Electrostatics of hemoglobins from measurements of the electric dichroism and computer simulations.

Hemoglobins from normal human cells, from sickle cells, and from horse were investigated by electrooptical methods in their oxy and deoxy forms. The reduced linear dichroism measured as a function of the electric field strength demonstrates the existence of permanent dipole moments in the range of 250-400 Debye units. The reduced limiting dichroism is relatively small (< or = 0.1); it is negative for hemoglobin from sickle cells and positive for the hemoglobins from normal human cells and from horse. The dichroism decay time constants are in the range from about 55 to 90 ns. Calculations of the electrooptical data from available crystal structures are given according to models of various complexity, including Monte Carlo simulations of proton fluctuations with energies evaluated by a finite difference Poisson-Boltzmann procedure. The experimental dipole moments are shown to be consistent with the results of the calculations. In the case of human deoxyhemoglobin, the root mean square dipole is higher than the mean dipole by a factor of about 4.5, indicating a particularly large relative contribution due to proton fluctuations. The ratio of the root mean square dipole to the mean dipole is much smaller (approximately 1.1 to approximately 1.5) for the other hemoglobin molecules. The calculations demonstrate that the dichroism decay time constants are not simply determined by the size/shape of the proteins, but are strongly influenced by the orientation of the dipole vector with respect to the axis of maximal absorbance. The comparison of experimental and calculated electrooptical data provides a useful test for the accuracy of electrostatic calculations and/or for the equivalence of structures in crystals and in solutions.     

The purple to blue transition of bacteriorhodopsin is accompanied by a loss of the hexagonal lattice and a conformational change

X-ray diffraction measurements show that in contrast to the purple membrane, the bacteriorhodopsin molecules are not organized in a hexagonal lattice in the deionized blue membrane. Addition of Ca2+ restores both the purple color and the normal (63 A) hexagonal protein lattice. In the blue state, the circular dichroism spectrum in the visible has the typical exciton features indicating that a trimeric structure is retained. Time-resolved linear dichroism measurements show that the blue patch rotates in aqueous suspension with a mean correlation time of 11 ms and provide no evidence for rotational mobility of bacteriorhodopsin within the membrane. The circular dichroism spectra of the blue and the Ca2+-regenerated purple state in the far-UV are different, indicating a small change in secondary structure. The thermal stability of the blue membrane is much smaller than that of the purple membrane. At pH 5.0, the irreversible denaturation transition of the blue form has a midpoint at 61 degrees C. The photocycle of the blue membrane (lambda ex 590 nm) has an L intermediate around 540 nm whose decay is slowed down into the millisecond time range (5 ms). Light-dark adaptation in the blue membrane is rapid with an exponential decay time of 38 s at 25 degrees C. The purple to blue transition apparently involves a conformational change in the protein leading to a change in the aggregation state from a highly ordered and stable hexagonal lattice to a disordered array of thermally more labile trimers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrooptical analysis of alpha-chymotrypsin at physiological salt concentration

The electric dichroism of alpha-chymotrypsin has been measured in a buffer containing 0.1 M Na(+), 10 mM Mg(2+) and 25 mM Tris-cacodylate pH 7.2. The reduced dichroism as a function of the electric field strength can be represented by the orientation function for permanent dipoles and is not consistent with the orientation function for induced dipoles. After correction for the internal directing field, the dipole moment is 1.1 x 10(-27) Cm (+/- 10%), corresponding to 340 D, at 20 degrees C. The assignment of the permanent dipole moment is confirmed by the shape of the dichroism rise curves, which require two exponentials with amplitudes of opposite sign for fitting. The dichroism decay time constants measured in the range of temperatures between 2 and 30 degrees C indicate a temperature induced change of the structure, which is equivalent to an increase of the hydrodynamic radius from r = 26.6 A at 2 degrees C to 28.5 A at 30 degrees C. Our results demonstrate that electrooptical investigations of proteins with a high time resolution can be extended to physiological salt concentrations without serious problems by use of appropriate instruments.     

Dynamics of DNA condensation

The condensation of DNA induced by spermine and spermidine is investigated by equilibrium titrations and stopped-flow and field-jump experiments using scattered light detection. The spermine concentration required for the cooperative condensation process is measured at different DNA concentrations; these data are used to evaluate both the condensation threshold degree of spermine binding and the binding constant of spermine according to an excluded-site model. Stopped-flow measurements of the spermine-induced condensation demonstrate the existence of two processes: (1) A "fast" reaction is observed in the millisecond time range, when the reactant concentrations are around 1 microM; it is associated with a characteristic induction period and is assigned to the intramolecular condensation reaction. (2) A slow reaction with time constants of, e.g., 100 s strongly dependent upon both spermine and DNA concentrations is assigned to an intermolecular DNA association. The unusual time course of the intramolecular condensation reaction with the induction period provides evidence for a "threshold kinetics". During the induction period, spermine molecules are bound to DNA, but the degree of binding remains below the threshold value. As soon as the degree of ligand binding arrives at the threshold, the DNA is condensed in a relatively fast reaction. Model calculations of the spermine binding kinetics according to an excluded-site model demonstrate that the spermine molecules bound to DNA are mobile along the double helix. A comparison of the experimental data with the results of Monte Carlo simulations suggests a rate constant of approximately 200 s-1 for spermine movement by one nucleotide residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and phosphorylation of initiation factor eIF-2 from rabbit skeletal muscle

Core particle DNA unfolding and refolding are followed by stopped-flow circular dichroism technique. When core particles are dissociated in the stopped-flow cuvette, the high CD deviation corresponding to the dissociated state is reached in the first millisecond, which means that the dissociation process is completed within the dead time of the apparatus which is ～1 ms. The same conclusion can be drawn when core particles are reassociated, since the low CD value, typical of the associated state, is immediately reached. Similarly histone release from chromatin is a very fast process. We also include some points of discussion about core particle assembly process.     

Fluorescence-detected circular dichroism of ethidium in vivo and bound to deoxyribonucleic acid in vitro

Fluorescence-detected circular dichroism (FDCD) spectra are reported for ethidium in Escherichia coli cells and bound to E. coli DNA in vitro. FDCD bands are observed at 325 and 385 nm. These bands change amplitude as the ethidium to DNA ratio changes. Spectra are similar for in vivo and in vitro measurements. However, the bands at 325 and 385 nm disappear when ethidium binds to macromolecules without intercalating between base pairs. The results demonstrate that FDCD spectra can be measured in cell suspensions and indicate that ethidium binds to nucleic acids in E. coli cells by intercalation.     

Nanosecond fluorescence microscopy. Emission kinetics of fura-2 in single cells.

A microscope based time-correlated single photon counting instrument has been constructed to measure fluorescence intensity and emission anisotropy decays from fluorophores in single cells on a nanosecond time scale. The sample is excited and the emission collected using epi-illumination optics with frequency-doubled pulses from the cavity-dumped output of a synchronously pumped dye laser serving as an excitation source. Collection of decays from a single cell is possible due to the presence of an iris in the emission path that can be reduced to less than the diameter of a single cell. Using the instrument the decay of 60 nM 1,6-diphenyl-1,3,5-hexatriene was measured, demonstrating that adequate data for lifetime analysis can be recorded from fewer 10(3) molecules of the fluorophore in an illuminated volume of 23 fl. In addition, the intensity and anisotropy decays of fura-2 in single adherent cells and in suspensions of fura-2 loaded cells in suspension, although the relative amplitudes and decay constants vary somewhat from cell to cell. The results indicate that a significant but variable fraction of fura-2 is bound to relatively immobile macromolecular components in these cells.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.     

Structure of the Tet repressor and Tet repressor-operator complexes in solution from electrooptical measurements and hydrodynamic simulations

The Tet repressor protein and tet operator DNA fragments and their complexes have been analyzed by electrooptical procedures. The protein shows a positive linear dichroism at 280 nm, a negative linear dichroism at 248 nm, and a strong permanent dipole moment of 3.5 X 10(-27) C m, which is independent of the salt concentration within experimental accuracy. Its rotation time constant of 40 ns indicates an elongated structure, which is consistent with a prolate ellipsoid of 100 A for the long axis and 40 A for the short axis. The time constant can also be fitted by a cylinder of length 78 A and diameter 37 A, which is consistent with nuclease protection data reported on repressor-operator complexes, if the cylinder axis is aligned parallel to the DNA axis. Addition of tetracycline induces changes of the limit dichroism but very little change of the rotation time constant. The rotation time constants observed for the operator DNA fragments show some deviations from the values expected from their contour length; however, these deviations remain relatively small. Formation of repressor-operator complexes leads to some increase of the DNA rotation time constants. Simulations by bead models demonstrate that these time constants can be explained without any major change of the hydrodynamic dimension of the components. The data for the complexes are fitted by bead models with smooth bending of the DNA corresponding to a radius of curvature of 500 A, but at the given accuracy, we cannot rule out that the DNA in the complex remains straight or is bent to a smaller radius of approximately 400 A. Thus, binding of the Tet repressor, which is a helix-turn-helix protein as judged from its sequence, to its operator seems to induce minor bending but does not induce strong bending of the DNA double helix.     

Ethidium dimer: a new reagent for the fluorimetric determination of nucleic acids.

A dimeric derivative of ethidium is used for fluorimetric assay of nucleic acids. Due to the very high binding affinity of this derivative for DNA and RNA, a significant increase of sensitivity of the ethidium fluorimetric technique for nucleic acids determination is obtained. Using a commercially available instrument, DNA concentrations in the nanogram per milliliter range are determined. In addition, we have found that an acridine ethidium dimer can also be used for a sensitive fluorimetric assay of DNA concentration, while simultaneously providing a determination of the DNA base composition.     

Cytochrome oxidase rotates in the inner membrane of intact mitochondria and submitochondrial particles

A transient dichroism is detected after photolysis by a linearly polarized laser flash of the cytochrome oxidaseCO complex in bovine heart mitochondria, rat heart mitochondria, and bovine heart submitochondrial particles. A decay in the absorption anisotropy is characterized by a time constant of about 300 to 400 mus in both mitochondria and submitochondrial particles. Since vesicle tumbling in the time range less than 5 ms can be excluded in these experiments, we conclude that cytochrome oxidase rotates in the mitochondrial membrane with a relaxation time of several hundred microseconds. However, it is likely that only about one-half of cytochrome oxidase contributes to the observed decay, the remainder being relatively immobile.     

Quenched flow analysis of exocytosis in Paramecium cells: time course, changes in membrane structure, and calcium requirements revealed after rapid mixing and rapid freezing of intact cells

Synchronous exocytosis in Paramecium cells was analyzed on a subsecond time scale. For this purpose we developed a quenched flow device for rapid mixing and rapid freezing of cells without impairment (time resolution in the millisecond range, dead time approximately 30 ms). Cells frozen at defined times after stimulation with the noncytotoxic secretagogue aminoethyldextran were processed by freeze substitution for electron microscopic analysis. With ultrathin sections the time required for complete extrusion of secretory contents was determined to be less than 80 ms. Using freeze-fracture replicas the time required for resealing of the fused membranes was found to be less than 350 ms. During membrane fusion (visible 30 ms after stimulation) specific intramembranous particles in the cell membrane at the attachment sites of secretory organelles ("fusion rosette") disappear, possibly by dissociation of formerly oligomeric proteins. This hitherto unknown type of rapid change in membrane architecture may reflect molecular changes in protein-protein or protein-lipid interactions, presumably crucial for membrane fusion. By a modification of the quenched flow procedure extracellular [Ca++] during stimulation was adjusted to less than or equal to 3 x 10(-8) M, i.e., below intracellular [Ca++]. Only extrusion of the secretory contents, but not membrane fusion, was inhibited. Thus it was possible to separate both secretory events (membrane fusion from contents extrusion) and to discriminate their Ca++ requirements. We conclude that no Ca++ influx is necessary for induction of membrane fusion.     

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.     

Thermal denaturation of the DNA-ethidium complex. Redistribution of the intercalated dye during melting.

The temperature dependence of the circular dichroism of the DNA-ethidium bromide complex at elevated temperatures provides evidence that the optical activity of the complex near 307 nm originates from interactions between intercalated dye molecules while the optical activity near 515 nm results from singly intercalated ethidium bromide molecules. The behavior of the circular dichroism of the complex at elevated temperatures also explains the higher ellipticities near 307 nm which characterize complexes formed between ethidium bromide and denaturated DNA. Finally the circular dichroism data indicate that the melting of the complex takes place in a stepwise manner with some DNA regions, probably AT-rich regions, dissociating first. The implications of these findings regarding the inhibiting effect of ethidium bromide on the function of DNA polymerase are examined.     

Macromolecular crowding extends the range of conditions under which DNA polymerase is functional

The nick-translation reaction of E. coli DNA polymerase I (Pol I) was used as a model system to demonstrate the ability of macromolecular crowding to alter the response of an enzyme to a number of basic parameters, such as pH, temperature or inhibitors. In the presence of high concentrations of non-specific polymers, nick translation occurred under a variety of otherwise strongly inhibitory conditions. The conditions tested included a range of pH values or temperatures or inhibitory concentrations of urea, formamide or ethidium bromide. These crowding effects are accentuated at higher ionic strengths, suggesting their origin in increased binding between the polymerase and its DNA template-primer under crowded conditions. Kinetic measurements were consistent with such a mechanism.     

A fluorescence photobleaching study of the microsecond reorientational motions of DNA.

We have conducted a polarized fluorescence photobleaching recovery (FPR) study of the rotational dynamics of ethidium azide labeled DNA. Polarized photobleaching experiments provide data on microsecond and millisecond molecular reorientation that complement the information available from nanosecond fluorescence depolarization studies. In polarized FPR experiments an anisotropic angular concentration of fluorophore is created by bleaching dye molecules in a preferred orientation with a short, intense pulse of polarized light. The sample is then weakly illuminated, and the temporal variation in the emitted fluorescence is monitored. The fluorescence signal will systematically change as molecules undergo post-bleach reorientation and the angular distribution of dye tends toward isotropy. We have observed that the time dependence of our microsecond FPR curves is also determined in part by nonrotational phenomena. To isolate the reorientational recovery we conduct our FPR experiments in two modes (called parallel and perpendicular) that differ only in the polarization of the bleaching light. A quotient function, R(t), is constructed from the data obtained in these two modes; the variation with time of this new quantity is governed solely by processes that are sensitive to the polarization of the incident light (e.g., molecular rotation). It is found experimentally that R(t) remains constant, as expected, for rotationally restricted DNA systems despite a temporal recovery in the parallel and perpendicular FPR curves. We also follow the dynamics of solutions of phage lambda DNA as revealed in the temporal dependence of R(t). This DNA system rotationally relaxes after approximately 100 microseconds and the dye/DNA complex reorients substantially during the 10-microseconds bleach period. Our FPR data are interpreted in terms of dynamic models of DNA motion.     

Quaternary dynamics of αB-crystallin as a direct consequence of localised tertiary fluctuations in the C-terminus

The majority of proteins exist in vivo within macromolecular assemblies whose functions are dependent on dynamical processes spanning a wide range of time scales. One such assembly is formed by the molecular chaperone αB-crystallin that exists in a variety of exchanging oligomeric states, centred on a mass of approximately 560 kDa. For many macromolecular assemblies, including αB-crystallin, the inherent dynamics, heterogeneity and high mass contribute to difficulties in quantitative studies. Here, we demonstrate a strategy based on correlating solution-state nuclear magnetic resonance spectroscopy and mass spectrometry data to characterize simultaneously the organization and dynamics of the polydisperse αB-crystallin ensemble. We show that protomeric dimers assemble into oligomers via the binding of extended C-termini, with each monomer donating and receiving one terminus. Moreover, we establish that the C-termini undergo millisecond fluctuations that regulate the interconversion of oligomeric forms. The combined biophysical approach allows construction of an energy profile for a single monomer that completely describes the equilibrium dynamics of the ensemble. It also facilitates an analysis of dynamics spanning the millisecond to hour time scales and secondary to quaternary structural levels, and provides an approach for, obtaining simultaneously detailed structural, thermodynamic and kinetic information on a heterogeneous protein assembly.     

Optical Anisotropy of Chromatin. Flow Linear Dichroism and Electric Dichroism Studies

Abstract

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentraitons by the use of flow linear dichroism (LD) and electric dichroism.

We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA—2 mM NaCl and close positive values in the range of 2–100 mM NaCl. Mg²⁺ cations, in contrast to Na⁺ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5–10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers.

The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA—for chromatins with the linker DNA of 10–30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible.

Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin.

Presented resutls explain the contradictory data obtained by electrooptical and hydrooptical methods.