetic relaxation dispersion (NMRD) behavior that allows to investigate structural and functional properties. Here, we report a thermodynamic analysis of NMRD profiles of heme-HSA between 20 and 60 °C to characterize its hydration. NMRD profiles, all showing two Lorentzian dispersions at 0.3 and 60 MHz, were analyzed in terms of modulation of the zero field splitting tensor for the S = ⁵/₂ manifold. Values of correlation times for tensor fluctuation (τ_v) and chemical exchange of water molecules (τ_M) show the expected temperature dependence, with activation enthalpies of −1.94 and −2.46 ± 0.2 kJ mol⁻¹, respectively. The cluster of water molecules located in the close proximity of the heme is progressively reduced in size by increasing the temperature, with ΔH = 68 ± 28 kJ mol⁻¹ and ΔS = 200 ± 80 J mol⁻¹ K⁻¹. These results highlight the role of the water solvent in heme-HSA structure-function relationships.     

Molecular mobilities in chromaffin granules magnetic field dependence of proton T1 relaxation times

The magnetic field dependence of the NMR spin-lattice relaxation time of water protons in intact bovine chromaffin vesicles has been studied over the range 1.00–23.49 kG. The T₁ relaxation time shows a dispersion a t field values near 20 kG. The observed proton resonance arises mainly from solvent protons (¹H₂O), but the relaxation rate, which is a weighted average over all sites with which the solvent protons rapidly exchange (i.e., NH and OH protons), is dominated by exchangeable protons in the most slowly moving soluble component. The field dependence of the T₁ dispersion demonstrates the existence of a site of exchangeable protons for which τ_r = 1.9±0.5 ns at 3°C. This site is assigned to ATP and cationic groups to which its phosphate esters are complexed, since previously measured correlation times of epinephrine and the chromogranin backbone are nearly an order of magnitude too short to explain the T₁ dispersion. Quantitative estimates of the relative numbers of exchangeable protons on the different soluble components support this interpretation. The temperature dependence of T₁ of the peak due to exchangeable protons has also been measured over a temperature range ?3 to 25°C. T₁ lengthens by about 30% over this range and exhibits no discontinuous behavior, as would be expected if a gel transition or structural alterations in the storage complex occurred. T₁ lengthens by less than 10% in chromaffin granule pastes that have been maintained at 25°C for 24 h, indicating considerable thermal stability in the storage complex. Possible effects on the solvent T₁ due to paramagnetic ions have been considered with the conclusion that they are probably negligible or of minor significance.     

Temperature and field dependent 1H-NMR relaxation data as probes of prostaglandin motional parameters in aqueous media

Proton resonance correlation times (τ_eff) for PGF₂α and a more rigid analog have been derived from the field-strength dependence of spinlattice relaxation times (T_1D) using 200 and 500 MHz observation. Those hydrogens showing τ_eff less than the value calculated for whole molecule tumbling (which applies for H-5 → H-15) also show a significantly greater temperature dependence for T_1D at 500 MHz. Minor wagging may occur at the C-7 and C-10 methylenes, and gradually increasing segmental motion is observed toward both side chain termini. A current model for the aqueous geometry of PGF₂α is developed from this data and studies of relaxation rate changes upon specific deuteration.     

Analysis of Sub-τc and Supra-τc Motions in Protein Gβ1 Using Molecular Dynamics Simulations

The functions of proteins depend on the dynamical behavior of their native states on a wide range of timescales. To investigate these dynamics in the case of the small protein Gβ1, we analyzed molecular dynamics simulations with the model-free approach of nuclear magnetic relaxation. We found amplitudes of fast timescale motions (sub-τ_c, where τ_c is the rotational correlation time) consistent with S² obtained from spin relaxation measurements as well as amplitudes of slow timescale motions (supra-τ_c) in quantitative agreement with S² order parameters derived from residual dipolar coupling measurements. The slow timescale motions are associated with the large variations of the ³J couplings that follow transitions between different conformational substates. These results provide further characterization of the large structural fluctuations in the native states of proteins that occur on timescales longer than the rotational correlation time.     

Hydration properties of adenosine phosphate series as studied by microwave dielectric spectroscopy

Hydration properties of adenine nucleotides and orthophosphate (Pi) in aqueous solutions adjusted to pH = 8 with NaOH were studied by high-resolution microwave dielectric relaxation (DR) spectroscopy at 20 °C. The dielectric spectra were analyzed using a mixture theory combined with a least-squares Debye decomposition method. Solutions of Pi and adenine nucleotides showed qualitatively similar dielectric properties described by two Debye components. One component was characterized by a relaxation frequency (f_c = 18.8-19.7 GHz) significantly higher than that of bulk water (17 GHz) and the other by a much lower f_c (6.4-7.6 GHz), which are referred to here as hyper-mobile water and constrained water, respectively. By contrast, a hydration shell of only the latter type was found for adenosine (f_c ~ 6.7 GHz). The present results indicate that phosphoryl groups are mostly responsible for affecting the structure of the water surrounding the adenine nucleotides by forming one constrained water layer and an additional three or four layers of hyper-mobile water.     

Dielectric properties of developing rabbit brain at 37 degrees C

The dielectric properties of developing rabbit brain were measured at 37 degrees C between 10 MHz and 18 GHz using time domain and frequency domain systems. The results show a variation with age of the dielectric properties of brain. An analysis of the data suggests that the water dispersion in the brain of newly born animals can be represented by a Debye equation. This dispersion increases in complexity with age, and there is evidence of a smaller additional relaxation process centered around 1 GHz. It is concluded that the principal contribution to this subsidiary dispersion region arises from water of hydration.     

Kinetics of polymerization of deoxyhemoglobin S and mixtures of hemoglobin A and hemoglobin S at high hemoglobin concentrations

Transverse water proton relaxation times (T₂) have been measured as a function of time after deoxygenation of solutions containing hemoglobin S. The shortened T₂ values observed upon deoxygenation of hemoglobin S result from an increase in the correlation time (τ_c) of the water fraction irrotationally bound to deoxyhemoglobin S as it polymerizes. Therefore, the change in τ_c as a function of time after deoxygenation can be used to measure the rate of polymer formation. The change in τ_c observed is reasonably fit by the first-order equation τ = τ₀ (1 ? e^?kt) + τ_oxy. At a total hemoglobin concentration of approximately 300 mg/ml, the pseudo-first-order rate constant in a heterozygous AS sample is 25 times slower than in a homozygous S sample, k = 0.019 and 0.47 s^?1, respectively. Since the transit time for an erythrocyte in vivo is approximately 15 s, these results suggest that the heterozygous A/S erythrocyte would traverse the circulation and become reoxygenated before extensive polymerization and, therefore, cell sickling could occur. For the homozygous S/S erythrocyte, there is ample time for polymerization and for cell sickling during circulation.     

Detection of nanosecond time scale side-chain jumps in a protein dissolved in water/glycerol solvent

In solution, the correlation time of the overall protein tumbling, τ _R , plays a role of a natural dynamics cutoff—internal motions with correlation times on the order of τ _R or longer cannot be reliably identified on the basis of spin relaxation data. It has been proposed some time ago that the ‘observation window’ of solution experiments can be expanded by changing the viscosity of solvent to raise the value of τ _R . To further explore this concept, we prepared a series of samples of α-spectrin SH3 domain in solvent with increasing concentration of glycerol. In addition to the conventional ¹⁵N labeling, the protein was labeled in the Val, Leu methyl positions (¹³CHD₂ on a deuterated background). The collected relaxation data were used in asymmetric fashion: backbone ¹⁵N relaxation rates were used to determine τ _R across the series of samples, while methyl ¹³C data were used to probe local dynamics (side-chain motions). In interpreting the results, it has been initially suggested that addition of glycerol leads only to increases in τ _R , whereas local motional parameters remain unchanged. Thus the data from multiple samples can be analyzed jointly, with τ _R playing the role of experimentally controlled variable. Based on this concept, the extended model-free model was constructed with the intent to capture the effect of ns time-scale rotameric jumps in valine and leucine side chains. Using this model, we made a positive identification of nanosecond dynamics in Val-23 where ns motions were already observed earlier. In several other cases, however, only tentative identification was possible. The lack of definitive results was due to the approximate character of the model—contrary to what has been assumed, addition of glycerol led to a gradual ‘stiffening’ of the protein. This and other observations also shed light on the interaction of the protein with glycerol, which is one of the naturally occurring osmoprotectants. In particular, it has been found that the overall protein tumbling is controlled by the bulk solvent, and not by a thin solvation layer which contains a higher proportion of water.     

Proton magnetic relaxation and anion effect in solutions of acid ferricytochrome c.

Measurements of the longitudinal relaxation rates of water protons in aqueous solutions of ferricytochrome c and their temperature dependence, were used for the elucidation of the heme iron ligands at acid pH. The relaxation rates increased with a decrease in pH and pK values of 2.5 and 4.48 were evaluated for the aqueous and 6 m urea solutions, respectively. The results at acid pH are compatible with a structure in which two water molecules exchange rapidly between the coordination sphere of high spin heme iron and the bulk. They suggest that concomitantly with the low-high spin transition the histidine-18 and methionine-80 iron bonds break simultaneously. Addition of various anions, including methanesulfonate at pH 1.95 caused a 85% decrease in the net longitudinal relaxation rate. However, neither the chemical shift nor the width of the methyl proton nmr line of methanesulfonate in solution of acid ferricytochrome c were affected indicating that the effect of anions is not due to a direct binding to the heme iron. The relaxation mechanism of the water molecules in the first coordination sphere of the ferric ion in acid cytochrome c is discussed. It appears that the longitudial relaxation rate is modulated by the electronic correlation time of the ferric ion which was calculated to be τ_s = 6 × 10^?11 sec at 60 MHz.     

Dielectric relaxation of DNA solutions. III. Effects of DNA concentration, protein contamination, and mixed solvents

As a continuation of previous papers [Biopolymers (1976) 15 , 879; (1978) 17 , 1508], the low-frequency dielectric relaxation of DNA solutions was studied with a four-electrode cell and the simultaneous two-frequency measurement. Below a critical concentration, the dielectric relaxation time agrees with the rotational relaxation time estimated from the reduced viscosity and is almost independent of DNA concentration C_p, and the dielectric increment is proportional to C_p. The critical concentration is approximately 0.02% of DNA for molecular weight M_r 2 × 10⁶ and 0.2% for M_r 4.5 × 10⁵ in 1 mM NaCl. Dielectric relaxations are compared for samples before and after deproteinization, and the protein contamination is found to have a minor effect on the dipole moment of DNA. The effect of a mixed solvent of water and ethanol on the dielectric relaxation of DNA is well interpreted in terms of changes in viscosity and the dielectric constant of the solvent, assuming that the relaxation arises from rotation of the molecule with a quasi-permanent dipole due to counterion fluctuation.     

Water and Backbone Dynamics in a Hydrated Protein

Rotational immobilization of proteins permits characterization of the internal peptide and water molecule dynamics by magnetic relaxation dispersion spectroscopy. Using different experimental approaches, we have extended measurements of the magnetic field dependence of the proton-spin-lattice-relaxation rate by one decade from 0.01 to 300 MHz for ¹H and showed that the underlying dynamics driving the protein ¹H spin-lattice relaxation is preserved over 4.5 decades in frequency. This extension is critical to understanding the role of ¹H₂O in the total proton-spin-relaxation process. The fact that the protein-proton-relaxation-dispersion profile is a power law in frequency with constant coefficient and exponent over nearly 5 decades indicates that the characteristics of the native protein structural fluctuations that cause proton nuclear spin-lattice relaxation are remarkably constant over this wide frequency and length-scale interval. Comparison of protein-proton-spin-lattice-relaxation rate constants in protein gels equilibrated with ²H₂O rather than ¹H₂O shows that water protons make an important contribution to the total spin-lattice relaxation in the middle of this frequency range for hydrated proteins because of water molecule dynamics in the time range of tens of ns. This water contribution is with the motion of relatively rare, long-lived, and perhaps buried water molecules constrained by the confinement. The presence of water molecule reorientational dynamics in the tens of ns range that are sufficient to affect the spin-lattice relaxation driven by ¹H dipole-dipole fluctuations should make the local dielectric properties in the protein frequency dependent in a regime relevant to catalytically important kinetic barriers to conformational rearrangements.     

Dielectric behavior of DNA solution at radio and microwave frequencies (at 20 degrees C).

The dielectric constant and conductivity of calf thymus DNA were investigated at frequencies between 0.1 MHz and 70 GHz. This work is to investigate the dielectric properties of DNA in low gigahertz region and also to study whether the dielectric behavior of the water is affected by the presence of highly charged DNA. The results of these measurements indicate the presence of two anomalous dispersions, the one between 1 MHz and 1 GHz and the second one above 1 GHZ. The dispersion at low frequencies is likely to arise from polar groups in the DNA molecule. The relaxation behavior of unbound water in DNA solution is only slightly affected by the presence of DNA at concentrations below 1%.     

Hydration analysis of Pseudomonas aeruginosa cytochrome c551 upon acid unfolding by dielectric relaxation spectroscopy

Dielectric relaxation (DR) study was performed to reveal the hydration change of Pseudomonas aeruginosa ferric cytochrome c551 (PA c551) in dilute aqueous solutions upon the acid unfolding which undergoes a two-state transition. The DR spectrum of a small spherical region containing a PA c551 molecule and its surrounding water shell was derived from the solution and solvent spectra by dielectric mixture theories. The derived spectrum was well-fitted with a sum of a Debye relaxation component (C1) with a DR frequency around 4.7 GHz and the bulk solvent component (CB). Upon acid unfolding, the DR amplitude of CB decreased with decreasing pH in an inverse manner to that of C1, while the total DR amplitude was almost constant. It indicates that C1 is due to the hydration water of PA c551. Little change in the DR frequency of C1 and a 1.7-fold increase in hydration number were observed.     

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.     

Magnetic Resonance Water Proton Relaxation in Protein Solutions and Tissue: T1ρ Dispersion Characterization

Background

Image contrast in clinical MRI is often determined by differences in tissue water proton relaxation behavior. However, many aspects of water proton relaxation in complex biological media, such as protein solutions and tissue are not well understood, perhaps due to the limited empirical data.

Principal Findings

Water proton T₁, T₂, and T_1ρ of protein solutions and tissue were measured systematically under multiple conditions. Crosslinking or aggregation of protein decreased T₂ and T_1ρ, but did not change high-field T₁. T_1ρ dispersion profiles were similar for crosslinked protein solutions, myocardial tissue, and cartilage, and exhibited power law behavior with T_1ρ(0) values that closely approximated T₂. The T_1ρ dispersion of mobile protein solutions was flat above 5 kHz, but showed a steep curve below 5 kHz that was sensitive to changes in pH. The T_1ρ dispersion of crosslinked BSA and cartilage in DMSO solvent closely resembled that of water solvent above 5 kHz but showed decreased dispersion below 5 kHz.

Conclusions

Proton exchange is a minor pathway for tissue T₁ and T_1ρ relaxation above 5 kHz. Potential models for relaxation are discussed, however the same molecular mechanism appears to be responsible across 5 decades of frequencies from T_1ρ to T₁.     

Relaxation dynamics of a protein solution investigated by dielectric spectroscopy

In the present work, we provide a dielectric study on two differently concentrated aqueous lysozyme solutions in the frequency range from 1MHz to 40GHz and for temperatures from 275 to 330K. We analyze the three dispersion regions, commonly found in protein solutions, usually termed β-, γ-, and δ-relaxations. The β-relaxation, occurring in the frequency range around 10MHz and the γ-relaxation around 20GHz (at room temperature) can be attributed to the rotation of the polar protein molecules in their aqueous medium and the reorientational motion of the free water molecules, respectively. The nature of the δ-relaxation, which is often ascribed to the motion of bound water molecules, is not yet fully understood. Here we provide data on the temperature dependence of the relaxation times and relaxation strengths of all three detected processes and on the dc conductivity arising from ionic charge transport. The temperature dependences of the β- and γ-relaxations are closely correlated. We found a significant temperature dependence of the dipole moment of the protein, indicating conformational changes. Moreover we find a breakdown of the Debye-Stokes-Einstein relation in this protein solution, i.e., the dc conductivity is not completely governed by the mobility of the solvent molecules. Instead it seems that the dc conductivity is closely connected to the hydration shell dynamics.     

The state of water in biological systems as studied by proton and deuterium relaxation

Careful experiments on the measurement of the intensity of the deuterium NMR signal for ²H₂O in muscle and in its distillate were performed, and they showed that all ²H₂O in muscles is “NMR visible.”The spin-lattice relaxation time (T₁) of the water protons in the muscle and liver of mice and in egg white has been studied at six frequencies ranging from 4.5 to 6.0 MHz over the temperature range of +37 to −70°C. T₁ values of deuterons in ²H₂O of gastrocnemius muscle and liver of mice have been measured at three frequencies (4.5, 9.21 and 15.35 MHz) over the temperature range of +37 to −20°C. Calculations on T₁ for both proton and deuteron have been made and compared with the experimental data. It is suggested that the reduction of the T₁ values compared to pure water and the frequency dependence of T₁ are due to water molecules in the hydration layer of the macromolecules, and that the bulk of water molecules in the biological tissues and egg white undergoes relaxation like ordinary liquid water.     

Dielectric spectroscopy of plant protoplasts

The relative permittivity and conductivity of the mesophyll protoplasts isolated from Brassica campestris leaves and Tulipa gesneriana petals were measured over a frequency range from 1kHz to 500 MHz.These protoplasts showed a broad dielectric dispersion, which was composed of three subdispersions, termed β₁-, β₂-, and β₃-dispersion in increasing order of frequency.The three subdispersions were assigned to the Maxwell-Wagner dispersion caused by charging processes at the interfaces of the surface and internal membranes; the plasma membrane, the tonoplast, and the membranes of cytoplasmic organelles (e.g., chloroplasts, granules, etc) primarily contribute to the β₁-, β₂-, and β₃-dispersion, respectively. The whole dielectric dispersion curve was satisfactorily interpreted in terms of a spherical cell model taking a large vacuole and cytoplasmic organelles into account. Using this model the capacitances of the plasma membranes and the tonoplasts were estimated to be 0.6-0.7 μF/cm² and 0.9-1.0 μF/cm², respectively.