The molecular details of collagen hydration

Nuclear magnetic resonance and dielectric data on hydrated collagen are interpreted in terms of Ramachandran's hydration model. It is found that all data are compatible with this model, indicating two specific binding sites per three amino acids in the threefold collagen helix. Sorption data have been interpreted according to the multilayer theory of Guggenheim and used to derive the fraction of bound water in the primary sites. From magnetic resonance anisotropies structural details of the position of the water molecules can be derived under the assumption that both sites are equally occupied. The residence time of a water molecule in one of these sites in moderately hydrated collagen (45 g H₂O/100 g collagen) is 1.2 × 10^?6 sec. The remainder of the water is weakly bound and consists of rapidly exchanging species with rotational correlation time shorter than 10^?10 sec. The sites are 50% occupied at a water content of 10 g/100 g collagen and may contribute significantly to the stability of the collagen threefold helix.     

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.     

Dielectric studies on water in solutions of purified lecithin vesicles

The complex permittivity of sonicated aqueous solutions of purified dimyristoylphosphatidylcholine has been measured as a function of frequency between 3 kHz and 40 GHz. The dielectric spectrum of the samples shows two dispersion/absorption regions, one centered at about 80 MHz the other at about 20.GHz (30°C). Otherwise than in previous studies no additional dispersion/absorption process has been found at frequencies below 10 MHz.The complex dielectric spectrum of the samples is discussed with respect to the dynamical state of solvent water in solutions of single-bilayer vesicles. The main relaxation time of the solvent water, τ₁ ((2πτ₁)^?1 ≈ 20 GHz), is smaller than that of pure water, τ_W, at the same temperature. This effect results from the action of internal depolarizing fields which obviously overcompensate and enhancement of τ₁ due to specific solute/solvent interactions (hydration) as had been previously found with micellar solutions of lysolecithins.It cannot be excluded, that some solvent water shows unusual dynamical behaviour. If there exists a substantial amount of such motionally perturbed water, however, it must be characterized by a relaxation time close to that of the phosphorylcholine zwitterions, τ₂ ((2πτ₂)^?1 ≈ 80 MHz).     

Evaluation of the water environments in deoxygenated sickle cells by longitudinal and transverse water proton relaxation rates

The longitudinal and transverse water proton relaxation rates of oxygenated and deoxygenated erythrocytes from both normal adults and individuals with sickle cell disease were measured as a function of temperature at two different frequencies. The simplest model which fits all of the data consists of three different environments for water molecules. The majority of the water (98%) has a correlation time indistinguishable from bulk water (3 × 10^?11 sec). Secondly, there is a small amount of water (1.3–1.5%) present which has a correlation time of 2–4 × 10 ^?9 sec and is apparently independent of the erythrocyte sample studied. Presumably this water is the hydration sphere around the hemoglobin molecules and its correlation time is significantly slower than bulk water. The third environment contains approximately 0.2% of the water present and has a correlation time≥ 10^?7 sec. This third environment is considered tightly bound to the hemoglobin because the water proton correlation time is very similar to the expected rotational correlation time for the hemoglobin molecules. The value of the transverse relaxation rate, f_b(T_2b)^?1, for the tightly bound water fraction decreases in oxy ($\text{S}\text{S}$), deoxy ($\text{A}\text{A}$), and oxy ($\text{A}\text{A}$) erythrocyte samples as the temperature is increased as expected for a rotational correlation time process. In dramatic contrast,f_b (T_2b)^?1 increases almost linearly as the temperature is increased over the whole 4 ° to 37 °C temperature range in samples of deoxy ($\text{S}\text{S}$) erythrocytes. The observation suggests a continual increase in the formation of deoxyhemoglobulin S polymers rather than a sudden transition from a homogeneous solution of deoxyhemoglobin S molecules to a solid gel.     

Acoustical properties of aqueous solutions of oxygenated and deoxygenated hemoglobin

The acoustical relaxation spectrum of aqueous solutions of oxygenated and deoxygenated hemoglobin solutions was investigated in the frequency range 0.5–1000 MHz. The abrupt high frequency cut off of the broad absorption spectrum is consistent with a heavily weighted, shortest relaxation time of 2 × 10^?9 sec. Both the high-frequency cut-off and high frequency residual absorption appear to be slightly different for oxygenated and deoxygenated hemoglobin solution.     

Microwave dielectric study on bound water of globule proteins in aqueous solution

A dielectric relaxation peak due to bound water of globule proteins in aqueous solution was observed at first by the use of a time domain reflectometry. This peak locates around 100 MHz as well as that of the aqueous DNA solution and the moist collagen, and has a relaxation strength in proportion to surface of the globule protein except for trypsin and pepsin of hydrolase. It is suggested that this peak is caused by orientation of bound water molecules on the protein surface. The number of bound water molecules estimated is in good agreement with that obtained by other method such as x-ray analysis. The solution exhibits another peak below 100 MHz, which is caused by the rotation of globule protein supplemented by migration of the counterion. Its relaxation time is completely proportional to the molecular weight of the protein. © 1994 John Wiley & Sons, Inc.     

Effect of concentration on the aggregation of poly(γ-benzyl-L-glutamate) in dioxane: Critical concentration and maximum length for linear head to tail aggregates

Dielectric relaxation studies in the frequency range 100 Hz to 2 MHz of poly(γ-benzyl-L -glutamate) in dioxane have been carried out over a range of concentration 10^?4–10^?2g/g. The structure of aggregates is analyzed in terms of dipole moment and relaxation time. A critical concentration (? 10^?3 g/g for the studied molecular weights) has been determined below which the aggregates are found to have linear head to tail type structure. Above the critical concentration a different structure of aggregates is apparent which could not be fully analyzed by these measurements alone. Possible forms of aggregation above the critical concentration are discussed. Formation of long range order which would lead to nematic liquid crystalline phase at higher concentrations has been discussed as one of the possible explanations for the observed behavior above the critical concentration. Maximum length of linear head to tail type aggregates for poly(γ-benzyl-L -glutamate) in dioxane as determined from these results correspond to an α-helix of molecular weight 210,000. A slight difference in the purity of dioxane has been shown to have an influence on the reproducibility of the state of aggregation as well as on the rate of disaggregation on dilution.     

Mobility of water bound to biological membranes: A proton NMR relaxation study

Water proton nuclear magnetic resonance relaxation measurements have been obtained for aqueous suspensions of red cell membranes. These data support a model in which water molecules are exchanging rapidly between a bound phase with restricted motions and a free phase with dynamic properties similar to liquid water. From this model and these data, estimates are obtained for the relaxation time for bound phase water. Possible relaxation mechanisms for bound phase water are discussed and some support is found for an intermolecular interaction modulated by translational motions characterized by a diffusion constant of 10^?9 cm²/s.     

Determination of the relaxation time of the helix–coil transition of poly(γ-benzyl-L-glutamate) by the nmr technique

NMR measurements of poly(γ-benzyl-L -glutamate) are reported in several different strengths of magnetic field to determine the relaxation time of the helix–coil transition. Nmr spectra of various samples had line shapes varying from the double to single, depending on the extent of the polydispersity of the sample. This result indicated that the correct line shape of a polypeptide is obscured in the overlapping of multipeaks, which are due to the heterogeneity of the molecular weight in the sample. Thus, the conventional line-shape analysis could not be applied to the kinetic study of the helix–coil transition of polypeptides without consideration of this polydispersity effect on the line shape. To overcome this difficulty, we measured linewidths of nmr spectra for fairly monodisperse samples, using various nmr spectrometers, having field strengths from 60 to 220 MHz. The results were analyzed by a quadratic equation, which involves an additional term proportional to the frequency difference of two sites. The equation differs from the conventional quadratic equation, usually utilized in the case of the fast-exchange limit, only in this additional term. This modification is required to evaluate correctly the unusual broadening of the linewidth resulting from the polydispersity effect and to determine the relaxation time reflected in nmr. Nmr spectra of three samples (DP-35, 85, and 250) were measured by 220-, 100-, and 60-MHz spectrometers in trifluoroacetic acid/chloroform at 28°C and linewidths were analyzed. Relaxation times of the helix–coil transition obtained at the transition midpoint are 2.5 × 10^?4, 7 × 10^?4, and 1.1 × 10^?3 sec, for DP-35, 85, and 250, respectively.     

The dynamics of the DNA hydration shell at gigahertz frequencies

We have used Brillouin scattering to measure the linewidths and frequencies of GHz acoustic phonons in Na- and Li-DNA films as a function of temperature between 300 and 140 K for samples that were dry, lightly, and heavily hydrated. The linewidths decrease with falling temperature and water contents, indicating that coupling to a water relaxation is the main source of phonon damping. The strength of the relaxation was determined using measurements of the phonon linewidth as a function of frequency, and confirmed by comparison of measured and calculated spectral profiles. The relaxation strength is anisotropic, being greater for phonons propagating perpendicular to the helix axis. The hydrated DNA exhibits both a rapid relaxation (≤ 10^?11 s per radian) giving rise to a classical f² damping, and a slower motion with a relaxation time that varies from ～ 4 × 10^?11 s per radian (primary hydration shell) to ～ 2 × 10^?12 s per radian (secondary hydration shell) at room temperature. In the frequency interval that bounds these relaxation times (～ 4 to 80 GHz) we expect degrees of freedom associated with the primary hydration shell to be important. The sample with primary hydration follows a simple Arrhenius behavior with ΔH ～ 5 kcal mole^?1. The effective activation energy for the sample with secondary hydration is somewhat higher (indicating a more cooperative water relaxation) and varies strongly with temperature. The elastic moduli change much more than can be accounted for by relaxation, indicating the importance of water motion in softening interatomic potentials. The extent of the softening caused by the “unfreezing” of water motion is similar to the degree of softening caused by hydrating the sample.     

Dielectric behaviour of frozen DNA in solution

In this article, measurements are reported on ice and frozen DNA solutions between 100 Hz–10 MHz. Pure ice is shown to exhibit single relaxation behaviour, which confirms previous work taken over a more restricted frequency range. The frozen DNA solution displays double‐dispersion behaviour. One dispersion centred around 3 kHz is due to a defect mechanism while the other, centred around 2 MHz, may be attributed to counterion flow through the water immediately adjacent to the DNA molecule. Bioelectromagnetics 20:40–45, 1999. © 1999 Wiley‐Liss, Inc.     

Rotational and translational water diffusion in the hemoglobin hydration shell: dielectric and proton nuclear relaxation measurements.

The dynamic properties of water in the hydration shell of hemoglobin have been studied by means of dielectric permittivity measurements and nuclear magnetic resonance spectroscopy. The temperature behavior of the complex permittivity of hemoglobin solutions has been measured at 3.02, 3.98, 8.59, and 10.80 GHz. At a temperature of 298 K the average rotational correlation time tau of water within a hydration shell of 0.5-nm thickness is determined from the activation parameters to be 68 +/- 10 ps, which is 8-fold the corresponding value of bulk water. Solvent proton magnetic relaxation induced by electron-nuclear dipole interaction between hemoglobin bound nitroxide spin labels and water protons is used to determine the translational diffusion coefficient D(T) of the hydration water. The temperature dependent relaxation behavior for Lamor frequencies between 3 and 90 MHz yields an average value D(298K) = (5 +/- 2) x 10(-10)m2 s-1, which is about one-fifth of the corresponding value of bulk water. The decrease of the water mobility in the hydration shell compared to the bulk is mainly due to an enhanced activation enthalpy.     

Microwave dielectric study on hydration of moist collagen

Two dielectric relaxation peaks were found in moist collagen by the time domain reflectometry. The low-frequency peak around 100 MHz moves little as the water content is varied. Its relaxation strength depends on the content and vanishes for completely dried collagen. This process is concluded to be due to water molecules strongly bound to the tropocollagen. Amount of the bound water is estimated as 0.12 g water/g collagen. Twenty-one water molecules are bound to one repeat of the triple helix. The existence of stringlike water chains is suggested. If the water content is less than 0.5 g water/g collagen, the high frequency peak locates between those of bound and bulk water. Water among the tropo-collagen is weakly bound to the collagen. In the higher region it does not change much with the content, being close to that of bulk water. The bulk water appears in this region.     

Conformational and kinetic features of the morphine-Mn(II) interaction as probed by nuclear paramagnetic resonance investigations

The nature of binding between manganese ions and morphine was studied using Fourier transform proton nuclear magnetic resonance techniques. Proton relaxation times in the presence of Mn(II) ions were determined together with their temperature dependence. Slow exchange conditions were observed for the NCH₃ group, while fast exchange conditions applied for all the other protons. The rotational correlation time of the complex was approximated by that of the free morphine molecule, as measured by selective and nonselective proton relaxation rate measurements. The distances between the metal ion and proton nuclei of morphine were evaluated on the basis of an association constant, measured from water proton spin-lattice relaxation rate binding studies. The results indicate that the metal binds directly to the two oxydryls with K_ass = 9.7 × 10^?3.The rate constant for the interaction of Mn(II) with the opiate is 2.25 × 10⁴ sec^?1 at 27°C, as determined from the temperature dependence of longitudinal relaxation rate of the NCH₃ group.     

Molecular dynamics of hinge-bending motion of IgG vanishing with hydrolysis by papain

We have performed dielectric relaxation measurements via a time domain reflectometry (TDR) method to study dynamic behaviors of the segmental flexibility of immunoglobulin G (IgG) in aqueous solution without antigen binding. In general, an intermediate relaxation process due to bound water is observed around 100 MHz at 25 degrees C for common proteins between two relaxation processes due to overall rotation and reorientation of free water. However, the intermediate process observed around 6 MHz for IgG was due to both bound water and hinge-bending motion. The apparent activation energy of 33 kJ/mol was larger than 27 kJ/mol for only bound water, and the relaxation strength was about five times as large as expected for bound water. The shape of the relaxation curve was very broad and asymmetric. These characteristic differences arising from the hinge-bending motion of IgG disappeared for fragments decomposed from IgG hydrolyzed by papain, since the hinge-bending motion did not exist in this case. We have separated the relaxation processes due to hinge-bending motion and bound water for IgG and obtained the Fab-Fab angle of IgG as about 130 degrees by Kirkwood's correlation parameter and the activation energy of 34 kJ/mol for hinge-bending motion.     

Water molecule binding and lifetimes on the DNA duplex d(CGCGAATTCGCG)2

Summary Measurements of the water proton spin-lattice relaxation rate for aqueous solutions of the palindromic dodecamer, d(CGCGAATTCGCG)₂, are reported as a function of the magnetic field strength. The magnitude of the relaxation rates at low magnetic field strengths and the shape of the relaxation dispersion curve permit assessment of the number of water molecules which may be considered bound to the DNA for a time equal to or longer than the rotational correlation time of the duplex. The data are examined using limiting models that arbitrarily use the measured rotational correlation time of the polynucleotide complex as a reference point for the water molecule lifetime. If it is assumed that water molecules are bound at DNA sites for times as long as or longer than the rotational correlation time of the duplex, then the magnitude of the relaxation rates at low field require that there may be only two or three such water sites. However, if the lifetime constraints is relaxed, and we assume that the number of water molecules bound to the DNA is more nearly the number identified in the X-ray structures, then the average water molecule lifetime is on the order of 1 ns. Measurements of ¹H NOESY spectra demonstrate that some water molecules must have lifetimes sufficiently long that negative Overhauser effects are observed. Taken together, these results suggest a distribution of water molecule lifetimes in which most of the DNA-bound water molecule lifetimes are shorter than the rotational correlation time of the duplex, but where some have lifetimes of at least 1 ns under these concentrated conditions.Abbreviations DNA deoxyribonucleic acid - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy     

31P nmr spin-lattice relaxation studies of deoxyoligonucleotides

Temperature-dependent conformational transitions of deoxyoligonucleotides have been monitored by measuring ³¹P chemical shifts, spin-lattice relaxation times (T₁), and ³¹P-{H} nuclear Overhauser enhancements (NOEs). The measured NOE ranged from 30 to 80%, compared to the theoretical maximum of 124% for a dipolar relaxation mediated by rapid isotropic rotation. The observed 3′-5′ phosphate diester ³¹P T₁ showed a similar temperature dependence over the range 2–75°C for both double- and single-stranded oligonucleotides, and for dinucleotides. The results show that dipole–dipole interactions dominate the internucleotide phosphate relaxation rate in oligonucleotides. The same is true of terminal phosphate groups at low temperature; but at higher temperature another process, possibly due to contamination by paramagnetic ions, becomes dominant. The rotational correlation time τ_R calculated from the dipole–dipole relaxation rate of the internucleotide phosphate in d(pA)₂ at 16°C is τ_R = 5.0 × 10^?10 sec, implying a Stokes radius for isotropic rotation of 7.6 Å. The T₁ and NOE values for the double-helical octanucleotide d(pA)₃pGpC(pT)₃ are consistent with dominance of dipole–dipole relaxation and isotropic rotation of a sphere of radius 14 Å, a reasonable dimension for the double helix. Activation energies for the rotation of dinucleotides range from 4 to 6 kcal/mol, close to the value of 4 kcal/mol expected for isotropic rotation. In order to test the possible effect of internal motion of correlation time τ_G on the results, we considered a model in which the nucleotide chain rotates about the P-O bonds. Comparison of the calculation with our experimental results shows that internal motion with τ_G ? 10^?9 sec, as found from other studies to be present for large nucleic acids, would not influence out T₁ and NOE values enough to be distinguished from isotropic rotation. However, we can conclude that τ_G cannot be as fast as 10^?10 sec, even for dinucleotides.     

81 Br-NMR studies of carbonic anhydrase

⁸¹Br nmr measurements have been made on high (bovine; BCA) and low (human-B; HCAB) specific activity forms of carbonic anhydrase and on a chemically modified form of the human enzyme (carboxyamidomethyl; CAM-HCAB). The high specific activity form of the enzyme, BCA, exhibits a ⁸¹Br line broadening which is determined by the lifetime of Br^? bound to the zinc ion of the enzyme. The low specific activity form of the enzyme, HCAB, under similar conditions of concentration, pH, etc., does not exhibit a ⁸¹Br nmr line broadening. $\text{Cl}{}^{\mathrm{?}}\text{Br}{}^{\mathrm{?}}$ competitive binding studies, using ³⁵Cl nmr, suggests that the failure to observe ⁸¹Br broadening is due to an increase in the lifetime of a zinc bound Br^?. An increase in this lifetime by a factor of 10–100 over that exhibited by BCA is sufficient to abolish the line broadening. A modified form of HCAB, CAM-HCAB, does, however, exhibit a ⁸¹Br nmr line broadening. Estimates of the lifetime of zinc bound Br^?, τ_M, are 4 × 10^?7 sec. for CAM-HCAB at pH 8 and 1 × 10^?7 sec. for BCA at pH 7. The lifetime for Br^? bound to HCAB is estimated to be ≥10^?6 sec.     

Infinite dilution viscoelastic properties of poly(gamma-benzyl-L-glutamate) in m-cresol.

The real and imaginary parts of complex viscosity, η′ and η″, are measured for dilute solutions of poly(γ-benzyl-L -glutamate) in m-cresol, a helicogenic solvent. The frequency range is 2.2–525 kHz; the concentration range 0.2–5 g/dl; the temperature 30°C, and the molecular weights M_r are 6.4 × 10⁴–17 × 10⁴. The dispersion curve of extrapolated intrinsic dynamic viscosity [η′] of samples with M_r > 10⁵ is interpreted in terms of three mechanisms appearing from low to high frequencies: end-over-end rotation, flexural deformation, and side-chain motion. For a sample with M_r < 10⁵, the flexural relaxation disappears and a plateau of [η′] is distinctly observed between rotational and side-chain relaxations. Rotational relaxation times of all the samples obey the Kirkwood–Auer theory. The strong concentration dependence of rotational relaxation time is explained by collisions of molecules rather than association. Flexural relaxation times calculated from a theory by assuming the persistence length as 1200 Å are consistent with observed dispersion curves of [η′].     

Observation of unfreezable water in aqueous solution of globule protein by microwave dielectric measurement

A freezing process analyzed by the dielectric method on aqueous solution of albumin has revealed water structure around protein molecule. A relaxation peak due to bound water attached on the protein surface around 100 MHz at room temperature was found. It could be seen commonly in globule proteins. Another peak due to a different kind of unfreezable water was found around 1 GHz at ?6°C. The amount of this water is estimated as 0.36 g water/g protein and in good agreement with that obtained by differential scanning calorimetry and nmr measurements. The water molecules form a shell layer around the protein molecule. © 1995 John Wiley & Sons, Inc.