Intrinsic proton relaxation parameters of hydrated polyglycine from two‐dimensional time domain NMR

Proton two‐dimensional time domain nmr involving T₁, T_1ρ, T_1D, and T₂ measurements was applied to hydrated polyglycine powders. The results were analyzed for magnetization exchange and found to be consistent with a general three‐site (glycine–water–glycine) exchange model. The intrinsic glycine and water proton relaxation parameters as well as the three exchange rates were obtained. Estimates of correlation times for water molecule motion at hydration sites are presented. © 1999 John Wiley & Sons, Inc. Biopoly 50: 630–640, 1999     

Hydration of Escherichia coli lipids: Deuterium T1 relaxation time studies of phosphatidylglycerol,phosphatidylethanolamine and phosphatidylcholine

The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic $\text{1,2-}\text{dioleoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ in H₂O/²H₂O mixtures (9:1, v/v) were investigated with ²H-NMR. Comparison of the ²H₂O spin lattice relaxation time ($\text{T}{}_1$) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H₂O systems. Two distinct hydration regions could be discerned in the $\text{T}{}_1$ relaxation time profile. (1) A minimum of 11–16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of $\text{T}{}_1\text{≈ 90}\text{ms}$. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with $\text{T}{}_1\text{= 500}\text{ms}$ and hydration water with $\text{T}{}_1\text{= 80–120}\text{ms}$. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.     

NMR study on molecular mobility of DNA molecules in solution

The molecular mobility of calf thymus DNA molecules in solution has been discussed in terms of correlation time τ calculated from measurements of longitudinal T₁ and transverse T₂ magnetic relaxation times. The influence of DNA concentration and ionic strength of the solution upon freedom of movement of DNA molecules was studied for native and denatured DNA and also during thermal helix-coil transition. The dependence of τ values on temperature was carried out by comparing the values of correlation times τ_tat given temperature with the correlation time τ₂₀ at 20°C. The molecular rotation of DNA at 20°C and at higher ionic strength at 0.15 and 1.0.M NaCl is described by τ values of the order of 1.0–1.2 × 10^?8 and was reduced slightly with increase of temperature below the helix-coil transition. The molecular rotation of DNA in 0.02MNaCl was lower at 20°C as compared to DNA in solvents with higher NaCl concentrations and increases rapidly with increase of temperature in the range 20–60°C. The values of correlation time are characterized by fast increase at temperatures above the spectrophotometrically determined beginning of melting curve. The beginning of this increase is observed at about 65, 80, and 85°C for DNA in 0.02, 0.15, and 1.0MNaCl, respectively. Values of correlation time for denatured DNA are in all cases about 1.1–1.4 times that for native DNA. The obtained results are discussed in terms of conformation of DNA molecules in solution as well as in terms of water dipole binding in DNA hydration shells.     

Azaadamantyl nitroxide spin label: complexation with β-cyclodextrin and electron spin relaxation

Abstract

An iodoacetamide azaadamantyl spin label was studied in fluid solution and in 9:1 trehalose:sucrose glass. In 9:1 toluene:CH₂Cl₂ solution at 293 K, the isotropic nitrogen hyperfine coupling is 19.2?G, T₁ is 0.37 µs and T₂ is 0.30–0.35 µs. Between about 80 and 150 K 1/T_m in 9:1 trehalose:sucrose is approximately independent of temperature demonstrating that the absence of methyl groups decreases 1/T_m relative to that which is observed in spin labels with methyl groups on the alpha carbons. Spin lattice relaxation rates between about 80 and 293 K in 9:1 trehalose:sucrose are similar to those observed for other nitroxide spin labels, consistent with the expectation that relaxation is dominated by Raman and local mode processes. Although complexation of the azaadamantyl spin label with β-cyclodextrin slows tumbling in aqueous solution by about a factor of 10, it has little impact on 1/T₁ or 1/T_m in 9:1 trehalose:sucrose between 80 and 293 K.     

Mode‐coupling Smoluchowski dynamics of a double‐stranded DNA oligomer

The local dynamics of a double‐stranded DNA d(TpCpGpCpG)₂ is obtained to second order in the mode‐coupling expansion of the Smoluchowski diffusion theory. The time correlation functions of bond variables are derived and the ¹³C‐nmr spin–lattice relaxation times T₁ of different ¹³C along the chains are calculated and compared to experimental data from the literature at three frequencies. The DNA is considered as a fluctuating three‐dimensional structure undergoing rotational diffusion. The fluctuations are evaluated using molecular dynamics simulations, with the ensemble averages approximated by time averages along a trajectory of length 1 ns. Any technique for sampling the configurational space can be used as an alternative. For a fluctuating three‐dimensional (3D) structure using the three first‐order vector modes of lower rates, higher order basis sets of second‐rank tensor are built to give the required mode coupling dynamics. Second‐ and even first‐order theories are found to be in close agreement with the experimental results, especially at high frequency, where the differences in T₁ for ¹³C in the base pairs, sugar, and backbone are well described. These atomistic calculations are of general application for studying, on a molecular basis, the local dynamics of fluctuating 3D structures such as double‐helix DNA fragments, proteins, and protein–DNA complexes. © 1999 John Wiley & Sons, Inc. Biopoly 50: 613–629, 1999     

Conformation of tetranactin in solution.

The conformation of tetranactin, an ionophore, in chloroform was investigated by infrared and Raman spectra and by proton and ¹³C magnetic resonances. The infrared spectra show that the structure of its K⁺ complex in the solution is quite similar to that in crystals. The proton spin–spin coupling constants are explained well by assuming that the crystalline structure is retained in solution. The spin–lattice relaxation times of the ¹³C nuclei of the K⁺ complex indicate that its framework is rigid. The correlation time of the overall reorientation of the molecule was calculated to be 9 X 10^?11 sec. On the other hand, the conformation of the complexed form in chloroform differs from that in crystals. Despite the geometrical nonequivalence of the four subunits in the crystalline state, the nuclear magnetic resonance spectra show their magnetic equivalence in the solution. The proton spin–spin coupling constants have values that are averaged by rapid internal rotation. The spin–lattice relaxation times of the ¹³C nuclei in its framework are unexplained by the overall reorientation of the molecule, and reveal the existence of internal motion in the framework. The rate of the local motion of the framework is between 10²–10¹⁰ sec^?1. By comparison of the infrared spectra, it can be said that the mean conformation of the fluctuated framework of the uncomplexed tetranactin in the solution is similar to that of nonactin in the crystalline form, which has an S₄ symmetry axis through the center of the macrocyclic ring.     

Interactions of DNA with divalent metal ions. I. 31P-nmr studies

³¹P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg²⁺, Mn²⁺, and Co⁺², with the phosphate groups of DNA. Mg²⁺ is found to have no significant effect on any of the ³¹P-nmr parameters (chemical shift, line-width, T₁, T₂, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn²⁺ and Co²⁺, on the other hand, significantly change the ³¹P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn²⁺ and Co²⁺, respectively, corresponding to only 15 ± 5% of the total bound Mn²⁺ and Co²⁺ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the ³¹P relaxation rates of DNA in the absence and presence of Mn²⁺ and Co²⁺ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10^?7 and 1.4 × 10^?5 s for Mn²⁺ and Co²⁺, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg²⁺ ions is 3–4 times weaker than the binding of either Mn²⁺ or Co²⁺. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (～15%), the higher binding strength of Mn²⁺ and Co²⁺ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg²⁺ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ～100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.     

Nitroxide spin labels as EPR reporters of the relaxation and magnetic properties of the heme–copper site in cytochrome bo 3, E. coli

A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme–copper dinuclear site in Escherichia coli cytochrome bo _3, a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T ₁, of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T ₂, of the dinuclear coupled pair Fe(III)–Cu_B(II) in the oxidized form of QO that is too short to measure directly. In the case of the F′ state, the relaxation properties of the heme–copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu_B(II) coupled by approximately 1.5–3 cm⁻¹ to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine–histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements.     

Proton and phosphorus-31 magnetic relaxation studies on the interaction of polyriboadenylic acid with Mn2+

Proton and phosphorus-31 nuclear spin–lattice relaxation times T₁ and spin–spin relaxation times T₂ have been measured on the single-stranded polyriboadenylic acid [poly(A)]–Mn²⁺ system in a neutral D₂O solution in the temperature range 10°–90°C at 100 and 40.5 MHz, respectively, with the Fourier transform nmr method. Minimum values of T₁ have been found for all these nuclei, which have enabled the exact estimation of apparent distances from Mn²⁺ to H₂, H₈, H_1′, and the phosphorus nucleus to be 4.7, 4.1, 5.2, and 3.0 Å, respectively. The electron spin of Mn²⁺ penetrates into the phosphorus nucleus, giving ³¹P hyperfine coupling of more than 10⁶ Hz. Evidence of penetration of the electron spin into H₈ and H₂ is also obtained, suggesting direct coordination of nitrogen atoms of the adenine ring to the Mn²⁺ Ion. Combined with the result from proton relaxation enhancement of water, it is concluded that every Mn²⁺ ion added is bound directly to two phosphate groups with a Mn²⁺–phosphorus distance of 3.3 Å, while a part of the Mn²⁺ ions are simultaneouly bound to the adenine ring. It is estimated that 39 ± 13% and 13 ± 5% of Mn²⁺ are coordinated by N₇ and N₃ (or N₁), respectively. The motional freedom of poly(A) in the environment of the Mn²⁺ binding site has been found to be quenched to the extent that the rotational motion becomes several times slower than that of the corresponding Mn²⁺–free poly(A). The activation energies for the molecular motion are, however, practically unchanged from those for Mn²⁺–free poly(A), and are found to be 8.3, 8.5, 6.1, and 8.7 kcal/mol for H₈, H₂, H_1′, and phosphorus, respectively. T₂ of phosphorus is determined by the dissociation rate (k_?1) of Mn²⁺ from the phosphate group for the whole temperature range studied with activation enthalpy of 6.5 kcal/mol. The dissociation rates of Mn²⁺ from the adenine ring are also estimated from proton T₂ values below 50°C.     

Copper(II)-DNA denaturation. II. The model of DNA denaturation

In a continuing study of the denaturation of DNA as brought abought about by Cu(II) ions, results are presented for the dependence of T_m and τ (the terminal relaxation time) on ionic strength, pH, reactant concentrations, and temperature. Maximum stability of the double helix, as reflected by the longest relaxation times and highest T_m values, was observed between pH 5.3 and 6.2. Outside this range both T_m and τ decreased sharply. A second, faster relaxation time was deduced from the kinetic cureves. The apparent activation energies of the rapid and slow (“terminal”) relaxations were found to be 12 and 55 kcal/mole, respectively. Several lines of evidence led to the conclusions that (1) the rate-determining step in DNA denaturation, when occurring in the transition region, is determined by chemical events and (2) the interactions which are disrupted kinetically in the rate-determining step are those which account for the major portion of the thermal (T_m) stability of helical DNA.     

Sensing mammographic density using single-sided portable Nuclear Magnetic Resonance

This research paper presents a quantitative approach to sensing mammographic density (MD) using single-sided portable Nuclear Magnetic Resonance (NMR). It focuses on three main techniques: spin–lattice relaxation (recovery) time (T₁), spin–spin relaxation (decay) time (T₂), and Diffusion (D) techniques by testing whether or not the aforementioned techniques are in agreement with the gold standard and with each other when used for scanning breast tissue specimens with a variety of mammographic densities (MDs). The high mammographic density (HMD), intermediate MD, and low mammographic density (LMD) regions of each slice were identified according to the mammogram images. Subsequently, the grayscale values for these regions were quantified. One region was measured from the first sample while the remaining ones were measured from the second sample. The same areas were then exposed to portable NMR, and the sequences used as following: the stimulated echo sequence for diffusion (D), the Carr-Purcell-Meiboom-Gill (CPMG) sequence for T₂, and saturation recovery sequence for T₁. The correlations between the grayscale values and NMR techniques were strongly correlated. The Pearson correlation coefficient, R, of T₁ (%) versus grayscale value, D (%) versus grayscale value, and T₂ (%) versus grayscale value, was 0.91, 0.91, and 0.93, respectively. Furthermore, the relative water content of the breast slices based on T₁, T₂, and diffusion (D) measurements were strongly in agreement with each other. The Pearson correlation coefficient, R, of D (%) versus T₁ (%), D (%) versus T₂ (%), and T₁ (%) versus T₂ (%), was 0.984, 0.966, and 0.9868, respectively. The three pulse sequences can be employed in a portable NMR device to deliver continuous quantitative measurements of MD in breast tissue samples. As a result, the method demonstrated to be acceptable for determining the distribution of MDs among breast tissue samples without the need for additional qualitative analysis.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

Intermediates in the strand-separation transition of T7 DNA.

Sedimentation velocity runs as a function of temperature in the region of the alkaline helix-coil transition have enabled us to demonstrate the existence of stable two-stranded intermediates in the strand-separation process for T7 DNA. The strand-separation transition under these conditions has an intrinsic breadth of ～1°C, and within this temperature range (T_m + 2°C < T < T_m + 3°C) the intermediate forms are progressively converted (as a function of temperature) to single-stranded DNA. Parallel characterizations of the strand-separation transition by viscosity and absorbance–renaturation studies in the alkaline solvent are entirely consistent with the sedimentation experiments. Comparison of the experimental mean sedimentation coefficient of the intermediate forms with theoretical predictions for branched structures suggests that in these molecules the two strands are connected at a single point, not centrally located with respect to the ends of the molecule.     

Anisotropic rotational motions of poly(L-glutamic acid) in the alpha-helix conformation

The anisotropic rotational motion of the backbone and the side chains of poly(L -glutamic acid) in the α-helical structure was investigated using the ¹³C-T₁ and T₂ relaxation times of all carbon atoms with directly attached protons, obtained at a ¹³C-Larmor frequency of 67.89 MHz. The evaluation of the nmr data was carried out according to the previously derived anisotropic diffusion model, in which the macromolecule is considered a rigid rod. The rotation of the backbone is characterized by two diffusion constants, D₁ and D₃, describing the rotation perpendicular to and around the symmetry axis. The additional internal motion of the C^β-methylene group is described as a jump process with a jump rate, k₁, between two allowed rotametric states. Steric considerations indicate that the occupation of the third rotameric position is forbidden. The rotation of the C^γ-methylene group is decribed as a one-dimensional diffusion process around the C^β–C^γ bond. Investigation of the temperature dependence of the relaxation parameters led to the temperature dependence of the dynamic parameters. Activation energies were determined from these data. The dynamic parameters obtained for poly(L -glutamic acid) at 291 K are compared with the corresponding results of a previous study of poly(L -lysine). The development of an anisotropic diffusion model for the motions of the rod-shaped poly(L -lysine) α-helix and its application to the interpretation of the ¹³C-relaxation data of this molecule have already been published previously. In this model, both the overall molecular tumbling and the various internal motions have been characterized by diffusion constants or jump rates typical for each process. These dynamic parameters can be calculated from the spin–lattice relaxation times, the spin–spin relaxation times and the NOE factors of the C^α, C^β, and C^γ nuclei of the polypetide. In the present paper, we describe the application of the above-mentioned dynamic model to the interpretation of ¹³C-relaxation studies of a further homopolypeptide, poly(L -glutamic acid), in the α-helical structure. Furthermore, we studied the temperature dependence of the relaxation times of this polymer and determined the anisotropic diffusion parameters at each temperature. From their temperature dependence and from comparison of our present results with the data of our previous study of poly(L -lysine), we were able to derive new insights into the intramolecular diffusion processes and the excitation of various motions.     

Index to Authors,Volume 7

Abstract

The thermally stimulated depolarization current (TSDC) measurements in frozen aqueous solutions, gels and solid layers of NaDNA show typically up to three dipolar overlapping peaks in the low-temperature range of 80—;150 K. Up to four discrete relaxation peaks have been observed at higher temperatures above 150 K. The low-temperature TSDC peaks are due to the dipolar relaxations of free and loosely bound water which crystallizes. Part of bound water especially in the first hydration shell of DNA molecule is at low temperatures in the form of glass. The transition of this glass from solidlike behavior to liquidlike behavior observed mainly in gels and solid samples is associated with a previously founded TSDC relaxation peak. The peak is at its maximum at 165- 250 K depending on the sample humidity. Existence of this relaxation in the samples with water contents in a broad range confirms, that the slowly relaxing shell (minimally 5–7 water molecules/nucleotide) closely associated with DNA double helix retains its characteristics. Also another peak of the high-temperature band at 180–205 K which was observed in the samples at hydration 2–1800 g H₂O/g dry NaDNA is due to a relaxation in the sample volume. At the highest temperatures relax the space charges trapped at the electrodes.     

Interaction of the antimalarial drug fluoroquine with DNA,tRNA, and poly(A): 19F-NMR chemical-shift and relaxation,optical absorption,and fluorescence studies

The interaction of the fluorinated antimalarial drug fluoroquine [7-fluoro-4-(diethyl-amino-1-methylbutylamino)quinoline] with DNA, tRNA, and poly(A) has been investigated by optical absorption, fluorescence, and ¹⁹F-nmr chemical-shift and relaxation methods. Optical absorption and fluorescence experiments indicate that fluoroquine binds to nucleic acids in a similar manner to that of its well-known analog chloroquine. At low drug-to-base pair ratios, binding of both drugs appears to be random. Fluoroquine and chloroquine also elevate the melting temperature (T_m) of DNA to a comparable extent. Binding of fluoroquine to DNA, tRNA, or poly(A) results in a downfield shift of about 1.5 ppm for the ¹⁹F-nmr resonance. The chemical shift of free fluoroquine depends on the isotopic composition of the solvent (D₂O vs H₂O). The solvent isotope shift is virtually eliminated by fluoroquine binding to any one of the nucleic acids. ¹⁹F-nmr relaxation experiments were carried out to measure the spin-lattice relaxation time (T₁), ¹⁹F{¹H} nuclear Overhauser effect (NOE), off-resonance intensity ratio (R), off-resonance rotating-frame spin-lattice relaxation time (T), and linewidth for fluoroquine in the nucleic acid complexes. By accounting for intramolecular proton-fluorine dipolar and chemical-shift anisotropy contributions to the fluorine relaxation, all of the relaxation parameters for the fluoroquine–DNA complex can be well described by a motional model incorporating long-range DNA bending on the order of a microsecond and an internal motion of the drug on the order of a nanosecond. Selective NOE experiments indicate that the fluorine in the drug is near the ribose protons in the RNA complexes, but not in the DNA complex. Details of the binding evidently differ for the two types of nucleic acids. This study provides the foundation for an investigation of fluoroquine in intact cells.     

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.     

Determination of NOE "silent" dipolar interactions between magnetically equivalent nuclei: application to poly(dA).poly(dT)

The ratio of the spin–spin relaxation rate, R₂, to the selective spin–lattice relaxation rate, R, can be used to detect and determine the magnitude of dipolar interactions between magnetically equivalent unclei in macromolecules. We use this method to show that there is strong interaction between adjacent AH2 protons in poly(dA) · poly(dT). The largest possible AH2–AH2 distance that will account for the observed magnitude of the interaction is ～3.5 Å, provided the AH2 protons are located close (within 0.6 Å) to the helix axis. Structures with shorter AH2–AH2 separations could account for the nmr data, but they are rejected because they would reduce the interbase separation to unacceptably small values. A comparison of rates measured at 360 and 500 MHz shows that the chemical-shift anisotropy makes a negligible contribution to the observed relaxation rates.     

Thermodynamic analysis of sol–gel transition of gelatin in terms of water activity in various solutions

Sol–gel transition of gelatin was analyzed as a multisite stoichiometric reaction of a gelatin molecule with water and solute molecules. The equilibrium sol–gel transition temperature, T_t, was estimated from the average of gelation and melting temperature measured by differential scanning calorimetry. From T_t and the melting enthalpy, ΔH_sol, the equilibrium sol‐to‐gel ratio was estimated by the van't Hoff equation. The reciprocal form of the Wyman–Tanford equation, which describes the sol‐to‐gel ratio as a function of water activity, was successfully applied to obtain a good linear relationship. From this analysis, the role of water activity on the sol–gel transition of gelatin was clearly explained and the contributions of hydration and solute binding to gelatin molecules were separately discussed in sol–gel transition. The general solution for the free energy for gel‐stabilization in various solutions was obtained as a simple function of solute concentration. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 685–691, 2015.     

The state of water on hydrated collagen as studied by pulsed NMR

The spin-lattice relaxation time (T₁) of water adsorbed on collagen fibers was determined at six frequencies and temperatures varying from 25° to ?80°C. Care was taken to eliminate the contributions to the signal of protons other than those in the adsorbed water. Quantitative calculations were made on T₁ and the results were compared with the experimental data. It is suggested that a maximum of about 0.50–0.55 g water per g collagen forms a hydration layer, which cannot be frozen down to ?90°C and exhibits a distribution of motional correlation times. For collagen samples containing a larger quantity of adsorbed water, the additional water molecules behave like ordinary isotropic water, having a single correlation time and a freezing temperature of about ?10°C.