Hydrodynamic studies of a DNA-protein complex. Elongation of single stranded nucleic acids upon complexation with the gene 32 protein of phage T4 deduced from electric field-induced birefringence experiments

Short DNA and RNA fragments complexed with the helix destabilizing protein of bacteriophage T4, GP32, have been studied in solution by electric birefringence and circular dichroism. The birefringence of the complexes is positive and the magnitude indicates that the DNA and RNA fragments become linear and rigid upon protein binding. The field free decay is biphasic. On the basis of a rigid rod approximation the slow relaxation time leads to a base-base distance along the helix axis in the complex from 4.3 to 5.6 A, an elongation of at least 50% compared to single-stranded DNA.     

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).     

Linear dichroism of the complex between the gene 32 protein of bacteriophage T4 and poly(1,N6-ethenoadenylic acid)

We performed linear dichroism measurements in compressed polyacrylamide gels on the complex between the helix-destabilizing protein of bacteriophage T4, GP32 and poly(1,N6-ethenoadenylic acid), which is used as a model system for single-stranded DNA. A strong hyperchromism for poly(1,N6-ethenoadenylic acid) in the complex indicates a strongly altered conformation. The positive linear dichroism in the wavelength region where the bases absorb must be explained by a strong tilting of the bases in the complex. This finding is in accordance with results from earlier studies, using electric birefringence and circular dichroism measurements. Our measurements show that the angle between the bases and the local helix axis is 42(+/- 6)degrees. In addition, a pronounced contribution from the tryptophan residues of GP32 can be recognized, indicating that several of these residues have a specific orientation in the complex. The sign of the dichroism due to the tryptophan residues is the same as that due to the DNA bases. However, it is not sufficient to assume that all the observed dichroism is due to one or more intercalated tryptophan residues and there must be one or more additional tryptophan residues that make an angle of less than 40 degrees with the local helix axis. Some possible structures of the DNA-protein complex are discussed.     

An electro-optical study of the mechanisms of DNA condensation induced by spermine

Condensation of DNA by spermine has been studied by electric dichroism, electric birefringence and rotational relaxation times at 1 mM ionic strength. Using Manning's theory, we found that condensation occurs for a fraction of neutralized phosphate charges (r) equal to 0.90, in good agreement with previous studies using spermidine, synthetic polyamines and trivalent cations (e.g. Co(NH3)36 +, Tb3 +). Our results are compatible with the presence in solution of torus-shaped condensed structures in a narrow range of spermine concentration; further addition of the polyamine produced precipitation due to the self-aggregation of several toroids. For spermine concentrations lower than that required for collapse, important changes of the orientation mechanism in the electric field and of DNA stiffness were observed. Whereas free DNA was mainly oriented by a fast-induced polarizability mechanism, DNA-spermine complexes displayed an important permanent dipole component, in the spermine concentration range where extension of the DNA molecules was present. The birefringence relaxation times suggested that, in the first step, the stiffness of the DNA molecules increased, and then, at higher spermine concentration, bending of the DNA molecules occurred so that condensation into toroidal particles became possible.     

The conformation of the complex of the helix destabilizing protein GP32 of bacteriophage T4 and single stranded DNA

The conformation of single stranded polynucleotides is changed specifically upon binding of the helix destabilizing protein of bacteriophage T4 (GP32). On the basis of circular dichroism (CD) and absorption experiments it is shown that denaturing conditions and the binding of oligopeptides can not induce the altered conformation. On the contrary, according to the current CD and absorption theory, the optical properties of the complex can be explained by a specific, regular conformation, characterized by an appreciable tilt of the bases (less than or equal to -10 degrees) and either a small rotation per base or a small helix diameter. This conformation agrees nicely with the increase of the base-base distance in the complex as determined in solution by electric field induced birefringence measurements. Our calculations show that also the model proposed by Alma (Ph.D. Thesis Catholic University Nijmegen, The Netherlands (1982)) for the complex of the helix destabilizing protein of bacteriophage fd, in which the helix diameter is large and the bases are almost parallel to the helix axis, would agree with the CD- and absorption spectra of the GP32-complex. For the latter protein this model would have to be modified with regard to the axial increment of the bases which is much larger in the GP32-complexes.     

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.     

The Conformation of the Complex of the Helix Destabilizing Protein GP32 of Bacteriophage T4 and Single Stranded DNA

Abstract

The conformation of single stranded polynucleotides is changed specifically upon binding of the helix destabilizing protein of bacteriophage T4 (GP32). On the basis of circular dichroism (CD) and absorption experiments it is shown that denaturing conditions and the binding of oligopeptides can not induce the altered conformation. On the contrary, according to the current CD and absorption theory, the optical properties of the complex can be explained by a specific, regular conformation, characterized by an appreciable tilt of the bases (?—10°) and either a small rotation per base or a small helix diameter. This conformation agrees nicely with the increase of the base-base distance in the complex as determined in solution by electric field induced birefringence measurements. Our calculations show that also the model proposed by Alma (Ph.D. Thesis Catholic University Nijmegen, The Netherlands (1982)) for the complex of the helix destabilizing protein of bacteriophage fd, in which the helix diameter is large and the bases are almost parallel to the helix axis, would agree with the CD- and absorption spectra of the GP32-complex. For the latter protein this model would have to be modified with regard to the axial increment of the bases which is much larger in the GP32-complexes.     

Electric birefringence and dichroism of acridine orange and methylene blue complexes with polynucleotides

Electro-optic measurements are reported for the polynucleotides poly A, poly U, poly G, and NaDNA, and their complexes with acridine orange (AO). Measurements were also made on the methylene blue (MB) complex with NaDNA. Poly U, poly G, and NaDNA complexes with AO as well as the NaDNA–MB complex were found to exhibit positive birefringence and perpendicular dichroism indicating the dye molecules are oriented with their long axes perpendicular to the applied electric field. The opposite was found for the AO complex with poly A, which showed positive birefringence and parallel dichroism, indicating that in this case the dye molecules are oriented with their long axes parallel to the field.     

Optical Properties of DNA in Aqueous Solution

In the study of DNA electric birefringence, it is usual to use theories that consider that molecules in solution are small in relation to the light wavelength. In this work, we study the DNA electric birefringence using a broken-rod macroion (BRM) model composed of two cylindrical arms which does not restrict the size of the molecules. To achieve this, we include the inhomogeneity effect of the light electric field through the molecule and the interaction between its different parts. To analyze the interaction between a molecule and the incident beam of light, we apply the discrete dipole approximation (DDA), according to which each molecule is described as a finite array of electronic coupled oscillators. The electric birefringence is calculated from the oscillator polarizability. This is obtained from experimental data of electric birefringence saturation and from the increment of the solution refraction index in relation to that of the solvent. Furthermore, the oscillator polarizability is also estimated from DNA absorption spectrum using the Kronig–Kramers relations. This allows us to analyze the contributions of the different absorption bands of DNA to the electric birefringence. We analyze the influence of the inhomogeneity of the light electric field and of the intramolecular interactions in the characterization of DNA optical properties using electric birefringence measurements.     

Electro-optical studies on the electric field-induced helix-to-coil transition of poly(l-ornithine hydrobromide) in methanol/water mixtures

Transient electric birefringence and circular dichroism measurements have been made on poly(L-ornithine hydrobromide) in methanol/water mixtures of various compositions. The specific Kerr constant and the molar ellipticity at 222 nm underwent an abrupt change between 93 and 98% (v/v) methanol at 25°C corresponding to a solvent-induced helix-coil transition. Anomalous birefringence transients were observed at high electric fields between 96 and 98% (v/v) methanol, i.e. on the helix side of the transition region. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different solvent compositions could be superimposed on one another by horizontal and vertical shifts, except for the range where anomalous birefringence transient were observed. This behavior served to determine the threshold field strength. The results indicate that a conformational change from the charged helix to the charged coils is induced by high electric fields in this system, as in the cases of poly(L-lysine hydrobromide) and poly(L-αγ-diaminobutyric acid hydrochloride) in methanol/water mixtures.     

Transient electric birefringence of T-even bacteriophages. I. T4B in the absence of tryptophan and fiberless T4 particles

Measurements of the electric birefringence of suspensions of T4B in the absence of tryptophan and of fiberless T4 particles show that both kinds of particles are hydrodynamically equivalent. Their rotational diffusion coefficients corrected to 25°C and water viscosity (D_25,w) are 280 ± 9 sec^?1 and 295 ± 10 sec^?1, respectively. These corrected rotational diffusion coefficients are almost independent of buffer concentration and temperature. The sedimentation coefficient (s_20,w) of T4 B is equal to 1023 ± 12 S, a value which is likewise independent of buffer concentration. By analysis of the field strength dependence of the steady-state birefringence and by reversing pulse experiments it could be shown that the orientation in an electric field is largely due to a permanent dipole moment. This dipole moment is somewhat dependent on buffer concentration and amounts to about 24,000 debye for T4B and 95,000 debye for fiberless T4. An approximate calculation shows that the difference in dipole moment may be ascribed to positive charges on the fiber tip (at least ten per fiber), to negative charges along the fiber or (and) positive charges on the fiberless particle at those places where the fibers are attached in normal particles.     

Electric polarization of polyelectrolyte and colloid media: dielectric versus electro-optic approach

The theories of dielectric dispersion and of electric birefringence as a representative of electro-optic methods are considered and it is shown that they both depend in a similar way simply on the real part of the complex electric polarizability of the macromolecules or the particles. The latter also contains the permanent dipole moment. Experimental data on dielectric dispersion, electric birefringence and electric light scattering of strongly elongated, rod-like poly(tetrafluoroethylene) particles are compared and an attempt is made to extend the dielectric dispersion curve to lower frequencies using electric birefringence and electric light scattering data. Further, the experimental data on dielectric dispersion, electric light scattering, electro-orientation and dipolophoresis for the more complicated Escherichia coli particles are compared. Again, the possibility to extend the 10 kHz-100 MHz dielectric dispersion curve down below 1 Hz by using electric light scattering data is examined. The good matching of the dielectric dispersion and electric light scattering frequency curves found in the overlapping frequency range (10 kHz-5 MHz) essentially enhances the chance that dielectric dispersion below 1 MHz is related to alpha dispersion and not to electrode polarization. Thus it is not only possible to obtain additional information on the mechanism of polarization at lower-frequency dielectric dispersion, but also to extend our knowledge about the effective dielectric properties of biological complex fluids to frequencies essentially below 1 MHz. This could be important for the understanding of the effect of low-frequency electromagnetic fields on living matter.     

Influence of histones H1/H5 on the DNA coiling in the nucleosome — electric dichroism and birefringence study

Removal of histones H1 and H5 from chicken erythrocyte mononucleosomes results in a large increase of the negative electric birefringence and dichroism, and of the relaxation times, towards the values observed for mononucleosomal DNA. Cross-linking with dimethylsuberimidate does not yield important changes in the electro-optical properties of mononucleosomes, provided that the reaction is performed at low ionic strength. We suggest that in the absence of H1/H5 the linker DNA is flexible, and that this DNA tail is unwound at low ionic strength and responsible for most of the negative anisotropy of these particles. Bipolar pulse experiments revealed that the orientation mechanism of chromatosomes and H1/H5-depleted nucleosomes is predominantly of the induced dipole type.     

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.     

Myosin subfragment 1 has tertiary structural domains

Transient electrical birefringence measurements were made on skeletal muscle myosin subfragment 1 (S1) at 3.7 degrees C in 10 mM tris(hydroxymethyl)aminomethane-acetate and 0.10 mM MgCl2, pH 7.0. The specific birefringence for 4.5 microM S1 was determined from steady-state measurements to be (8.1 +/- 0.3) X 10(-7) (cm/statvolt)2. For electric fields in the range of 2.47-24.7 statvolts/cm, the alignment was due to a large permanent dipole moment for S1, estimated to be 8500 +/- 2000 D. The duration and the strength of the transient electric field was varied, and the temporal response of the decay of the birefringence signal was analyzed. The rate of rotational motion after the field was removed increased with increasing field strength for short (0.35-microseconds) pulses and decreased with increasing pulse lengths for all field strengths. The rate of decay from a steady-state birefringence signal was independent of field strength. A model of S1 structure is proposed, which is consistent with these data and most other data on S1 structure. In this model, S1 is composed of two tertiary structural domains that are connected by a flexible linkage with a substantial restoring force. The electric dipole moments on the two domains are arranged head to tail. The segmental movement of the domains is restricted to certain directions. The average conformation of the molecule is elongated, but it can be made more compact by the torque exerted by an electric field. The structural changes depend on the strength and duration of the pulse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electro-optical studies on synthetic polyelectrolytes. I. Ultraviolet electric dichroism of poly-N-alkylvinylpyridinium bromides in aqueous solution.

The electric dichroism of aqueous solutions of poly-2- and 4-vinylpyridinium bromides has been measured in the 220 to 300 nm region. From the knowledge of the assignments of the absorption bands, the orientation of the pyridinium ring with respect to the macromolecular axis was determined for the poly-N-butyl-4-vinylpyridium bromide. An estimation of the birefringence contribution in the visible region originating from these ultraviolet dichroic bands revealed that the negative birefringence observed mainly arose from electronic transitions at shorter wavelengths.     

Electric birefringence of polytetrafluoroethylene particles in agarose gels

Electric birefringence studies of strongly elongated, rod-like particles of polytetrafluoroethylene (PTFE) in agarose gels show that the negative effect observed by semi-diluted aqueous suspensions at low frequencies and at low electric field strengths (the so called "anomaly') disappears. The absolute value of the low frequency effect increases 3-4 times and the amplitude of modulation decreases faster compared to that of the suspensions. This together with decreased decay relaxation times in gels make the possibilty that the PTFE particles orientation in gels is not due to dipolar but to electrophoretic orientation mechanism quite probable. Similar change in the orientation mechanism could be expected also for suspensions of higher concentrations. The further elucidation of the orientation mechanism using fractions with lower polydispersity, broader ranges of experimental conditions (particle concentration, ionic strength and composition, electric field strengths, frequencies, etc.) could be interest for several fields: colloid electro-optics and especially that of concentrated colloids, pulsed field gel electrophoresis of DNA (and especially its sinusoidal biased field variant) and of nucleoprotein complexes and for the gel research.     

Interaction of ethidium bromide with DNA. Optical and electrooptical study

The binding of ethidium bromide to DNA has been studied by various optical methods. From fluorescence polarization studies, and film, electric linear dichroism, and circular dichroism spectra, we propose assignments of the absorption bands of the dye, which are discussed in connection with wave-mechanical calculations recently reported. The optical activity induced in the dye absorption bands upon binding to DNA was attributed to various origins depending on the electronic transition considered. The visible absorption band displayed a circular dichroism due to the asymmetry of the binding site and independent of the amount of binding. The transition identified at 378 nm from the circular dichroism and electric dichroism observations was thought to be due to a magnetic-dipole transition. It remained constant with increasing amounts of dye bound. The main ultraviolet band showed circular dichroism characteristics corresponding to exciton interactions between dye molecules bound to neighboring sites. The electric dichroism observed for the strongly bound dye molecules indicated that the phenanthridinium ring of ethidium bromide was probably not perfectly parallel to the DNA base planes. When the amount of dye bound to DNA exceeded the maximum amount compatible with the exclusion of adjacent binding sites, the electric dichroism decreased owing to the appearance of externally bound dye molecules with no contribution to the dichroism. Sonicated DNA was used to study the lengthening of the DNA molecule upon complexation. Although the viscosity of the complexes increased with the amount of binding, the rotational diffusion coefficient measured by the electric birefringence relaxation was not detectably affected. The absence of variation in the electric birefringence with the binding indicated that the DNA base stacking remained unaltered.     

The effects of an electric field on soluble collagen.

Electric birefringence decay curves of collagen suspended in aqueous buffered media were plotted as functions of pulse width and amplitude. They were then resolved into two components by means of an analog simulator. When these data were combined with the results of repeated pulsing, it was shown that an electric field promotes aggregation of collagen, although the variety of aggregate sizes falls within a fixed range. Observations of electric birefringence of dissolved collagen preparations as a function of ionic strengths tend to indicate that the bonding that occurs in an electric field is electrostatic.