Dynamic bending rigidity of DNA

Rapidly relaxing components in the decay of the transient electric dichroism of DNA restriction fragments were reported by Diekmann et al. [(1982) Biophys. Chem. 15, 263-270] and P?rschke et al. [(1987) Biopolymers 26, 1971-1974]. These are analyzed using a new normal mode theory for weakly bending rods and assigned to bending. The longest bending relaxation times for fragments with 95-250 base pairs coincide with the theoretical curve calculated for a dynamic bending rigidity corresponding to a dynamic persistence length Pd = 2100 A. Analysis of the relative amplitudes of fast and slow components following weak orienting pulses is also consistent with a rather large dynamic persistence length. The enhancement of the relative amplitude of the fast component in large electric fields is attributed to steady-state bending of initially perpendicular DNAs by the field. Several reasons are proposed why the dynamic bending rigidity is 4 times larger than the apparent static bending rigidity inferred from equilibrium persistence length measurements on the same fragments.     

Intramolecular interference effects in dynamic light scattering: rigid double spirals and superhelical DNAs

A theory is developed for dynamic light scattering (DLS) from rigid double spirals by treating an invisible rigid cylinder with two helical scattering stripes on opposite sides of its cylindrical surface. The exact initial, or first cumulant, diffusion coefficient Dapp (K) is obtained in terms of the translational diffusion coefficients (D parallel and D perpendicular) parallel and perpendicular to the symmetry axis, the rotational diffusion coefficients (DR parallel and DR perpendicular) around the symmetry and transverse axes, the length (L) and radius (b) of the cylindrical surface bearing the stripes, and the pitch (p). Interference effects, namely geometrical antiresonances, between strands, produce deep minima in the static structure factor S (K) and corresponding prominent peaks in Dapp (K). These peaks in Dapp (K) depend sensitively on the rotational dynamics around the symmetry axis, and nearly vanish when DR parallel = 0. Some results for single spirals are also presented. A simpler model in which scattering points are attached at opposite ends of an otherwise invisible thin rigid rod is also treated, and shown to exhibit modest minima in S (K) and corresponding maxima in Dapp (K). Confining this rod to a plane containing K enhances the amplitudes of the oscillations in S (K) and Dapp (K), as expected. Rigid double spirals are employed as crude models for interwound supercoiled DNAs in order to assess the possible occurrence of interference effects. Although native supercoiled DNAs exhibit a cylinder diameter that is much too small to exhibit geometrical antiresonances in the presently accessible range of K2, nearly relaxed supercoiled DNAs are predicted to exhibit their first maximum in Dapp (K) just inside this range. Previously reported data for the effect of Escherichia coli single-strand binding (ssb) protein on the DLS of supercoiled pBR322 DNA cannot be mimicked by a gradual homogeneous reduction of superhelix density with increasing ssb, but instead can be mimicked by inhomogeneous all-or-none binding in which uncomplexed native DNAs and nearly relaxed saturated ssb/DNA complexes coexist in varying proportions. Experimental Dapp (K) and S (K) data for a sample of relaxed pUC8 dimers display, respectively, a broad maximum and a corresponding minimum, in qualitative agreement with rough theoretical predictions.     

Dynamic bending rigidity of a 200-bp DNA in 4 mM ionic strength: a transient polarization grating study

DNA may exhibit three different kinds of bends: 1) permanent bends; 2) slowly relaxing bends due to fluctuations in a prevailing equilibrium between differently curved secondary conformations; and 3) rapidly relaxing dynamic bends within a single potential-of-mean-force basin. The dynamic bending rigidity (kappa(d)), or equivalently the dynamic persistence length, P(d) = kappa(d)/k(B)T, governs the rapidly relaxing bends, which are responsible for the flexural dynamics of DNA on a short time scale, t < or = 10(-5) s. However, all three kinds of bends contribute to the total equilibrium persistence length, P(tot), according to 1/P(tot) congruent with 1/P(pb) + 1/P(sr) + 1/P(d), where P(pb) is the contribution of the permanent bends and P(sr) is the contribution of the slowly relaxing bends. Both P(d) and P(tot) are determined for the same 200-bp DNA in 4 mM ionic strength by measuring its optical anisotropy, r(t), from 0 to 10 micros. Time-resolved fluorescence polarization anisotropy (FPA) measurements yield r(t) for DNA/ethidium complexes (1 dye/200 bp) from 0 to 120 ns. A new transient polarization grating (TPG) experiment provides r(t) for DNA/methylene blue complexes (1 dye/100 bp) over a much longer time span, from 20 ns to 10 micros. Accurate data in the very tail of the decay enable a model-independent determination of the relaxation time (tau(R)) of the end-over-end tumbling motion, from which P(tot) = 500 A is estimated. The FPA data are used to obtain the best-fit pairs of P(d) and torsion elastic constant (alpha) values that fit those data equally well, and which are used to eliminate alpha as an independent variable. When the relevant theory is fitted to the entire TPG signal (S(t)), the end-over-end rotational diffusion coefficient is fixed at its measured value and alpha is eliminated in favor of P(d). Neither a true minimum in chi-squared nor a satisfactory fit could be obtained for P(d) anywhere in the range 500-5000 A, unless an adjustable amplitude of azimuthal wobble of the methylene blue was admitted. In that case, a well-defined global minimum and a reasonably good fit emerged at P(d) = 2000 A and (1/2) = 25 degrees. The discrimination against P(d) values <1600 A is very great. By combining the values, P(tot) = 500 A and P(d) = 2000 A with a literature estimate, P(pb) = 1370 A, a value P(sr) = 1300 A is estimated for the contribution of slowly relaxing bends. This value is analyzed in terms of a simple model in which the DNA is divided up into domains containing m bp, each of which experiences an all-or-none equilibrium between a straight and a uniformly curved conformation. With an appropriate estimate of the average bend angle per basepair of the curved conformation, a lower bound estimate, m = 55 bp, is obtained for the domain size of the coherently bent state. Previous measurements suggest that this coherent bend is not directional, or phase-locked, to the azimuthal orientation of the filament.     

The distribution of end-to-end distances of the weakly bending rod model

The weakly bending rod model is an approximation to a worm-like chain in the limit where the ratio L(0)/P of the contour length L(0) to the persistence length P is not too large. The range of validity of the weakly bending rod model is investigated by deriving analytical expressions for its distribution of end-to-end distances P(L) and its moments and numerically comparing the results with corresponding values for the worm-like chain model. No general, closed form analytical expression for either P(L) or the average length of a worm-like chain exists, so those quantities are obtained by Monte Carlo simulations. Exact analytical expressions for and for the worm-like chain are employed in the comparison of the computed moments. Moments calculated for the approximate distributions of Daniels and Yamakawa and Stockmayer are also compared with the others. In addition, P(L) and its moments for the alternative model of Winkler et al. are compared with the others. The weakly bending rod model gives a reasonably good account of both P(L) and , m = 1,2,4, over the range L(0)/P /= 1.0. In contrast, the alternative model of Winkler et al. yields rather poor results in the rodlike domain.     

Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein

The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D0) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (Dapp) obtained from dynamic light scattering display the well-known increase with K2 (K = scattering vector), leveling off toward a plateau value (Dplat) at high K2. For both DNAs, the difference Dplat - D0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed.     

Dynamic light scattering studies of porcine submaxillary mucin fractions in dilute solution at intermediate scattering vectors

We report dynamic light scattering measurements over a wide range of scattering vectors for fractionated samples of porcine submaxillary mucin (PSM) glycoproteins in two different solvents: 0.1M NaCl, and 6M GdnHCl. The relaxation spectrum has been successfully resolved into a slow mode corresponding to pure translational diffusion and a fast mode containing information on the relaxation times for intramolecular motion. Analysis of the slow mode permits a light scattering evaluation of the polydispersity of these high molecular weight mucin glycoprotein fractions. Determination of the longest intramolecular relaxation times tau 1 shows that these are much longer for the PSM fractions in 0.1M NaCl compared to 6M GdnHCl. These data are consistent with earlier studies showing that the chain conformation is the same in both solvents, but that in 0.1M NaCl, the PSM glycoprotein undergoes a self-association process that is end-to-end in nature. Since the tau 1 value is intimately related to the viscoelastic behavior of PSM solutions and gels, it is interesting to speculate that the end-to-end association process may be physiologically important.     

Dynamics of superhelical DNA studied by photon correlation spectroscopy

We have conducted photon correlation spectroscopy (PCS) studies on the plasmid pUC8 (2717 bp) in order to elucidate the internal dynamics of this superhelical DNA. We confirm that the first-order autocorrelation function of the scattered light from pUC8 solutions can be separated into two distinct exponential decay components, as first shown by Lewis et al. (R. Lewis, J.H. Huang and P. Pecora, Macromolecules 18 (1985) 944). A thorough analysis of the dependence on scattering vector K of the rates and amplitudes of the two components enables us to assign the slowly relaxing part to the center-of-mass diffusion of the DNA, while the faster component corresponds to rotational, bending and twisting motions of the superhelix. For larger K values the internal motions can be formally expressed in terms of an 'internal diffusion coefficient' Di, whose value of 2.0-2.5 X 10(-11) m2 s-1 is approximately equal to the translational diffusion coefficient predicted for a stiff DNA piece of the persistence length, 65 nm. Comparison of our measured Di values to those predicted from a recent theory of circular worm-like coils (K. Soda, Macromolecules 17 (1984) 2365) shows that the internal motions are faster than the theoretical values. One of the reasons for this discrepancy could be that the theory does not take into account torsional motions, which contribute significantly to the internal dynamics (J.C. Thomas, S.A. Allison, C.J. Appelof and J.M. Schurr, Biophys. Chem. 12 (1980) 177). At low K values, the fast relaxation of superhelical pUC8 is no longer proportional to K2, but reaches a constant value as K approaches zero. This behavior, not seen for the linearized DNA, can be interpreted in terms of rotational diffusion of a flexible rod-like molecule (T. Maeda and S. Fujime, Macromolecules 17 (1984) 2381) and supports an interwound rod-like structure for pUC8 DNA with an average end-to-end distance of 220 nm.     

Nuclear magnetic resonance of the filamentous bacteriophage fd.

The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy. 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus. There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA. 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations. The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions. The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus. Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings.     

The internal dynamics of gene 32 protein-DNA complexes studied by quasi-elastic light scattering

The hydrodynamic properties of large homodisperse single stranded DNAs complexed with the helix destabilizing protein of phage T4, the product of gene 32 (GP32), have been measured. The results suggest a size of the binding site between 8 and 10 nucleotides/GP32 molecule, in reasonable agreement with earlier work on a complex between GP32 and single stranded 145 base DNA. From static light scattering experiments it is concluded that the persistence length of these complexes is about 30 nm, distinctly smaller than the generally accepted value for double stranded DNA. The quasi-elastic light scattering properties of the DNA-GP32 complexes were determined. The variation of the apparent translation diffusion coefficient Dapp with the scattering vector q was analyzed using the discrete ISMF and Rouse-Zimm models [S.C. Lin et al., Biopolymers 17 (1978) 425]. The model parameters that followed from the fit of Dapp versus q2 and from an extensive global analysis of the actually measured autocorrelation functions agreed with the notion that these DNA-protein complexes are indeed rather flexible. The continuous Soda model [K. Soda, Macromolecules 17 (1984) 2365] could successfully explain the variation of Dapp versus q2, assuming a persistence length of 30 nm and a base-base distance in the complex of 0.44 nm.     

Analysis of DNA bending by transient electric birefringence

Transient electric birefringence has been used to analyze DNA bending in six restriction fragments containing 171, 174, 207, 263, 289, and 471 bp in three different low ionic strength buffers. The target fragments contain sequences corresponding to the apparent bend centers in pUC19 and Litmus 28, previously identified by the circular permutation assay (Strutz, K.; Stellwagen, N. C. Electrophoresis 1996, 17, 989-995). The target fragments migrate anomalously slowly in polyacrylamide gels and exhibit birefringence relaxation times that are shorter than those of restriction fragments of the same size, taken from nonbent regions of the same plasmids. Apparent bend angles ranging from 30 degrees to 41 degrees were calculated for the target fragments by tau-ratio method. The bend angles of four of the target fragments were independent of temperature from 4 degrees C to 20 degrees C, but decreased when the temperature was increased to 37 degrees C. The bend angles of the other two target fragments were independent of temperature over the entire range examined, 4 degrees -37 degrees C. Hence, the thermal stability of sequence-dependent bends in random-sequence DNA is variable. The bend angles of five of the six target fragments were independent of the presence or absence of Mg2+ ions in the solution, indicating most of the target fragments were stably bent or curved, rather than anisometrically flexible. Restriction fragments containing 219 and 224 bp, with sequences somewhat offset from the sequence of the 207 bp fragment, were also studied. Comparison of the tau-ratios of these overlapping fragments allowed both the bend angle and bend position to be independently determined. These methods should be useful for analyzing sequence-dependent bending in other random-sequence DNAs.     

Normal mode theory for the Brownian dynamics of a weakly bending rod: Comparison with Brownian dynamics simulations

A normal mode theory is developed for the Brownian dynamics of weakly bending rods with preset hydrodynamic interactions. The rod is replaced by a chain of contiguous spheres whose radius is chosen to yield the appropriate uniform translational and rotational diffusion coefficients. Despite the inclusion of preset hydrodynamic interactions in the dynamical operator, its normal modes are not coupled by the potential energy, so their amplitudes remain pairwise “orthogonal” under equilibrium averaging. The uniform translational and rotational diffusion coefficients obtained from Langevin theory are shown to be identical to those obtained from the Kirkwood algorithm, despite their rather different appearance. An expression is given for the mean squared angular displacement 〈Δ_xm(t)²〉 of the mth bond vector around the instantaneous x axis (perpendicular to the end-to-end vector z). Necessary algorithms are presented for the numerical evaluation of all quantities. The normal mode theory is compared with Brownian dynamics simulations for the same model by examining 3〈Δ_xm(t)²〉 for the central bond vector of rods comprising 10 and 30 subunits with various persistence lengths. The normal mode theory works very well for all times for L/P ? 0.6, where P = κ/k_BT is the persistence length and κ is the bending rigidity. With increasing flexibility, the domain of validity of the normal mode theory is restricted to shorter times, where violations of the weak bending approximation are less severe. However, increasing the length of the rod from 10 to 30 subunits yields improved agreement with the simulations for the same and even longer times. This latter effect is tentatively attributed to the greater fluctuating tension in the longer chains, which acts to retard the rotational relaxation in the simulations, but is not taken into account in the present normal mode theory.     

Backbone motions in a crystalline protein from field-dependent 2H-NMR relaxation and line-shape analysis

We have used 2H-nmr to study backbone dynamics of the 2H-labeled, slowly exchanging amide sites of fully hydrated, crystalline hen egg white lysozyme. Order parameters are determined from the residual quadrupole coupling and values increase from S2 = 0.85 at 290 K to S2 = 0.94 at 200 K. Dynamical rates are determined from spin-lattice relaxation at three nmr frequencies (38.8, 61.5, and 76.7 MHz). The approach used here is thus distinct from solution nmr studies where dynamical amplitudes and rates are both determined from relaxation measurements. At temperatures below 250 K, relaxation is independent of the nmr frequency indicating that backbone motions are fast compared to the nmr frequencies. However, as the temperature is increased above 250 K, relaxation is significantly more efficient at the lowest frequency, which shows, in addition, the presence of motions that are slow compared to the nmr frequencies. Using the values of S2 determined from the residual quadrupole coupling and a model-free relaxation formalism that allows for fast and slow internal motions, we conclude that these slow motions have correlation times in the range of 0.1 to 1.0 microsecond and are effectively frozen out at 250 K where fast motions of the amide planes with approximately 15 ps effective correlation times and 9 degrees rms amplitudes dominate relaxation. The fast internal motions increase slightly in amplitude as the temperature rises toward 290 K, but the correlation time, as is also observed in solution nmr studies of RNase H, is approximately constant. These findings are consistent with hypotheses of dynamic glass transitions in hydrated proteins arising from temperature-dependent damping of harmonic modes of motion above the transition point.     

Structural study of hinge bending in L-arabinose-binding protein

The L-arabinose-binding protein of Escherichia coli is a periplasmic component of the bacterial L-arabinose transport system. The three-dimensional structure of the molecule has been determined by x-ray diffraction and shown to have two globular domains and a connecting hinge. Theoretical study of the flexibility of the hinge using computer simulation showed that the hinge is quite permissive in that only moderate increases in the internal energy are required for opening the cleft where the L-arabinose-binding site is located. In this study, the structural changes that accompany the hinge bending are analyzed. The results show that bending-induced stresses are accommodated by coupled action of covalent and noncovalent forces within the protein molecule. Strains in internal coordinates (bond lengths, bond angles, and torsional angles) are distributed throughout the hinge region after structural relaxation. The pattern of structural changes within a hinge strand upon bending and relaxation depends in large degree on its geometric relationship with the bending axis (e.g. distance and orientation) and the atomic packing of its immediate environment. The distributed structural changes result in a characteristic zigzag pattern for the directional change at each residue in the hinge strands.     

Electron spin-lattice relaxation of the S0 state of the oxygen-evolving complex in photosystem II and of dinuclear manganese model complexes

The temperature dependence of the electron spin-lattice relaxation time T1 was measured for the S0 state of the oxygen-evolving complex (OEC) in photosystem II and for two dinuclear manganese model complexes by pulse EPR using the inversion-recovery method. For [Mn(III)Mn(IV)(mu-O)2 bipy4]ClO4, the Raman relaxation process dominates at temperatures below 50 K. In contrast, Orbach type relaxation was found for [Mn(II)Mn(III)(mu-OH)(mu-piv)2(Me3 tacn)2](ClO4)2 between 4.3 and 9 K. For the latter complex, an energy separation of 24.7-28.0 cm(-1) between the ground and the first excited electronic state was determined. In the S0 state of photosystem II, the T1 relaxation times were measured in the range of 4.3-6.5 K. A comparison with the relaxation data (rate and pre-exponential factor) of the two model complexes and of the S2 state of photosystem II indicates that the Orbach relaxation process is dominant for the S0 state and that its first excited state lies 21.7 +/- 0.4 cm(-1) above its ground state. The results are discussed with respect to the structure of the OEC in photosystem II.     

Electrostatic contribution to the bending of DNA

A model is derived that accounts for the short-range electrostatic contribution to the bending of DNA molecule in solution and in complexes with proteins in terms of the non-linear Poisson-Boltzmann equation. We defined that the short-range electrostatic interactions depend on the changes of the polyion surface charge density under deformation, while the long-range interactions depend on the bending-induced changes in distances between each two points along the polyion axis. After an appropriate simplification of the Poisson-Boltzmann equation, the short-range term is calculated separately giving the lower limit for the electrostatic contribution to the DNA persistence length. The result is compared with the theoretical approaches developed earlier [M. Fixman, J. Chem. Phys. 76 (1982) 6346; M. Le Bret, J. Chem. Phys. 76 (1982) 6243] and with the experimental data. The conclusion is made that the results of Fixman-Le Bret, which took into account both types of the electrostatic interactions for a uniformly bent polyion, give the upper limit for the electrostatic persistence length at low ionic strength, and the actual behavior of the DNA persistence length lies between two theoretical limits. Only the short-range term is significant at moderate-to-high ionic strength where our results coincide with the predictions of Fixman-Le Bret. The bending of DNA on the protein surface that is accompanied by an asymmetric neutralization of the DNA charge is also analyzed. In this case, the electrostatic bending energy gives a significant favorite contribution to the total bending energy of DNA. Important implications to the mechanisms of DNA-protein interactions, particularly in the nucleosome particle, are discussed.     

The structure of vesicular stomatitis virus membrane. A phosphorus nuclear magnetic resonance approach.

The proton decoupled 40.48 M Hz 31P NMR spectrum of intact and unperturbed membrane-enclosed vesicular stomatitis virus (sterotype Indiana) exhibited two distinct maxima. These can be resolved into a narrow, symmetric line and a broad asymmetric line. The 31P NMR spectrum of a multilamellar (unsonicated) preparation of the extracted viral lipids exhibited a line shape similar to that of the intact virus. A sonicated vesicle preparation of the extracted viral lipids exhibited a narrow symmetric line. The narrow component in the intact virus spectrum may be attributed to small membrane fragments. Phospholipase C digestion of the intact virus resulted in substantial reduction in intensity of both components which suggests that much of the contribution to both peaks is due to phosphate in the phospholipid polar head groups. The phospholipid phosphates in both sonicated and unsonicated preparations of the extracted viral lipids exhibited substantially longer relaxation times than did those in the intact virus. The short relaxation time emanating from the intact virus preparation is caused by immobilization of the phospholipid head groups which could be due to lipid-protein interactions. Trypsin treatment of vesicular stomatitis virions, which results in complete removal of the exterior hydrophilic segment of the membrane glycoprotein, increased the 31P relaxation time to a value similar to that observed in the protein-free total lipid extracts; this finding provides supporting evidence for the role of virus glycoprotein in shortened relaxation times. A reversible temperature-dependent change in apparent line width and absence of an effect of cholesterol on the 31P phospholipid spectrum were also demonstrated.     

Protein-induced bending of the simian virus 40 origin of replication

A 3.5 S protein, isolated from mammalian nuclei, specifically binds to DNA fragments containing the simian virus 40 (SV40) origin of replication. Two distinct nucleoprotein complexes are formed, a complex with high electrophoretic mobility carrying probably only one protein molecule, and a complex with reduced electrophoretic mobility carrying probably two protein molecules per DNA fragment. Band shift competition as well as methylation interference assays locate the binding site of the protein in the A + T-rich "late" region of the origin between SV40 nucleotides 13 and 35. The late origin binding (LOB) protein and T antigen bind simultaneously to adjacent sites in the origin. Using circularly permuted DNA fragments of identical lengths we show that the LOB protein induces pronounced bending of the origin fragment. The bending center maps at the 5' end of the adenine tract with one bound protein molecule and at the 3' end when two LOB proteins are bound to one origin fragment.     

The structure of vesicular stomatitis virus membrane A phosphorus nuclear magnetic resonance approach

The proton decoupled 40.48 M Hz ³¹P NMR spectrum of intact and unperturbed membrane-enclosed vesicular stomatitis virus (serotype Indiana) exhibited two distinct maxima. These can be resolved into a narrow, symmetric line and a broad asymmetric line. The ³¹P NMR spectrum of a multilamellar (unsonicated) preparation of the extracted viral lipids exhibited a line shape similar to that of the intact virus. A sonicated vesicle preparation of the extracted viral lipids exhibited a narrow symmetric line. The narrow component in the intact virus spectrum may be attributed to small membrane fragments. Phospholipase C digestion of the intact virus resulted in substantial reduction in intensity of both components which suggests that much of the contribution to both peaks is due to phosphate in the phospholipid polar head groups.The phospholipid phosphates in both sonicated and unsonicated preparations of the extracted viral lipids exhibited substantially longer relaxation times than did those in the intact virus. The short relaxation time emanating from the intact virus preparation is caused by immobilization of the phospholipid head groups which could be due to lipid-protein interactions. Trypsin treatment of vesicular stomatitis virions, which results in complete removal of the exterior hydrophilic segment of the membrane glycoprotein, increased the ³¹P relaxation time to a value similar to that observed in the protein-free total lipid extracts; this finding provides supporting evidence for the role of virus glycoprotein in shortened relaxation times. A reversible temperature-dependent change in apparent line width and absence of an effect of cholesterol on the ³¹P phospholipid spectrum were also demonstrated.     

Anisotropic viscoelastic properties of cortical bone

Relaxation Young's modulus of cortical bone was investigated for two different directions with respect to the longitudinal axis of bone (bone axis, BA): the modulus parallel (P) and normal (N) to the BA. The relaxation modulus was analyzed by fitting to the empirical equation previously proposed for cortical bones, i.e., a linear combination of two Kohlraush-Williams-Watts (KWW) functions (Iyo et al., 2003. Biorheology, submitted): E(t)=E0 (A1 exp[-(t/tau1)beta]+(1-A1) exp[-(t/tau2)gamma]), [0 < A1, beta, gamma < 1], where E0 is the initial modulus value E0. Tau1 and tau2(>tau1) are characteristic times of the relaxation, A1 is the fractional contribution of the fast relaxation (KWW1 process) to the whole relaxation process, and beta and gamma are parameters describing the shape of the relaxation modulus. In both P and N samples, the relaxation modulus was described well by the empirical equation. The KWW1 process of a P sample almost completely coincided with that of an N sample. In the slow process (KWW2 process), there was a difference between the relaxation modulus of a P sample and that of an N sample. The results indicate that the KWW1 process in the empirical equation represents the relaxation in the collagen matrix in bone and that the KWW2 process is related to a higher-order structure of bone that is responsible for the anisotropic mechanical properties of bone.