Light scattering of aqueous solutions of DNA, poly(acrylic acid), and tobacco mosaic virus under alternating electric field

Two new techniques, amplitude modulation (AM) and frequency modulation (FM) of an electric field, are developed for the light-scattering study of polymer solutions under ac electric fields. The AM technique makes it possible to observe accurately the frequency dependence of the intensity changes of scattered light due to the electric field. The FM one allows us to obtain directly the frequency derivative of the intensity change. The techniques are applied to DNA, poly(acrylic acid), and tobacco mosaic virus in the frequency range from 10 Hz to 100 kHz. A low-frequency relaxation is found for both DNA and poly(acrylic acid). The obsersved relaxation time of DNA agrees with that in the dielectric relaxation of DNA, which has been attributed to the rotation of the molecule with a quasipermanent dipole. In the case of poly(acrylic acid), the relaxation strength increases with increasing degree of neutralization. TMV at a concentration of 0.1% exhibits a negative relaxation at low frequencies, which indicates the rotation of TMV aggregate with a permanent dipole along its minor axis.     

Structural aspects and orientation mechanism of mitochondrial F1 adenosinetriphosphatase. Evidence for a negative electric birefringence due to a permanent moment perpendicular to the long axes of the particle

The electric birefringence technique was used to investigate the steady-state birefringence, the orientational relaxation time, and the orientation mechanism of pig heart mitochondrial F1 adenosine-5'-triphosphatase (F1-ATPase). The electrooptical properties of this enzyme in solution were studied as functions of pH, protein concentration, and applied electric field. The F1-ATPase exhibits a surprising negative electric birefringence with a specific Kerr constant of -1.5 X 10(-3) esu cgs. The field-independent relaxation time was found to be 0.65 +/- 0.05 microseconds, corresponding to a rotational diffusion constant of 2.55 X 10(5) s-1. The overall size and shape of F1-ATPase have been calculated from both translational and rotational diffusion constants. The enzyme may be assumed to be an oblate ellipsoid of revolution with dimensions of about 170 X 170 X 70 A. The orientation mechanism of F1-ATPase was analyzed by fitting experimental birefringence rising curves with theoretical rising functions. The ratio of the permanent to induced dipole moment is found to be very high; therefore, the birefringence of F1-ATPase is due to a strong permanent dipole moment in a direction perpendicular to the long axes of the particle. These particular electric properties can be explained by the oligomeric structure of the protein and seem likely to play a role in its mechanism of functioning.     

Electric birefringence studies of rabbit skeletal tropomyosin

The electric birefringence of rabbit skeletal tropomyosin and its nonpolymerizable derivative was studied as a function of protein concentration, pulse length, and electric field strength. Analyses of the zero field birefringence decay curves show that nonpolymerizable tropomyosin, which has had on average six C-terminal residues removed, and tropomyosin are both well approximated by rigid cylinders in solution at low salt concentrations at 20°C. The measured relaxation times for the monomers of polymerizable and nonpolymerizable tropomyosin are 1.5 ± 0.4 μs and 1.30 ± 0.2 μs, respectively, in good agreement with the values calculated from the known dimensions. For tropomyosin the electrical pulse induces the formation of linear dimers. Orientation occurs primarily by a permanent dipole mechanism. Permanent and induced dipole moments were calculated from reversing field experiments and from the saturation of the birefringence. Removal of the six C-terminal residues decreases the measured permanent dipole moment by 9.5%, from 6300 to 5700 Debyes, which is in good agreement with the 7% decrease calculated for permanent dipole contributions arising from the peptide dipoles and from the asymmetric distribution of the formal charges. This change is due primarily to the removal of Asp 280.     

Effect of aliphatic alcohols on the conformation of poly (l-ornithine hydrobromide) as studied by square-pulse and reversing-pulse electric birefringence

The electric birefringence and circular dichroism spectra of poly(l-ornithine hydrobromide) have been measured in ethanol/water, 2-propanol/water and tertiary butyl alcohol/water mixtures of various compositions. This charged polypeptide underwent a transition from the coil conformation to the helical conformation at high alcohol content in every case tested. Anomalous birefringence signals, indicative of a field-induced helix-to-coil transition. were observed at high electric fields only in the case of ethanol/water mixtures. The reversing-pulse electric birefringence of this polypeptide has been studied in ethanol/water mixtures and in neutral aqueous solution. Upon rapid reversal of the pulse field, no transient could be observed. This confirms that the electric-field orientation of poly(l-ornithine hydrobromide) results predominantly from the contribution of the counterion-induced dipole moment, regardless of its molecular conformations. It is very probable that the backbone permanent dipole moment of the helical conformation is largely suppressed by the counterion-induced dipole moment in the ionized form.     

Dichroism of TMV in pulsed electric fields

The linear dichroism induced in a solution of electrically anisotropic molecules by a pulsed electric field has been studied. Equations have been obtained which express the dichroism as a function of dipole moment, excess polarizability, field strength, and the angle α between the dipole moment and the transition moment for the absorption band. These expressions have been related to the experimentally observed difference signal in such a way that when the dichroism is measured as a function of field strength the permanent moment, excess polarizability and angle a can be determined. Experiments have been carried out on tobacco mosaic virus (TMV), which is similar in its properties to the theoretical model. The polarizability anisotropy and rotary diffusion constant for the monomer and dimer of TMV have been obtained from these experiments. In addition to the molecular parameters mentioned above, the saturated electric dichroism of the virus was measured as a function of wave length and the presence of an n–π* transition in the tryptophan spectrum was indicated. Further experiments measuring dichroism as a function of pH demonstrated the general denaturation of the virus at high pH (10–11) but also the existence of a stable fraction which is not fragmented even at the high pH involved.     

Electric birefringence of myosin subfragments.

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.     

Electric birefringence of myosin subfragments

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.     

Electric properties of macromolecules. IX. Dipole moment, polarizability, and optical anisotropy factor of collagen in solution from electric birefringence

The electric birefringence of collagen solutions has been measured over a wide range of field strength with the pulse technique. The soluble collagen was from rat tail tendon. The solvent used was dilute acetic acid. Very pronounced saturation of the electric birefringence was observed, permitting calculation of the optical anisotropy factor. The Kerr constant was determined by extrapolation to zero field strength. From the dependence on field strength of the birefringence, the permanent dipole moment and the anisotropy of polarizability were separately determined. The contribution of the former to the Kerr constant was found to be twice as large as that of the latter. The same conclusion was obtained from the initial slope of the rise curves of the birefringence at low fields. The permanent dipole moment was 1.5 × 10⁴ Debye, and the anisotropy of polarizability was about 3 × 10^?15 cm.³. The magnitude of the latter indicates that the ion atmosphere polarization is important. Effects of added salt and thermal denaturation on the electric birefringence were explored.     

Light chain-dependent myosin structural dynamics in solution investigated by transient electrical birefringence.

The technique of transient electrical birefringence was used to compare some of the electric and structural dynamic properties of myosin subfragment 1 (S1(elc, rlc)), which has both the essential and regulatory light chains bound, to S1(elc), which has only an essential light chain. The rates of rotational Brownian motion indicate that S1(elc, rlc) is larger, as expected. The permanent electric dipole moment of S1(elc, rlc) is also larger, indicating that the regulatory light chain portion of S1(elc, rlc) has a dipole moment and that it is aligned head-to-tail with the dipole moment of the S1(elc) portion. The permanent electric dipoles decrease with increasing ionic strength, apparently because of ion binding to surface charges. Both S1(elc, rlc) and S1(elc) have intrinsic segmental flexibility, as detected by the ability to selectively align segments with a brief weak electric field. However, unlike S1(elc), which can be structurally distorted by the action of a brief strong electric field, S1(elc, rlc) is stiffer and cannot be distorted by fields as high as 7800 V/cm applied to its approximately 8000 D permanent electric dipole moment. The S1 . MgADP . Pi analog S1 . MgADP . Vi is smaller than S1 . MgADP, for both S1(elc, rlc) and S1(elc). Interestingly, the smaller, stiffer S1(elc, rlc) . MgADP . Vi complex retains intrinsic segmental flexibility. These results are discussed within a framework of current hypotheses of force-producing mechanisms that involve S1 segmental motion and/or the loss of cross-bridge flexibility during force production.     

Electric birefringence study of the purple membrane of Halobacterium halobium

An electric birefringence study was carried out on aqueous suspensions of the purple membrane of Halobacterium halobium. In addition to the characterization of both native and modified membrane samples, the dependence of electric birefringence on pH and ionic strength was also investigated. The results indicate that purple membrane shows electric birefringence at a field strength as low as 200 V/cm. The permanent dipole moment and polarizability ranged from 20,500 debyes and 1.01 × 10^?14 cm³ for a purple membrane concentration of 0.40 mg/mL to 41,000 debyes and 2.05 × 10^?14 cm³ for a concentration of 0.80 mg/mL. It was also found that removal of the retinyl group of bacteriorhodopsin substantially decreases but does not eliminate the electric birefringence of the membrane. The solubilization of the membrane by Triton X-100, however, completely abolishes the electric birefringence. These experiments indicate that there is an interaction between adjacent bacteriorhodopsin molecules within the purple membrane via the retinyl chromophore moiety that builds up the permanent dipole moment. They also suggest that there are two types of response when purple membrane suspensions are placed in an electric field. One is an alignment of the disk-shaped particles with the field. The other is a stacking of the particles following their alignment by the electric field, which is promoted by the induced dipole moment.     

Simultaneous measurement of electric birefringence and dichroism. A study on photosystem 1 particles.

We have developed a straightforward method to separate linear-dichroism and birefringence contributions to electric-field induced signals in a conventional birefringence setup. The method requires the measurement of electric birefringence for three different angular positions of the analyzer. It is demonstrated that the presence of linear dichroism can significantly influence the measured signals and lead to completely erroneous calculations of the birefringence signal and field-free decay times if its contribution is not taken into account. The new method is used to determine electric birefringence and linear dichroism of trimeric Photosystem 1 complexes from the cyanobacterium Synechocystis PCC 6803 in the detergents n-dodecyl-beta-D-maltoside and n-octyl-beta-D-glucoside. It is concluded that the orientation of the particles in the field is predominantly caused by a permanent electric dipole moment that is directed parallel to the symmetry axis of the particles. Comparison of the decay times obtained with dodecylmaltoside and octylglucoside supports a model in which the thickness of the disc-like complexes remains similar (7-8 nm) upon replacing dodecylmaltoside by octylglucoside, whereas the diameter increases from 14.4 +/- 0.2 to 16.6 +/- 0.2 nm because of an increased thickness of the detergent layer. This change in diameter is in good agreement with electron-microscopy results on Photosystem 2 complexes in dodecylmaltoside and octylglucoside (Dekker, J. P., E. J. Boekema, H. T. Witt, and M. Rögner. 1988. Biochim. Biophys. Acta 936:307-318). The value of approximately 16.6 nm for the diameter of Photosystem 1 trimers in dodecylmaltoside is in good agreement with recent results obtained from electron microscopy in combination with extensive image analysis (Kruip, J., E. J. Boekema, D. Bald, A. F. Boonstra, and M. Rögner. 1993. J. Biol. Chem. 268:23353-23360).     

Dielectric relaxation and orientation of DNA molecules

A conductivity dispersion has been measured at very low frequencies (VLF) on several concentrated DNA solutions. By measuring simultaneously their electric birefringence decay, it is shown that the dielectric relaxation (which is related to the conductivity dispersion) is due to the molecular orientation. Different polarization mechanisms are discussed. It is concluded that the DNA polarizability measured in the VLF range can only be explained by the orientation of a permanent ionic dipole. It is suggested that such permanent dipoles could be caused by small differences in the ionic composition between the two molecular “ends;” the difference could either be stable (asymmetrical localization of protein impurities for instance) or transient (fluctuating dipoles explained by the Kirkwood-Schumaker theory).     

Electrical and hydrodynamical properties of polypeptides in solution. I. Poly(L-glutamic acid) in methanol-water mixtures

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol–water mixtures has been measured by the use of the rectangular pulse technique at 25°C. The permanent dipole moment, the anisotropy of electrical polarizability, and the optical anisotropy factor of PLGA in solution were obtained from the dependence of the steady-state birefringence on the electric field strength. Further, the mean length of PLGA in solution was calculated by a parameter method developed for analyzing the decay curve of electric birefringence. The permanent dipole moment per unit length obtained from these studies was 2.9₆, 2.4₈, 2.3₀, 2.6₆ D/Å in pure methanol, 10, 30, and 50 vol-% water, respectively. The increase of water content caused the decrease of the mean length and broadened the length distribution of PLGA. These results are discussed in relation to the viscosity and the electrical conductivity of PLGA solutions.     

The structural organization of oligonucleosomes

We have used electric birefringence to study the structure of oligonucleosomes and to show the influence of histone H1 depletion on their conformation in solution. Measurements are made at low ionic strength on monodisperse samples containing up to 8 nucleosomes. For each oligomer, having H1 or not, the analysis of both relaxation and orientation times gives information about the particle's orientation mechanism through the ratio r of permanent over induced dipole terms. For native oligomers, the data confirm the previous finding of a discontinuity in hydrodynamic behavior between pentamer and heptamer: the rotational times are multiplied by 10 and r increases from 0.2 to 0.7 showing the appearance of a non-negligible contribution of a permanent dipole to the orientation mechanism. We suggest a model for the hexanucleosome at low ionic strength and discuss its implications for the higher-order structure of chromatin. The treatment for H1 depletion abolishes the transitions in electro-optical properties: the value of r remains constant, r = 0.15, and both rotational times increase progressively with the number of nucleosomes in the chain. That reflects an important unfolding of oligonucleosomal structure which we attributed to the unwinding of DNA tails and internucleosomal segments. The disc planes of nucleosomes become closely parallel to the nucleosomal chain axis.     

Transient electric birefringence studies of xanthan solutions

Transient electric birefringence studies have been performed on heat denatured xanthan in 4 m urea. The induced birefringence was positive, the Kerr law was obeyed at low field amplitudes and the birefringence saturated at high fields. The orientation mechanism appears to be mainly induced dipolar in character and the magnitude of the induced dipole moment can be explained on the basis of counterion polarization. The molecules behave as independent rods of mean length 0.65 μm with no evidence for ‘hindered rotation’ in moderately concentrated solutions. The molecular rigidity is attributed to extension of the polyanion due to charge charge repulsions or steric hindrance due to the side chains.     

The Structural Organization of Oligonucleosomes

Abstract

We have used electric birefringence to study the structure of oligonucleosomes and to show the influence of histone H1 depletion on their conformation in solution. Measurements are made at low ionic strength on monodisperse samples containing up to 8 nucleosomes. For each oligomer, having H1 or not, the analysis of both relaxation and orientation times gives information about the particle's orientation mechanism through the ratio r of permanent over induced dipole terms. For native oligomers, the data confirm the previous finding of a discontinuity in hydrodynamic behavior between pentamer and heptamer: the rotational times are multiplied by 10 and r increases from 0.2 to 0.7 showing the appearance of a non-negligible contribution of a permanent dipole to the orientation mechanism. We suggest a model for the hexanucleosome at low ionic strength and discuss its implications for the higher-order structure of chromatin.

The treatment for H1 depletion abolishes the transitions in electro-optical properties: the value of r remains constant, r=0.15, and both rotational times increase progressively with the number of nucleosomes in the chain. That reflects an important unfolding of oligonucleosomal structure which we attributed to the unwinding of DNA tails and internucleosomal segments. The disc planes of nucleosomes become closely parallel to the nucleosomal chain axis.     

Electric birefringence studies on polyglutamic acid.

The electric birefringence for the aqueous solution of poly-L -glutamic acid (PGA) in the helical form was studied. PGA samples were fractionated by gel column chromatography. PGA showed a positive electric birefringence. The permanent dipole moment of the PGA molecule was suggested to be largely suppressed. The measurements of the intrinsic Kerr constants for various molecular lengths showed that the electric anisotropy (polarizability) of PGA is proportional to the 1.5 power of the length. The electric birefrigence measurement was also carried out in the helix–coil transition region. The Kerr constant of PGA was largely reduced on going from the helical form to the coiled form.     

Anisotropic electric properties of purple membrane and their change during the photoreaction cycle

Purple membrane suspension shows two different orientations in electric fields of different frequencies. The orientation at low frequencies (less than or equal to approximately 10 Hz), with the membrane surface perpendicular to the electric field, is due to permanent dipole moment of the membrane and the orientation at high frequencies (greater than or equal to approximately 100 Hz), with the surface parallel to the electric field, is due to induced dipole moment. By quantitative analysis of these orientations, we determined the permanent dipole moment and the polarizability. Both values varied according to the membrane size: the permanent dipole moment ranged from 500 kD to 10 MD and was proportional to the square of the diameter of the membrane. The polarizability ranged from 1 X 10(-13) to 1 X 10(-11)cm3 and was proportional to the third to fourth power of the diameter. Because the permanent dipole moment was proportional to the area of the membrane, we could determine permanent dipole moment per bacteriorhodopsin. By determining the actual membrane size under electron microscopy, we got 98 D/bacteriorhodopsin. We also concluded that the direction of the permanent dipole moment was from the cytoplasmic to the extracellular side. These values, however, were strongly dependent on the ionic strength in the medium, suggesting a screening effect due to counter ions near the membrane surface. We evaluated the screening effect and showed about a four-charge difference between the two sides of the purple membrane. Under illumination, we found that the permanent dipole moment decreased from 98 to 63 D/bacteriorhodopsin. From the best-oriented sample, we also concluded that the angle of retinal against the axis normal to the membrane surface was greater than 68.6 degrees.     

Electric birefringence of restriction enzyme fragments of DNA: optical factor and electric polarizability as a function of molecular weight

The electric birefringence of restriction enzyme fragments of DNA has been investigated as a function of DNA concentration, buffer concentration, and molecular weight, covering a molecular weight range from 80 to 4364 base pairs (bp) (6 × 10⁴–3 × 10⁶ daltons). The specific birefringence of the DNA fragments is independent of DNA concentration below 20 μg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent. At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field at which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law region the rise of the birefringence is accurately symmetrical with the decay for fragments ≤ 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated from a 1/E extrapolation of the high field birefringence data is ?0.028, independent of molecular weight; if a 1/E² extrapolation is used, the optical factor is ?0.023. The induced polarizability, calculated from the Kerr constant and the optical factor, is proportional to the square of the length of the DNA fragments, and inversely proportional to temperature. Saturation curves for DNA fragments ≤ 161 bp can be described by theoretical saturation curves for induced dipole orientation. The saturation curves of larger fragments are broadened, because of a polarization term which is approximately linear in E, possibly related to the saturation of the induced dipole in high electric fields. This “saturated induced dipole” is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp sample is decreased 6°C in an electric field of 8 kV/cm, because the lower charge density of the coil form of DNA makes it more stable in an electric field than the helix form.     

The optical birefringence of DNA solutions induced by oscillatory electric and hydrodynamic fields

The optical birefringence induced in DNA solutions by both oscillating hydrodynamic fields (flow birefringence) and oscillating electric fields (Kerr effect) is measured over a wide frequency range. The observed frequency response of the birefrigence is compared with theories for rigid ellipsoidal particles and for Gaussian chains. DNA at 6 × 10⁵ molecular weight is found to exhibit rigid particle hydrodynamic behavior, while DNA at 5 × 10⁶ molecular weight behaves like a flexible chain. Characterization of the hydrodynamic relaxation spectra for the DNA's by oscillatory flow birefringence allows precise comparison between theory and the experimental Kerr effect response. The dielectric model for DNA contains both permanent and dispersionless induced dipole moments. The dielectric behavior of DNA has the character of a permanent dipole but with anomalous low-frequency dispersion in the Kerr effect. The existing theories do not adequately describe this dispersion. A fluctuation dipole mechanism with relaxation times comparable to those associated with the hydrodynamic motion could possibly demonstrate the observed polar behavior.