An unusual electrooptical effect observed for DNA fragments and its apparent relation to a permanent electric moment associated with bent DNA

Dichroism decay curves of DNA fragments with chain lengths in the range of 179-256 bp show an amplitude inversion suggesting the existence of a positive dichroism component, when these fragments are dissolved at monovalent salt concentrations above approx. 5 mM and are exposed to field pulses with amplitudes and/or lengths above critical values. At the critical values, the unusual dichroism is reflected by an apparent acceleration of the decay curves, which can be fitted by single exponentials with time constants much below the values expected from the DNA contour lengths. The critical pulse amplitudes and lengths decrease with increasing DNA chain length and increasing salt concentration. The experimental data are consistent with results obtained by hydrodynamic and electric model calculations on smoothly bent DNA double helices. The DNA is represented by a string of overlapping beads, which is used to calculate the rotational diffusion tensor and the center of diffusion. The distribution of phosphate charges is asymmetric with respect to this center and thus gives rise to a substantial permanent dipole moment. The magnitude of this dipole moment is calculated as a function of DNA curvature and is used together with experimental values of polarizabilities for simulations of dichroism decay curves. The curves simulated for bent DNA show the same phenomenon as observed experimentally. The ionic strength dependence of the unusual dichroism is explained by an independently observed strong decrease of the polarizability with increasing salt concentration. The field strength dependence is probably due to field-induced bending of double helices driven by the change of the dipole moment. Although our calculations are on rigid models of DNA and thus any flexibility of the double helix has not been considered, we conclude that the essential part of our experimental results can be explained by our model.     

Dielectric properties of proteins from simulation: the effects of solvent, ligands, pH, and temperature

We have used a standard Fr?hlich-Kirkwood dipole moment fluctuation model to calculate the static dielectric permittivity, epsilon(0), for four different proteins, each of which was simulated under at least two different conditions of pH, temperature, solvation, or ligand binding. For the range of proteins and conditions studied, we calculate values for epsilon(0) between 15 and 40. Our results show, in agreement with prior work, that the behavior of charged residues is the primary determinant of the effective permittivity. Furthermore, only environmental changes that alter the properties of charged residues exert a significant effect on epsilon. In contrast, buried water molecules or ligands have little or no effect on protein dielectric properties.     

The dipole moment of the electron carrier adrenodoxin is not critical for redox partner interaction and electron transfer

Dipole moments of proteins arise from helical dipoles, hydrogen bond networks and charged groups at the protein surface. High protein dipole moments were suggested to contribute to the electrostatic steering between redox partners in electron transport chains of respiration, photosynthesis and steroid biosynthesis, although so far experimental evidence for this hypothesis was missing. In order to probe this assumption, we changed the dipole moment of the electron transfer protein adrenodoxin and investigated the influence of this on protein-protein interactions and electron transfer. In bovine adrenodoxin, the [2Fe-2S] ferredoxin of the adrenal glands, a dipole moment of 803 Debye was calculated for a full-length adrenodoxin model based on the Adx(4-108) and the wild type adrenodoxin crystal structures. Large distances and asymmetric distribution of the charged residues in the molecule mainly determine the observed high value. In order to analyse the influence of the resulting inhomogeneous electric field on the biological function of this electron carrier the molecular dipole moment was systematically changed. Five recombinant adrenodoxin mutants with successively reduced dipole moment (from 600 to 200 Debye) were analysed for their redox properties, their binding affinities to the redox partner proteins and for their function during electron transfer-dependent steroid hydroxylation. None of the mutants, not even the quadruple mutant K6E/K22Q/K24Q/K98E with a dipole moment reduced by about 70% showed significant changes in the protein function as compared with the unmodified adrenodoxin demonstrating that neither the formation of the transient complex nor the biological activity of the electron transfer chain of the endocrine glands was affected. This is the first experimental evidence that the high dipole moment observed in electron transfer proteins is not involved in electrostatic steering among the proteins in the redox chain.     

Conformational flexibility of the furanose ring in DNA and its dipole moment

The influence of conformational rearrangement of the furanose ring in DNA on its dipole moment is studied. The dipole moment of the deoxyribose molecule as a function of its puckered state is calculated by the quantum-mechanical method using the MINDO/3 approximation. The values of the dipole moment and its components are obtained at various magnitudes of the pseudorotation phase angle. The C3'-endo in equilibrium C2'-endo conformational transition of deoxyribose is shown to be accompanied by the change in the dipole moment up to 3D. The results obtained are used to explain the structural properties of the DNA hydration shell.     

Dielectric behavior of polyelectrolytes. IV. Electric polarizability of rigid biopolymers in electric fields

The equilibrium Kerr effect of a system of mobile charges constrained to the surface of biomacromolecules is calculated. Cylindrical and spherical geometries are considered. For the cylinder we determine the anisotropy of electric polarizability as a function of length, temperature, and number of charged species in the low-field regime, and the fraction of the maximum induced dipole in the field direction for higher electric fields. The results are compared to experimental data for DNA oligomers taken from the literature. With spherical geometry we calculate the fractional induced dipole moment as a function of electric field strength and from this deduce the orientation function. The field dependence of the orientation function is compared to experimental data in the literature for bovine disk membrane vesicles.     

Conformational Flexibility of the Furanose Ring in DNA and its Dipole Moment

Abstract

The influence of conformational rearrangement of the furanose ring in DNA on its dipole moment is studied. The dipole moment of the deoxyribose molecule as a function of its puckered state is calculated by the quantum-mechanical method using the MINDO/3 approximation. The values of the dipole moment and its components are obtained at various magnitudes of the pseudorotation phase angle. The C3′-endo = C2′-endo conformational transition of deoxyribose is shown to be accompanied by the change in the dipole moment up to 3D. The results obtained are used to explain the structural properties of the DNA hydration shell.     

Analysis of distribution of ligands adsorbed on DNA fragments

The effect of finite fragment length on the distribution pattern of bound protein along the DNA fragment is considered. If the size of the binding site for a ligand on DNA is comparable with the length of the DNA fragment fluctuations in the amount of ligand bound to the fragment create some difficulties for evaluating the distribution pattern of ligand on DNA. A mathematical approach is developed which enables one to calculate the distribution pattern of ligand on DNA provided that the number of bound ligand on the DNA fragment is known. Expression are also obtained to treat the effects of fluctuations in the number of ligand molecules bound to the DNA fragment on the distribution pattern of ligand. A new procedure is proposed which may be useful for locating the preferable binding sites for ligand on DNA on the basis of footprinting experiments.     

Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1.

By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first bead is immobilized by the optical tweezers and the second by the micropipette. Movement of the micropipette allows manipulation and stretching of the DNA molecule, and the force exerted on it can be monitored simultaneously with the optical tweezers. We used this setup to study elongation of dsDNA by RecA protein and YOYO-1 dye molecules. We found that the stability of the different DNA-ligand complexes and their binding kinetics were quite different. The length of the DNA molecule was extended by 45% when RecA protein was added. Interestingly, the speed of elongation was dependent on the external force applied to the DNA molecule. In experiments in which YOYO-1 was added, a 10-20% extension of the DNA molecule length was observed. Moreover, these experiments showed that a change in the applied external force results in a time-dependent structural change of the DNA-YOYO-1 complex, with a time constant of approximately 35 s (1/e2). Because the setup provides an oriented DNA molecule, we determined the orientation of the transition dipole moment of YOYO-1 within DNA by using fluorescence polarization. The angle of the transition dipole moment with respect to the helical axis of the DNA molecule was 69 degrees +/- 3.     

The binding capacity is a probability density function.

The binding capacity of a system, or equivalently, the fluctuations of the number of ligands bound around the average value defined by the binding isotherm, can be regarded as a probability density function for the chemical potential of the ligand. The first moment of this density function is the mean ligand activity as defined by Wyman and gives the average free energy (in kT units) of binding per site. The second moment is directly related to the cooperativity of the system. These and higher moments can be obtained from numerical integration of experimental data in a direct way. An analytical expression for the moment generating function shows that the N independent coefficients of the partition function of a system containing N sites are uniquely defined by the first N moments of the binding capacity.     

A model for the calculation of the dielectric dispersion and the dipole moment of globular proteins in solution

We describe a new procedure whereby the magnitude of the dielectric dispersion of a solution of globular protein molecules can be calculated. The protein molecule is considered to have spherical symmetry and the charged residues are thought to be situated in a medium whose dielectric constant increases continuously as a function of the distance from the centre of mass. The dipole moment of the protein in the solution is made up of two parts: the intrinsic dipole moment due to the charge distribution of the protein and the dipole moment due to polarization of the medium and the ionic cloud. When the model is applied to solutions of cytochrome c it is found that polarization of the medium results in a decrease in the dielectric dispersion amplitude. The mean square dipole moment calculated with the help of this method indicates that the fluctuation of the configurations cannot be responsible for the large dispersion in the megahertz region.     

Ligand binding distributions in nucleic acids

We illustrate a new method for the determination of the complete binding polynomial for nucleic acids based on experimental titration data with respect to ligand concentration. From the binding polynomial, one can then calculate the distribution function for the number of ligands bound at any ligand concentration. The method is based on the use of a finite set of moments of the binding distribution function, which are obtained from the titration curve. Using the maximum-entropy method, the moments are then used to construct good approximations to the binding distribution function. Given the distribution functions at different ligand concentrations, one can calculate all of the coefficients in the binding polynomial no matter how many binding sites a molecule has. Knowledge of the complete binding polynomial in turn yields the thermodynamics of binding. This method gives all of the information that can be obtained from binding isotherms without the assumption of any specific molecular model for the nature of the binding. Examples are given for the binding of Mn(2+) and Mg(2+) to t-RNA and for the binding of Mg(2+) and I(6) to poly-C using literature data.     

Steric effects on multivalent ligand-receptor binding: exclusion of ligand sites by bound cell surface receptors.

Steric effects can influence the binding of a cell surface receptor to a multivalent ligand. To account for steric effects arising from the size of a receptor and from the spacing of binding sites on a ligand, we extend a standard mathematical model for ligand-receptor interactions by introducing a steric hindrance factor. This factor gives the fraction of unbound ligand sites that are accessible to receptors, and thus available for binding, as a function of ligand site occupancy. We derive expressions for the steric hindrance factor for various cases in which the receptor covers a compact region on the ligand surface and the ligand expresses sites that are distributed regularly or randomly in one or two dimensions. These expressions are relevant for ligands such as linear polymers, proteins, and viruses. We also present numerical algorithms that can be used to calculate steric hindrance factors for other cases. These theoretical results allow us to quantify the effects of steric hindrance on ligand-receptor kinetics and equilibria.     

Electric dipole theory and thermodynamics of actomyosin molecular motor in muscle contraction

Movements in muscles are generated by the myosins which interact with the actin filaments. In this paper we present an electric theory to describe how the chemical energy is first stored in electrostatic form in the myosin system and how it is then released and transformed into work. Due to the longitudinal polarized molecular structure with the negative phosphate group tail, the ATP molecule possesses a large electric dipole moment (p(0)), which makes it an ideal energy source for the electric dipole motor of the actomyosin system. The myosin head contains a large number of strongly restrained water molecules, which makes the ATP-driven electric dipole motor possible. The strongly restrained water molecules can store the chemical energy released by ATP binding and hydrolysis processes in the electric form due to their myosin structure fixed electric dipole moments (p(i)). The decrease in the electric energy is transformed into mechanical work by the rotational movement of the myosin head, which follows from the interaction of the dipoles p(i) with the potential field V(0) of ATP and with the potential field Psi of the actin. The electrical meaning of the hydrolysis reaction is to reduce the dipole moment p(0)-the remaining dipole moment of the adenosine diphosphate (ADP) is appropriately smaller to return the low negative value of the electric energy nearly back to its initial value, enabling the removal of ADP from the myosin head so that the cycling process can be repeated. We derive for the electric energy of the myosin system a general equation, which contains the potential field V(0) with the dipole moment p(0), the dipole moments p(i) and the potential field psi. Using the previously published experimental data for the electric dipole of ATP (p(0) congruent with 230 debye) and for the amount of strongly restrained water molecules (N congruent with 720) in the myosin subfragment (S1), we show that the Gibbs free energy changes of the ATP binding and hydrolysis reaction steps can be converted into the form of electric energy. The mechanical action between myosin and actin is investigated by the principle of virtual work. An electric torque always appears, i.e. a moment of electric forces between dipoles p(0) and p(i)(/M/ > or = 16 pN nm) that causes the myosin head to function like a scissors-shaped electric dipole motor. The theory as a whole is illustrated by several numerical examples and the results are compared with experimental results.     

Circular dichroism to determine binding mode and affinity of ligand-DNA interactions

Circular dichroism (CD) is a useful technique for an assessment of DNA-binding mode, being a more accessible, low-resolution complement to NMR and X-ray diffraction methods. Ligand-DNA interactions can be studied by virtue of the interpretation of induced ligand CD signals resulting from the coupling of electric transition moments of the ligand and DNA bases within the asymmetric DNA environment. This protocol outlines methods to determine the binding mode and affinity of ligand-DNA interactions and takes approximately 7.5 h.     

The apparently symmetrical hexagonal bilayer hemoglobin from Lumbricus terrestris has a large dipole moment

The giant approximately 3.6 MDa hexagonal bilayer hemoglobin (HBL Hb) from Lumbricus terrestris consists of 12 213-kDa dodecamers of four globin chains ([b + a + c]3[d]3) tethered to a central scaffold of approximately 36 non-globin, linker subunits L1-L4 (24-32 kDa). Three-dimensional reconstructions obtained by electron cryomicroscopy showed it to have a D6 point-group symmetry, with the two layers rotated approximately 16 degrees relative to each other. Measurement of the dielectric constants of the Hb and the dodecamer over the frequency range 5-100 kHz indicated relaxation frequencies occurring at 20-40 and 300 kHz, respectively, substantially lower than the 700-800 kHz in HbA. The dipole moments calculated using Oncley's equation were 17,300 +/- 2300 D and 1400 D for the Hb and dodecamer, respectively. The approximately threefold higher dipole moment of the dodecamer relative to HbA is consistent with an asymmetric shape in solution suggested by small-angle X-ray scattering. Although a two-term Debye equation and a prolate ellipsoid of revolution model provided a good fit to the experimental dielectric dispersion of the dodecamer, a three-term Debye equation based on an oblate ellipsoid of revolution model was required to fit the asymmetric dielectric dispersion curve of the Hb: the required additional term may represent either an induced dipole moment or a substructure which rotates independently of the main permanent dipole component of the Hb. The D6 point-group symmetry implies that the dipole moments of the dodecamers cancel out. Thus, in addition to a possible contribution from fluctuations of the proton distribution, the large dipole moment of the Hb may be due to an asymmetric distribution of the heterogeneous linker subunits.     

The electric dipole moment of rhodopsin solubilized in Triton X-100.

The electric dipole moment of solubilized rhodopsin was determined with dielectric dispersion measurements. Rhodopsin was extracted from disc membranes of cattle rod outer segments with the nonionic detergent Triton X-100. The dipole moment of rhodopsin at its isoionic point in the detergent micelle is 720 D (150 charge-A). This value is comparable to dipole moments of nonmembrane proteins, especially those which tend to aggregate or polymerize. Flash irradiation of the rhodopsin results in an increase in the dipole moment of about 25 D (5 charge-A). The light-induced increase in dipole moment appears to be composed of two parts--a faster component related to a change in the number of protons bound by rhodopsin and a slower component apparently independent of the change in proton binding.     

Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching

We used single DNA molecule stretching to investigate DNA intercalation by ethidium and three ruthenium complexes. By measuring ligand-induced DNA elongation at different ligand concentrations, we determined the binding constant and site size as a function of force. Both quantities depend strongly on force and, in the limit of zero force, converge to the known bulk solution values, when available. This approach allowed us to distinguish the intercalative mode of ligand binding from other binding modes and allowed characterization of intercalation with binding constants ranging over almost six orders of magnitude, including ligands that do not intercalate under experimentally accessible solution conditions. As ligand concentration increased, the DNA stretching curves saturated at the maximum amount of ligand intercalation. The results showed that the applied force partially relieves normal intercalation constraints. We also characterized the flexibility of intercalator-saturated dsDNA for the first time.     

Covalent hybridization of CNT by thymine and uracil: A computational study

We have investigated the electronic and structural properties of covalent functionalization of the tip of (5,0) carbon nanotube (CNT) by di-keto and keto-enol forms of thymine (T) and uracil (U) nucleobases. Density functional theory (DFT) calculations have been performed to optimize the investigated structures and to calculate the properties such as dipole moment, bond length, band gap, total energy, binding energy and quadrupole coupling constant. The results indicated that, due to the functionalization of CNT by T and U, the hybrids exhibit new properties in which they are similar in both types of CNT-T and CNT-U hybrids.     

The dichroism of DNA in electric fields

We have studied the dichroism of various samples of calf thymus DNA (of molecular weight from 3 × 10⁵ to 7 × 10⁶) in pulsed electric fields. The results may be summarized as follows:

• 1 We find that calf thymus DNA behaves in electrical orientation as if it possessed a large permanent dipole moment. This apparent moment is sensitive to such effects as Mg⁺⁺ binding which lower the net charge on DNA.
• 2 The limiting dichroism at infinite field corresponds to an angle of at least 80% between the transition moments at 265 nm and the helix axis, and could be consistant with a number of known forms of DNA. This result is independent of DNA molecular weight. There is evidence that the conformation may be different in 80% ethanol.
• 3 The dichroism relaxation curves contain a component with a relaxation time of about 8 μsec, which is nearly independent of molecular weight, and a longest component which behaves either according to the Broersma theory for low-molecular-weight samples, or the Zimm-Rouse theory at high molecular weights.

     

Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA

We have investigated the physical binding of pyrene and benzo[a]pyrene derivatives to denatured DNA. These compounds exhibit a red shift in their absorbance spectra of 9 nm when bound to denatured calf thymus DNA, compared to a shift of 10 nm when binding occurs to native DNA. Fluorescence from the hydrocarbons is severely quenched when bound to both native and denatured DNA. Increasing sodium ion concentration decreases binding of neutral polycyclic aromatic hydrocarbons to native DNA and increases binding to denatured DNA. The direct relationship between binding to denatured DNA and salt concentration appears to be a general property of neutral polycyclic aromatic hydrocarbons. Absorption measurements at 260 nm were used to determine the duplex content of denatured DNA. When calculated on the basis of duplex binding sites, equilibrium constants for binding of 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydro-benzo[a]pyrene to denatured DNA are an order of magnitude larger than for binding to native DNA. The effect of salt on the binding constant was used to calculate the sodium ion release per bound ligand, which was 0.36 for both native and denatured DNA. Increasing salt concentration increases the duplex content of denatured DNA, and it appears that physical binding of polycyclic aromatic hydrocarbons consists of intercalation into these sites.