Multiple conformational states of the hammerhead ribozyme, broad time range of relaxation and topology of dynamics.

The dynamics of a hammerhead ribozyme was analyzed by measurements of fluorescence-detected temperature jump relaxation. The ribozyme was substituted at different positions by 2-aminopurine (2-AP) as fluorescence indicator; these substitutions do not inhibit catalysis. The general shape of relaxation curves reported from different positions of the ribozyme is very similar: a fast decrease of fluorescence, mainly due to physical quenching, is followed by a slower increase of fluorescence due to conformational relaxation. In most cases at least three relaxation time constants in the time range from a few microseconds to approximately 200 ms are required for fitting. Although the relaxation at different positions of the ribozyme is similar in general, suggesting a global type of ribozyme dynamics, a close examination reveals differences, indicating an individual local response. For example, 2-AP in a tetraloop reports mainly the local loop dynamics known from isolated loops, whereas 2-AP located at the core, e.g. at the cleavage site or its vicinity, also reports relatively large amplitudes of slower components of the ribozyme dynamics. A variant with an A-->G substitution in domain II, resulting in an inactive form, leads to the appearance of a particularly slow relaxation process (tau approximately 200 ms). Addition of Mg(2+) ions induces a reduction of amplitudes and in most cases a general increase of time constants. Differences between the hammerhead variants are clearly demonstrated by subtraction of relaxation curves recorded under corresponding conditions. The changes induced in the relaxation response by Mg(2+) are very similar to those induced by Ca(2+). The relaxation data do not provide any evidence for formation of Mg(2+)-inner sphere complexes in hammerhead ribozymes, because a Mg(2+)-specific relaxation effect was not visible. However, a Mg(2+)-specific effect was found for a dodeca-riboadenylate substituted with 2-AP, showing that the fluorescence of 2-AP is able to indicate inner sphere complexation. Amplitudes and time constants show that the equilibrium constant of inner sphere complexation is 1.2, corresponding to 55% inner sphere state of the Mg(2+) complexes; the rate constant 6.6 x 10(3) s(-1) for inner sphere complexation is relatively low and shows the existence of some barrier(s) on the way to inner sphere complexes.

Dynamics of the RNA hairpin GNRA tetraloop.

The dynamics of RNA hairpin tetraloops of the GNRA type [sequence G- any ribonucleotide (N)-purine (R)-A] was analyzed by fluorescence spectroscopy and by fluorescence-detected temperature-jump relaxation, using RNA oligomers with 2-aminopurine (2AP) substituted in two different positions of the loop sequence, Gp2APpApA (HP1) and GpAp2APpA (HP2), as indicator. The fluorescence of HP1 is much higher than that of HP2, indicating a lower degree of 2AP-stacking in HP1. Addition of Mg(2+) or Ca(2+) ions leads to an increase of fluorescence in HP1, whereas a decrease of fluorescence is observed in HP2. In both cases at least two ion-binding equilibria are required to fit titration data. T-jump experiments using fluorescence detection show a relaxation process with a time constant of 22 micros for HP1, whereas two relaxation processes with time constants 5 and 41 micros, are found for HP2. These results clearly demonstrate the existence of more than the single conformation state detected by NMR analysis. The T-jump amplitudes decrease with increasing bivalent ion concentration, indicating that one of the states is favored in the presence of bivalent ions. The loop relaxation processes are slower than standard stacking processes, probably because of activation barriers imposed by a restricted mobility of loop residues, and are assigned to a stacking rearrangement, probably between the 5' and the 3'-side. A similar process has been observed previously for the anticodon loop of tRNA(Phe). The rate constants of the transition are in the range of 10(4) s(-1) in the case of HP1. The data demonstrate the existence of structures that are not resolved by standard NMR because of fast exchange and are not found by X-ray analysis because of restrictions by crystal packing.

Global structure and flexibility of hairpin ribozymes with extended terminal helices.

Global structure and flexibility of three different hairpin ribozyme constructs have been analyzed by measuring their electric dichroism decay in various buffers at temperatures between 2 and 30 degrees C. The hairpin ribozyme is characterized by two independently folding domains A and B that are connected through a hinge and have to interact to enable catalysis. The analyzed constructs feature extended terminal helices 1 and 4 with 27 and 25 bp, respectively, to increase the sensitivity of the molecular rotational diffusion time constants with respect to the interdomain bending angle. Constructs HP1 and HP2 cannot cleave because of a G+1A change at the 3'-side of the cleavage site; in HP1 the helices 2 and 3 that flank the hinge form a continuous double helical segment; in HP2 and HP3, a six nucleotide bulge confers flexibility to the expected bending site; HP3 is a cleavable form of HP2 with a G+1-base. For comparison, a standard RNA double helix with 72 bp was included in our analysis. The dichroism decay curves of the hairpin constructs after pulses of low electric field strengths can be fitted to single exponentials taus, whereas the curves after pulses of high field strengths require two exponentials. In all cases, time constants increase with RNA concentration, indicating intermolecular interactions. Extrapolation of the tausvalues measured in standard buffer (50 mM Tris (pH 7.5) and 12 mM MgCl2) to zero RNA concentration provide values of 112, 93, and 73 ns for HP1, HP2 and HP3, respectively, at 30 degrees C, indicating increasingly compact structures. The 72 bp RNA reference under corresponding conditions did not show a dependence of its decay time constant on the RNA concentration nor on the field strength; its time constant is 175 ns (standard buffer, 30 degrees C). The observation of two relaxation processes for the hairpin constructs at high field strengths indicates stretching to a more elongated state; the fast process with a time constant of the order of 50 ns is assigned to reversion of stretching, the slow process to overall rotation. The overall rotational time of the stretched state at 20 degrees C is close to that for a completely stretched rigid state; at 30 degrees C the experimental values are around 70 % of that expected for a completely stretched rigid state, indicating flexibility and/or residual bending. Bead models were constructed to simulate dichroism decay curves. The time constants observed for the 72 bp RNA are as expected for a rigid rod with a rise of 2.8 A per base-pair. Based on this rise per base-pair for models of a V and a Y-shape, we estimate average bending angles of 80(+/-20) degrees and 105 (+/-25) degrees, respectively, for the catalytically active hairpin ribozyme HP3. The energy required for stretching is of the order of the thermal energy.

Time-resolved analysis of macromolecular structures during reactions by stopped-flow electrooptics.

A stopped-flow field-jump instrument and its use for the analysis of macromolecular structure changes during reactions is described. The operation of the new instrument is simple and reliable, owing to a new type of cell construction with electrodes directly integrated in a quartz cuvette: major advantages are the relatively low demand on sample quantities and a high time resolution. The stopped flow is characterized by a dead time of approximately 0.5 ms. Electric field pulses with field strengths up to 20 kV/cm and rise times in the nanosecond range are applied at adjustable times after stop of the flow. The time resolution of the optical detection is up to the nanosecond time range. The instrument may be used for the combination of stopped flow with temperature-jump and field-jump experiments. A particularly useful new application is the analysis of macromolecular reactions by electrooptical measurements, because electrooptical data provide information about structures. This is demonstrated for the intercalation of ethidium into double-helical DNA. The transients, measured at 313 nm, where the signal is exclusively due to ethidium bound to the DNA, demonstrate a relatively high negative dichroism at 0.5 ms after mixing. The absolute value of this negative dichroism increases in the millisecond time range and approaches the equilibrium value within about a second. The dichroism decay time constants demonstrate a clear increase of the effective DNA length due to ethidium binding, already 0.5 ms after mixing; a further increase to the equilibrium value is found in the millisecond time range. The analysis of these data demonstrate the existence of up to three relaxation processes, depending on the conditions of the experiments. The dichroism amplitudes, together with the decay time constants, indicate that all the reaction states found in the present investigation are complexes with insertion of ethidium residues between basepairs. Moreover, the data clearly show the degree of intercalation in the intermediate states, which is very useful information for the quantitative assignment of the mechanism.

The cyclic AMP receptor promoter DNA complex: a comparison of crystal and solution structure by quantitative molecular electrooptics.

The complexes formed between the cyclic AMP receptor and three different promoter DNA fragments, including a synthetic 30 bp fragment with the sequence used for determination of the crystal structure, have been analysed in solution by measurements of the electric dichroism (ED) at an ionic strength of 105 mM, using a special instrument based on cable discharge. The ED of the protein is negligible and, thus, the ED of the complexes is determined by the DNA and its orientation relative to the protein. The complex formed between the cyclic AMP receptor with the 30 bp fragment is characterized by a positive ED, indicating that the electric dipole is perpendicular relative to the direction of the helix; moreover, the dipole changes its nature from an induced one for the free DNA to a permanent one of 3.0 x 10(-27) Cm for the complex; both the limiting value of the ED +0.3 and the dichroism decay time constant of 62 ns found for the complex (free DNA: 52 ns; 20 degrees C) demonstrate bending of the DNA double helix. All these parameters are calculated quantitatively from the crystal structure: bead model simulations are used to derive the coefficients of rotational diffusion and to define the center of diffusion, which is the reference for calculation of the dipole vector; the dipole vector is then the basis for calculation of the limit value of the dichroism; the time constants are derived from the diffusion coefficients of the bead model The calculated parameters are in very satisfactory agreement with the experimental ones, demonstrating agreement of the structures in the crystal and in solution with respect to their essential features. These results also demonstrate the utility of electrooptical procedures for a quantitative comparison of crystal or model structures with structures in solution. While the crystal structure has been determined only for the complex with the 30 bp promoter fragment, it has been relatively simple to extend measurements of the ED to complexes formed with a 40 bp and a 203 bp promoter fragment: the data obtained for a 40 bp fragment with the consensus binding sequence are quite similar to those obtained for the 30 bp promoter, whereas the data obtained for the 203 bp promoter clearly show a much higher degree of protein induced bending with a bending angle of approximately 180 degrees.

Macrodipoles. Unusual electric properties of biological macromolecules.

The wide range of different effects induced by electric fields in biological macromolecules is clearly due to the unusual quality and quantity of their electric parameters. A general concept for a quantitative description of the polarizability of macromolecules remains to be established. In the case of DNA, experimental data indicate the existence of an effective polarization length N(p); at chain lengths N < N(p) the polarizability increases with N(2), whereas saturation is approached at N > or = N(p). The polarization length decreases with increasing ionic strength in close analogy to the Debye length, but is approximately 10 times larger than the Debye length. The dynamics of DNA polarization at high field strengths has been observed in the ns time range and is consistent with biased field induced ion dissociation. In the range of chain lengths from approximately 400 to approximately 850 base pairs DNA molecules exhibit permanent dipole moments, which are in a preferentially perpendicular direction to the end-to-end-vector, leading to a positive electric dichroism. These results are consistent with a "frozen" ensemble of bent DNA configurations and provide evidence for the existence of slow, non-elastic bending transitions. The electric parameters of proteins are usually dominated by a permanent anisotropy of the charge distribution, corresponding to permanent dipole moments of the order of several hundred Debye up to about 1500 Debye. Relatively small dipole moments of protein monomers add up to millions of Debye, when these proteins are in a vectorial organization in membrane patches, as found for bacteriorhodopsin and Na (+)K (+)-ATPase . In these cases the dipole vector may support vectorial ion transport. It is remarkable that the dipole moments of proteins usually show a relatively small dependence on the salt concentration; a rational for these observations is provided by a dipole potential at the plane of shear for rotational diffusion, which is defined in close analogy to the zeta-potential for translational diffusion. Symmetry breaking leading to huge electric dipole moments may be expected for mixed lipid vesicles: according to model calculations the phase separation of lipid components with and without net charges may lead to very high dipole moments; the expectation has been verified experimentally for vesicles containing DMPA and DMPC. The state of these systems should be extremely sensitive to electric fields. In summary, there is an unusual wide variation of electric parameters associated with biological macromolecules and with biomolecular assemblies, which is the basis for the complexity of different phenomena induced by electric fields in biological systems.

Electrostatics and electrodynamics of bacteriorhodopsin.

The stationary electric dichroism of bacteriorhodopsin is in qualitative, but not quantitative, agreement with the orientation function for disks having a permanent dipole directed perpendicular to the plane and an induced dipole in the plane. Fits of the orientation function to data measured at low field strengths demonstrate: an increase of the permanent dipole moment mu with the square of the disk radius r2, whereas the polarizability alpha increases with r4; the ionic strength dependence is small for mu and clearly stronger for alpha; the permanent dipole moment is 4x10(6) D at r = 0.5 micron. According to the risetime constants, the induced dipole does not saturate and increases to 4x10(8) D at 40 kV/cm and r = 0.5 micron. The data indicate that the permanent dipole is not of some interfacial character but is due to a real assymetry of the charge distribution. The experimental dipole moment per protein monomer is approximately 55 D, whereas calculations based on the structure of Grigorieff et al. (Grigorieff, N., T.A. Ceska, K.H. Downing, J.M. Baldwin, and R. Henderson. 1996. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J. Mol. Biol. 259:393-421) provide a dipole moment of approximately 570 D. The difference is probably due to a nonsymmetric distribution of charged lipid residues. It is concluded that experimental dipole moments reflect the mu-potential at the plane of shear for rotational diffusion, in analogy to the sigma-potential used for translational diffusion. It is suggested that the permanent dipole of bacteriorhodopsin supports proton transport by attraction of protons inside and repulsion of protons outside of the cell. Dichroism rise curves at field strengths between E = 150 and 800 V/cm reveal an exponential component with time constants tau 3r in the range between 1 and 40 ms, which is not found in Brownian dynamics simulations on a disk structure using hydrodynamic and electric parameters characteristic of bacteriorhodopsin disks. The experimental data suggest that this process reflects a cooperative change of the bacteriorhodopsin structure, which is induced already at a remarkably low field strength of approximately 150 V/cm.

Mg(2+)-dependent conformational changes in the hammerhead ribozyme.

Conformational changes of the hammerhead ribozyme were examined by fluorescence changes of 2-aminopurine riboside incorporated either in the substrate or in the ribozyme. Fluorescence changes could be observed for both the substituted substrate and ribozyme upon complex formation, indicating a different environment for the 2-aminopurine in the complex. Ribozyme-substrate constructs for ciscleavage containing 2-aminopurine at various sites were used for the determination of binding constants of Mg2+ and Ca2+. Depending upon the site of 2-aminopurine substitutions, the fluorescence intensity upon addition of Mg2+ or Ca2+ was reduced by 0-50%. The measurements were performed in high ionic strength buffers such that base pairing in the helical regions is expected to be complete. With three of the ribozymes, the dependence of the fluorescence emission as a function of Mg2+ concentration could be fitted by single binding processes, whereas for the two remaining ribozymes a second binding process needed to be included. The binding constants range from 7600 M-1 down to 12 M-1 in 75 mM Tris-HCl (pH 7.5) and indicate the presence of multiple binding sites in the ribozymes with varying degrees of affinity toward the metal ions. Mg2+ binding constants determined in the same buffer from the Mg2+ dependence of the cleavage rate are of the order of 100 M-1; thus, Mg2+ sites directly involved in catalysis are of intermediate affinity. The ribozyme containing 2-aminopurine in loop III demonstrated the highest binding constant whereas the ribozyme with a 2-aminopurine next to a 2'-deoxy-2'-aminocytosine at the cleavage site exhibited only low metal ion affinity. The data obtained for Ca2+ are very similar to those found for Mg2+. This approach provides a first set of data describing a Mg2+ binding topography to hammerhead RNA molecules and should be useful for the analysis of other RNA molecules.

Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase.

Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.

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.

Electric parameters of Na+/K(+)-ATPase by measurements of the fluorescence-detected electric dichroism.

The electric parameters of Na+/K(+)-ATPase labeled by FITC have been characterized by measurements of the fluorescence-detected electric dichroism. The fluorescence emission was measured with polarizers at the magic angle and the light for excitation was usually polarized parallel to the field vector. The FITC-Na+/K(+)-ATPase preparations exhibit a negative electric dichroism at field strengths up to about 600 V/cm and a positive dichroism at higher field strengths. Pulse reversal experiments reveal a dominant permanent electric moment at low electric field strengths and an increasing contribution from an induced electric moment at higher field strengths. The dichroism rise curves and the transients upon pulse reversal show two relaxation processes with opposite amplitudes, whereas the dichroism decay curves in most cases can be represented by single exponentials at a reasonable accuracy. The amplitude A2 associated with the slower of the rise processes is dominant at low field strengths and also approaches saturation already at low field strengths. The dependence of A2 on the electric field strength is consistent with the orientation function for permanent dipoles and cannot be represented by the orientation function for induced dipoles. The fitted permanent dipole moment is in the range of 3.5 x 10(-24) Cm [1 x 10(6) D] and shows only a relatively small decrease with increasing ionic strength. The stationary values of the electric dichroism up to field strengths E < or = 800 V/cm can be represented with high accuracy by an orientation function for disk-shaped particles with a permanent moment along the particle symmetry axis and an induced moment along the semi-major axis. The permanent electric moment determined according to this function is consistent with the one obtained from the amplitudes A2. In summary, our measurements indicate that Na+/K(+)-ATPase is associated with a large permanent electric moment directed perpendicular to the membrane plane. The dipole moment per ATPase monomer unit is estimated to be 1.4 x 10(-27) Cm [430 +/- 50 D].

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 structure of the RNA polymerase-promoter complex. DNA-bending-angle by quantitative electrooptics.

The complex formed between RNA polymerase holoenzyme from Escherichia coli and the strong promoter A1 from the phage T7 has been characterized by measurements of the electric dichroism. The dichroism decay time constant of a promoter DNA fragment with 126 bp increases upon binding of the polymerase, but the increase is less than expected for simple addition of the components at the known binding site. Our results demonstrate a protein-induced decrease of the hydrodynamic DNA dimensions, which is not a consistent with an increased flexibility, but indicates bending of the double helix with a relatively narrow distribution of bending angles. We have characterized the degree of DNA bending by bead model simulations and used, in addition to our present experimental data, the available information on the overall size and shape of the RNA polymerase, together with the location of the DNA bending center at the starting point of RNA synthesis. We conclude that the bending angle is 45 degrees (+/- 5 degrees).

Structure and dynamics of peptide-polynucleotide complexes.

The mode and the dynamics of LysTrpLys-binding to double helical DNA and to single stranded poly(A) has been analyzed by measurements of the chemical relaxation detected by fluorescence and of the rotational diffusion using the electric dichroism. The chemical relaxation, induced by electric field pulses, requires two exponentials for a satisfactory representation, indicating a two step reaction mechanism. The data are consistent with a bimolecular reaction step followed by a relatively slow intramolecular transition, which is expected to reflect "insertion" of the Trp-indole residues between the nucleic acid bases. The experimental data are analyzed quantitatively by global fitting with exact correction of the convolution due to the experimental device. In this procedure a complete set of relaxation curves is fitted directly to the reaction model and, thus artifacts resulting from erroneous assignments of coupled modes are avoided. According to this analysis the bimolecular reaction step is controlled by diffusion. The intramolecular transition in adenylate chains is found to be dependent on the chain length and on the ionic strength I: at I = 2.5 mM the "insertion" rate constant is 3 x 10(4) s-1 for the polymer and 2 x 10(5) s-1 for A(pA)19; the rate constant for poly(A) increases with increasing salt concentration. The corresponding "insertion" rate constant for DNA double helices with 30 kbp is 2.5 x 10(4) s-1. For DNA double helices we find again an increase of the "insertion" rate with increasing salt concentration and with decreasing chain length. The mode of LysTrpLys-binding to double helical DNA is compared with that of LysTyrLys, LysLeuLys and LysGlyLys by measurements of the rotational diffusion of complexes with restriction fragments of different chain lengths. The persistence lengths derived from these measurements do not reveal any special effects resulting from insertion of aromatic residues. Apparently "insertion" of indole rings into double helical DNA does not increase the length of the double helix, which may be attributed to a special form of insertion, e.g. partial insertion. According to these results the interaction of the indole residues of LysTrpLys with DNA double helices is not equivalent to e.g. intercalation of aromatic residues like ethidium-neither with respect to structure nor to dynamics.

Mechanism of intercalation into the DNA double helix by ethidium.

The mechanism of intercalation into DNA double helices by ethidium has been analyzed by temperature-jump relaxation and stopped-flow measurements using fluorescence detection. Artifacts due to field- or flow-induced alignment have been eliminated by measurements under magic angle conditions; the theoretical basis for suppression of orientation effects resulting from external forces is given for the case of fluorescence measurements. Excluded site effects have been avoided by restriction to low degrees of binding. The temperature-jump relaxation observed for ethidium binding to DNA could be described by single exponentials under most conditions. The reciprocal time constants increased linearly with the DNA concentration, leading to association rate constants of 2.7 x 10(6) M-1 s-1 at 12 degrees C. These rate constants are virtually independent of the DNA chain length for samples with 200, 500, 4228, and 30,000 base pairs, showing that the rate is controlled by reaction and not by a diffusive process. At high DNA concentrations around 200 microM, an additional relaxation effect with an amplitude opposite to the main one is observed which is probably due to some conformational change of the DNA-ethidium complex. The results obtained by stopped-flow measurements are consistent with those from T-jump measurements, but owing to higher amplitudes and better signal to noise ratios, the stopped-flow data clearly require two exponentials for satisfactory representation. The reciprocal time constants for both processes increase linearly with the DNA concentration. The simplest mechanism consistent with this result involves parallel formation of two different complexes with a direct transfer of ethidium between the binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Nature of protamine-DNA complexes. A special type of ligand binding co-operativity.

The mode of protamine binding to DNA double helices has been analyzed for the example of clupein Z from herring and DNA samples from bacteriophages lambda and PM2 by measurements of light-scattering intensities, ultracentrifugation and kinetics. The light-scattering intensity of DNA increases co-operatively at a threshold clupein concentration suggesting co-operative binding of clupein to double helices. These data are first analyzed in terms of a model with a transition at a threshold degree of binding. The parameters resulting from this analysis appear to be reasonable, but are shown to be in contrast with data on the absolute degree of clupein binding to DNA obtained by centrifugation experiments. An analysis of the kinetics associated with clupein binding to DNA by measurements of the time-dependence of light-scattering intensities in the time range of seconds demonstrates directly that clupein-induced intermolecular interactions of DNA molecules are essential. The rate constants of DNA association increase co-operatively at threshold clupein concentrations, which correspond to those observed in the equilibrium titrations. Above the threshold, the rate constants arrive at a level that is almost constant, but shows some decrease with increasing clupein concentrations. These results are described by a model with a monomer and a dimer state of DNA, which bind ligands with different affinities according to an excluded-site binding scheme. When the ligand binding constant is larger for the dimer than for the monomer state, as should be expected, binding of ligands drives the DNA from the monomer to the dimer state, even if the dimerization equilibrium in the absence of ligands is far in favor of the monomer. The transition from the monomer to the dimer state proves to be strongly co-operative. When the ligand concentration is increased to higher values, the dimers may be converted back to monomers due to an increased extent of ligand binding to the monomer state. The model is consistent with the available experimental data. The analysis of the data by the model indicates the existence of a reaction unit much below the DNA chain length, corresponding to about 80 nucleotide residues. The present model describes ligand driven intermolecular association; an analogous model is applicable to ligand driven intramolecular association. In summary, the co-operativity of clupein binding to DNA double helices is not due to nearest neighbor interactions, but results from thermodynamic coupling of clupein binding with clupein-induced DNA association.

Phase transition of dimyristoylphosphatidylglycerol bilayers induced by electric field pulses.

The phase transition of dimyristoylphosphatidylglycerol (DMPG) bilayers has been studied by measurements of light scattering under high electric field pulses. Midpoints of phase transitions have been identified by a clear discontinuity of field induced relaxation amplitudes. We show that the phase transition of DMPG suspensions in monovalent salt is virtually independent of the electric field strength up to approx. 35 kV/cm. A shift of the lipid phase by electric field pulses has been observed, however, for DMPG suspensions in the presence of Ca2+ ions. DMPG suspensions exhibit a jump of the phase transition temperature from 17 degrees C at Ca/DMPG molar ratios r less than 1/7 to 32 degrees C at r greater than 1/7. Field pulses of 60 to 100 microseconds applied to DMPG suspensions with Ca2+ at r greater than 1/7 induce discontinuities of relaxation amplitudes in the temperature range 15 to 22 degrees C in addition to the 'standard' one at 32 degrees C, when the electric field strength is above 15 kV/cm. These results indicate that electric field pulses induce a transition from the phase formed at 'high' Ca(2+)- to the one formed at 'low' Ca(2+)-ion concentrations. Our results are consistent with a dissociation field effect on Ca(2+)-lipid complexes which drives the phase transition.

Persistence length and bending dynamics of DNA from electrooptical measurements at high salt concentrations.

A new electrooptical apparatus has been used to characterize the dichroism decay time constants for a collection of nine blunt-ended DNA restriction fragments in the range of chain lengths from 41 to 256 base-pairs at physiological salt concentrations. The experimental data show an increase of rotational diffusion coefficients, when the monovalent salt concentration is increased from a few mM, used previously for standard electrooptical experiments, to the range of salt concentrations around 100 mM. The presence or absence of 10 mM Mg2+ in a buffer with 100 mM NaCl does not induce any large change of the rotational diffusion. Bending of double helices is reflected by a fast component in the dichroism decay for fragments greater than or equal to 90 bp; the time constant of the first bending mode is 7-9% relative to the time constant of overall rotational diffusion for fragments with 90 to 179 bp at the temperatures 2, 10 and 20 degrees C. Interpretation of the overall rotational diffusion time constants by different models on the hydrodynamics of flexible polymer chains leads to diverging values of the persistence length. The most accurate description is expected from a combination of the rotational diffusion coefficient for rigid rods given by Tirado and Garcia de la Torre (J. Chem. Phys. 73 (1980) 1986) with correction factors derived from Monte Carlo simulations (P.J. Hagerman and B.H. Zimm, Biopolymers 20 (1981) 1481). This model leads to 'average' values of the persistence length of 440, 400 and 380 A at the temperatures 20, 10 and 2 degrees C, respectively (in 110 mM Na+ and 10 mM Mg2+, pH 7.0); the hydrodynamic radius of the helix is approx. 12.5 A. The persistence lengths measured at various monovalent salt concentrations can be represented as a linear function of the reciprocal square root of the ionic strength. The rotational time constants measured for individual fragments at physiological salt show clearly larger deviations from the model average than corresponding time constants measured previously at low salt; 'apparent' persistence lengths of individual fragments as well as their temperature dependence show strong variations. Thus, it is hardly possible to define a 'standard' persistence length for mixed sequences--even though the sequences used in the present investigation do not show clear deviations from standard gel mobilities. These data indicate that formation of individual, sequence-directed structures of DNA fragments is favoured under physiological salt conditions.

Permanent dipole moment of tRNA's and variation of their structure in solution.

The structure of six different tRNA molecules has been analyzed in solution by electrooptical measurements and by bead model simulations. The electric dichroism measured as a function of the field strength shows that tRNA's are associated with substantial permanent dipole moments, which are in the range of 1 x 10(-27) cm(identical to 300 D; before correction for the internal directing field). Rotational diffusion time constants of tRNA molecules in their native state at 2 degrees C show a considerable variation. A particularly large value found for tRNA(Tyr) (50 ns) can be explained by its nine additional nucleotide residues. However, remarkable variations remain for tRNA molecules with the standard number of 76 nucleotide residues (tRNA(Phe) [yeast] 41.6 ns, tRNA(Val) [Escherichia coli] 44.9 ns, tRNA(Glu) [E. coli] 46.8 ns; tRNA(Phe) [E. coli] 48.3 ns). These variations indicate modulations of the tertiary structure, which may be due to a change of the L-hinge angle. Bead models are used to simulate both electric and hydrodynamic parameters of tRNA molecules according to the crystal structure of tRNA(Phe) (yeast). The asymmetric distribution of phosphate charges with respect to the center of diffusion leads, under the assumption of a constant charge reduction to 15% by ion condensation, to a theoretical dipole moment of 7.2 x 10(-28) cm, which is in reasonable agreement with the measurements. The dichroism decay curve calculated for tRNA(Phe) (yeast) is also consistent with the measurements and thus the structure in solution and in the crystal must be very similar in this case. However, our measurements also indicate that the structure of some other tRNA's in solution is different, even in cases with the same number of nucleotide residues.