On the mechanism of dielectric relaxation in aqueous DNA solutions.

The complex dielectric response of calf thymus DNA in aqueous saline solutions has been measured from 1 MHz to 1 GHz. The results are presented in terms of the relaxation of the incremental contributions to the permittivity and conductivity from the condensed counterions surrounding the DNA molecules. Measurements of the low-frequency conductivity of the samples also lends support to the condensed counterion interpretation.     

Dynamics of water in supercooled aqueous solutions of glucose and poly(ethylene glycol)s as studied by dielectric spectroscopy

The dielectric behaviour of aqueous solutions of glucose, poly(ethylene glycol)s (PEGs) 200 and 600, and poly(vinyl pyrrolidone) (PVP) has been examined at different concentrations in the frequency range of 10(6)-10(-3) Hz by dielectric spectroscopy and by using differential scanning calorimetry down to 77 K from room temperature. The shape of the relaxation spectra and the temperature dependence of the relaxation rates have been critically examined along with temperature dependence of dielectric strength. In addition to the so-called primary (alpha-) relaxation process, which is responsible for the glass-transition event at T(g), another relaxation process of comparable magnitude has been found to bifurcate from the main relaxation process on the water-rich side, which continues to the sub-T(g) region, exhibiting relaxation at low frequencies. The sub-T(g) process dominates the dielectric measurements in aqueous solutions of higher PEGs, and the main relaxation process is seen as a weak process. The sub-T(g) process was not observed when water was replaced by methanol in the binary mixtures. These observations suggest that the sub-T(g) process in the aqueous mixtures is due to the reorientational motion of the 'confined' water molecules. The corresponding dielectric strength shows a noticeable change at T(g), indicating a hindered rotation of water molecules in the glassy phase. The nature of this confined water appears to be anomalous compared to most other supercooled confined liquids.     

Water structures of differing order and mobility in aqueous solutions of schizophyllan, a triple-helical polysaccharide as revealed by dielectric dispersion measurements

Dielectric dispersion measurements were made on aqueous solutions of a triple-helical polysaccharide schizophyllan over a wide concentration range 10-50 wt % at -45 to +30 degrees C. In the solution state, three different water structures with the different relaxation times tau were found, namely, bound water (taul), structured water (taus), and loosely structured water (tauls) in addition to free water (tauP). Structured water is less mobile and loosely structured water is nearly as mobile as free water, but bound water with taul is much less mobile, thus taul > taus > tauls greater, similar tauP. The order-disorder transition accompanies the conversion between structured water and loosely structured water. However, the species with taus remains even in the disordered state and constitutes part of bound water in the entire temperature range. In the frozen state, in addition to bulk water formed by partial melting, two mobile species existed, which were assigned to liquidlike bound water and found to be a continuation of bound water in the solution state. These relaxation time data are discussed in connection with the entropy levels of the four structures deduced from heat capacity data (cf. Yoshiba, K.; et al. Biomacromolecules 2003, 4, 1348-1356).     

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

Selective incorporation of ¹³C into the methyl groupsof protein side chains is described as a means for simplifying themeasurement and interpretation of ¹³C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3-¹³C, 99%) as the sole carbon source in the growthmedia for protein overexpression in E. coli. This improved labeling schemeincreases the sensitivity of the relaxation experiments by approximatelyfivefold when compared to randomly fractionally ¹³C-labeledprotein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost.     

Motion of groups of atoms in DNA studied by molecular dynamics simulation

The analysis of Molecular Dynamics simulations of two double stranded oligonucleotides is presented in terms of motions of quasi rigid subunits. First, a strategy is presented for grouping atoms submitted to concerted internal motions. The method is based on the analysis of the interatomic distance RMS matrix. It is found that each nucleotide can reasonably be decomposed into 3 or 4 rigid groups of atoms depending on the tolerance of the definition of a rigid body. In the second part, the different kinds of motions of the subunits (deformation, translation and rotation) are studied in terms of correlation using the canonical correlation analysis of data. It is shown that the residual deformation of any subunit does not influence the translational and rotational motions of the others, except perhaps for long time dynamics. Received: 3 October 1997 / Revised version: 22 December 1997 / Accepted: 23 December 1997     

13C Nuclear magnetic relaxation study of segmental dynamics of hyaluronan in aqueous solutions

(13)C spin-lattice relaxation times (T(1)) and nuclear Overhauser enhancements (NOE) were measured as a function of temperature and magnetic field strength for the hetero-polysaccharide hyaluronan in water solutions. The relaxation data of the endocyclic ring carbons were successfully interpreted in terms of chain segmental motions by using the bimodal time-correlation function of Dejean de la Batie, Laupretre and Monnerie. On the basis of the calculated correlation times for segmental motion and amplitudes of librational motions of the C-H vectors at the various carbon sites of the HA repeating unit, we concluded that intramolecular hydrogen bonding of the secondary structure of HA plays a major role in the conformational flexibility of this carbohydrate molecule. The internal rotation of the free hydroxymethyl groups about the exocyclic C-5-C-6 bonds superimposed on segmental motion has been described as a diffusion process of restricted amplitude. The rate and amplitude of the internal rotation indicate that the hydroxymethyl groups are not involved in intramolecular hydrogen bonding. Finally, the motional parameters describing the local dynamics of the HA chain were correlated with the secondary structure of HA in aqueous solutions.     

Methyl motions in 13C-methylated concanavalin as studied by 13C magnetic resonance relaxation techniques

The carbon- 13 spin-lattice relaxation times and nuclear Overhauser enhancements of the N epsilon-monomethyllysine, N epsilon,N epsilon-dimethyllysine, and N alpha,N alpha-dimethylalanine resonances of 13C-methylated concanavalin A have been measured at three carbon frequencies and compared to the relaxation parameters predicted by several motional models. The experimental parameters cannot be reproduced by a simple dipolar relaxation model which includes isotropic reorientation of the protein plus free internal rotational diffusion of the methyl groups but are well predicted by a wobble in a cone model which includes isotropic reorientation of the protein at 33 ns, free internal rotational diffusion of the methyl groups, and a wobble diffusion which reflects the net motion of the amino acid side chains. The analysis indicates that the methylated epsilon-amino side chains exhibit only slightly more motional freedom than does the methylated N-terminal alpha-amino group and suggests some restriction of methyl group rotation in the dimethylamino residues.     

Internal motions of apo-neocarzinostatin as studied by 13C NMR methine relaxation at natural abundance

Summary Dynamics of the backbone and some side chains of apo-neocarzinostatin, a 10.7 kDa carrier protein, have been studied from ¹³C relaxation rates R1, R2 and steady-state ¹³C-{¹H} NOEs, measured at natural abundance. Relaxation data were obtained for 79 nonoverlapping C resonances and for 11 threonine C single resonances. Except for three C relaxation rates, all data were analysed from a simple two-parameter spectral density function using the model-free approach of Lipari and Szabo. The corresponding C–H fragments exhibit fast (_e < 40 ps) restricted libration motions (S²=0.73 to 0.95). Global examination of the microdynamical parameters S² and _e along the amino acid sequence gives no immediate correlation with structural elements. However, different trends for the three loops involved in the binding site are revealed. The -ribbon comprising residues 37 to 47 is spatially restricted, with relatively large _e values in its hairpin region. The other -ribbon (residues 72 to 87) and the large disordered loop ranging between residues 97–107 experience small-amplitude motions on a much faster (picosecond) time scale. The two N-terminal residues, Ala¹ and Ala², and the C-terminal residue Asn¹¹³, exhibit an additional slow motion on a subnanosecond time scale (400–500 ps). Similarly, the relaxation data for eight threonine side-chain C must be interpreted in terms of a three-parameter spectral density function. They exhibit slower motions, on the nanosecond time scale (500–3000 ps). Three threonine (Thr⁶⁵, Thr⁶⁸, Thr⁸¹) side chains do not display a slow component, but an exchange contribution to the observed transverse relaxation rate R2 could not be excluded at these sites. The microdynamical parameters (S², _e and R2_ex) or (S _infslow ^sup2 , S _inffast ^sup2 and _slow) were obtained from a straightforward solution of the equations describing the relaxation data. They were calculated assuming an overall isotropic rotational correlation time _e for the protein of 5.7 ns, determined using standard procedures from R2/R1 ratios. However, it is shown that the product (1–S²)× _e is nearly independent of _e for residues not exhibiting slow motions on the nanosecond time scale. In addition, this parameter very closely follows the heteronuclear NOEs, which therefore could be good indices for local fast motions on the picosecond time scale.     

Properties of aqueous solutions of lentinan in the absence and presence of zwitterionic surfactants

Morphological and conformational transitions of lentinan (LT), a β-glucan extracted from Shiitake mushrooms (Lentinula edodes), were investigated at different concentrations of aqueous NaOH, using Small Angle X-ray Scattering (SAXS) technique. At low NaOH_(aq) concentration LT chains are self-associated and adopt the triple helix form where as at higher NaOH concentrations the polymer chains undergo a transition to random coil chains. Also, the presence of fractal dimensions was observed through analysis of the exponential decay of the scattering intensity as a function of the scattering angle. In addition, the lateral radius of gyration was determined for LT in different concentrations of NaOH solution, indicating a rigid triple helix present as a small rod-like structure. Interactions of LT with two zwitterionic surfactants were investigated by surface tension, fluorescence, and static light scattering measurements. Experimental data showed that the formation of LT–(surfactant) complexes occurred through a cooperative process.     

The contribution of proton fluctuation to dielectric relaxation in protein solutions

Equations are derived which account for the effect of an applied electric field on the fluctuation of protons associated with a macromolecule. The contribution of this proton polarization to the complex permittivity of the macromolecules is evaluated in terms of its effect on both the dispersion due to proton fluctuation and the dispersion caused by dipolar rotation. An expression for the fluctuation correlation time τiδ is derived in terms of the mean lifetimes of the ionised and unionised state τ0 and τ+. If τiδ is very much less than the dipolar correlation time τi, the fluctuation dispersion will occur at much higher frequencies than the dispersion due to orientation polarization; hence the dispersions will be well separated. If τiδ » τi the two regions would overlap and would be indistinguishable as separate entities. At present insufficient data are available to test rigorously these conclusions but the potentialities of the theory in relation to small globular proteins is shown.     

Frequency dependence of the proton magnetic relaxation in aqueous solutions iof haemoproteins

The longitudinal proton magnetic relaxation times T₁ were measured for ferri (met)-and carbonmonoxy-bovine haemoglobin and equine myoglobin in 0.1 M KH₂PO₄ aqueous solutions near pH 6 at 5°C and 35°C from 1.5- to 60-MHz Larmor frequencies. It is concluded that the correlation time τ_C for the dipole–dipole interaction of electron and nuclear spins is in fact the electron (ferric) spin relaxation time τ_S being close to 1.5 × 10^?10 sec for both metHb and metMb at 5°C. At 35°C the paramagnetic relaxation rates are not determined solely by the relaxation of protons exchanging from the haem pocket with bulk solvent. Hence, τC at 35°C cannot be calculated from the dispersion data obtained at this temperature. The relevance of this for the determination of interspin distances r is discussed.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved 13C and 15N signals of [1-13C]Tyr-, [15N]Pro- and [1-13C]Val-, [15N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-13C]Val-, [15N]Pro signals to the specific residues in bR. The previous assignments of [1-13C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent 15N chemical shifts of Pro bonded to Val in the presence of 13C-15N correlation, although no assignment of peak is feasible for 13C nuclei not bonded to Pro. 13C or 15N spin-lattice relaxation times (T1), spin-spin relaxation times under the condition of CP-MAS (T2), and cross relaxation times (TCH and TNH) for 13C and 15N nuclei and carbon or nitrogen-resolved, 1H spin-lattice relaxation times in the rotating flame (1H T1 rho) for the assigned signals were measured in [1-13C]Val-, [15N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid beta-sheet in spite of longer loop and possesses large amplitude motions as revealed from 13C and 15N conformation-dependent chemical shifts and T1, T2, 1H T1 rho and cross relaxation times. In addition, breakage of the beta-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved ¹³C and ¹⁵N signals of [1-¹³C]Tyr-, [¹⁵N]Pro- and [1-¹³C]Val-, [¹⁵N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-¹³C]Val-, [¹⁵N]Pro signals to the specific residues in bR. The previous assignments of [1-¹³C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent ¹⁵N chemical shifts of Pro bonded to Val in the presence of ¹³C-¹⁵N correlation, although no assignment of peak is feasible for ¹³C nuclei not bonded to Pro. ¹³C or ¹⁵N spin-lattice relaxation times (T₁), spin-spin relaxation times under the condition of CP-MAS (T₂), and cross relaxation times (T_CH and T_NH) for ¹³C and ¹⁵N nuclei and carbon or nitrogen-resolved, ¹H spin-lattice relaxation times in the rotating flame (¹H T_1ρ) for the assigned signals were measured in [1-¹³C]Val-, [¹⁵N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid β-sheet in spite of longer loop and possesses large amplitude motions as revealed from ¹³C and ¹⁵N conformation-dependent chemical shifts and T₁, T₂, ¹H T_1ρ and cross relaxation times. In addition, breakage of the β-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

A proton magnetic relaxation study of ferrimyoglobin in aqueous ionic solutions

Proton magnetic longitudinal T₁ relaxation times have been measured for acid (horse) ferrimyoglobin solutions [0.1 M NaCl and KH₂PO₄, 2 M NaCl and 1 M MgCl₂] from 5°C to 35°C in dependence on myoglobin concentration up to 6 mM. The enhancement of the relaxation rate due to the paramagnetic haem iron. which is observed in this temperature range is compared with analogous data for the ferrihaemoglobin solution. The conclusion is that the protons exchanging from the haem pocket with bulk solvent are not those from the water molecule at the sixth ligand site of haem iron. The exchanging protons are more than 4 Å away from the haem iron being closer to it in ferrimyoglobin than in ferrihaemogiobin. This distance becomes larger in solutions with higher salt concentration, the largest difference between 0.1 M NaCl and 1 M MgCl₂ being over one Angstrom unit. This indicates a conformational change of the haem pocket, possibly its tightening.     

Ionization of the acid-base groups of polyene antibiotics in aqueous solutions]

The acid-base characteristics of polyenic antibiotics, such as nystatin, mycoheptin and levorin in aqueous solutions were studied. A special procedure provided the use of potentiometric titration for investigation of ionization of the groups of vater-insoluble substances. The ionization constants of the carboxylic and amine groups of the antibiotics at several temperatures were determined. It was found that ionization of the acid group did not practically depend on the temperature. At the same time the heat effect of the amine group ionization was significant and amounted to about 10 kcal/mole. Thermodynamic analysis of the ionization process of the polyenic antibiotics in aqueous solutions was performed. Integral components defining the process energetics were calculated.     

Membrane assembly of the 16-kDa proteolipid channel from Nephrops norvegicus studied by relaxation enhancements in spin-label ESR

The 16-kDa proteolipid from the hepatopancreas of Nephrops norvegicus belongs to the class of channel proteins that includes the proton-translocation subunit of the vacuolar ATPases. The membranous 16-kDa protein from Nephrops was covalently spin-labeled on the unique cysteine Cys54, with a nitroxyl maleimide, or on the functionally essential glutamate Glu140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). The intensities of the saturation transfer ESR spectra are a sensitive indicator of spin-spin interactions that were used to probe the intramembranous structure and assembly of the spin-labeled 16-kDa protein. Spin-lattice relaxation enhancements by aqueous Ni(2+) ions revealed that the spin label on Glu140 is located deeper within the membrane (around C9-C10 of the lipid chains) than is that on Cys54 (located around C5-C6). In double labeling experiments, alleviation of saturation by spin-spin interactions with spin-labeled lipids indicates that spin labels both on Cys54 and on Glu140 are at least partially exposed to the lipid chains. The decrease in saturation transfer ESR intensity observed with increasing spin-labeling level is evidence of oligomeric assembly of the 16-kDa monomers and is consistent with a protein hexamer. These results determine the locations and orientations of transmembrane segments 2 and 4 of the 16-kDa putative 4-helix bundle and put constraints on molecular models for the hexameric assembly in the membrane. In particular, the crucial DCCD-binding site that is essential for proton translocation appears to contact lipid.     

Calorimetric measurements of the transition enthalpy of DNA in aqueous urea solutions

The helix-coil equilibrium of DNA is delicately affected by the nature of the solvent. In this investigation the helical secondary structure was destabilized by an increasing concentration of urea. We found a linear dependence of the transition enthalpy deltaH on the urea concentration for calf thymus DNA as well as for salmon sperm DNA.