Kinetics of the B-A transition of DNA: analysis of potential contributions to a reaction barrier

Kinetics of the B-A transition of DNA: analysis of potential contributions to a reaction barrier Because of open problems in the relation between results obtained by relaxation experiments and molecular dynamics simulations on the B-A transition of DNA, relaxation measurements of the B-A dynamics have been extended to a wider range of conditions. Field-induced reaction effects are measured selectively by the magic angle technique using a novel cell construction preventing perturbations from cell window anisotropy. The kinetics was recorded for the case of poly[d(AT)] up to the salt concentration limit of 4.4 mM, where aggregation does not yet interfere. Now experimental data on the B-A dynamics are available for poly[d(AT)] at salt concentrations of 0.18, 0.73, 2.44 and 4.4 mM. In all cases, a spectrum of time constants is found, ranging from ~ 10 μs up to components approaching ~ 1 ms. The relatively small dependence of these data on the salt concentration indicates that electrostatic effects on the kinetics are not as strong as may be expected. The ethanol content at the transition center is a linear function of the logarithm of the salt concentration, and the slope is close to that expected from polyelectrolyte theory. The B-A transition dynamics was also measured in D O at a salt concentra- tion of 2.4 mM: the center of the transition is found at 20.0 mol/l H O and at 20.1 mol/l D O with an estimated accuracy of 2 2 ± 0.1 mol/l; the spectrum of time constants at the respective transition centers is very similar. The experimental results are discussed regarding the data obtained by molecular dynamics simulations. Keywords B-A transition · Kinetics · Magic angle measurements · Electrostatics · Solvent isotope effect Introduction sugar pucker is C2′-endo for the B and C3′-endo for the A form; (4) the hydration is more extensive in the B form and Looking at molecular models of the B- and A-form DNA more “economic” in the A form (Saenger et al. 1986). Dick- suggests that the transition between these well-ordered, erson and Ng ( 2001, 2002) concluded from their analysis of right-handed double-helical structures (Saenger 1984) is a the structures that there is a smooth transition between the B relatively simple rearrangement. The molecular details of B- and A forms without major activation barriers. and A-helix structures in crystals have been analyzed at high The equilibrium conditions for the existence of the B- resolution (Egli et al. 1998; Schneider et al. 1998; Dickerson and A-form DNA in solution have been characterized in and Ng 2001; Ng and Dickerson 2002). A short summary of detail (Ivanov et al. 1974). In general, the B form is observed the major differences: (1) the helix increase per base pair is in usual aqueous solutions, whereas formation of the A form 3.4 Å in the B form and ~ 2.9 Å in the A form; (2) the planes requires reduction of the water activity, e.g., by addition of of the base pairs are inclined with respect to the helix axis at ethanol. Because addition of ethanol not only favors the A angles of ~ 90° for the B and of 74° for the A form; (3) the form but also aggregation and precipitation of DNA, such reactions had to be avoided by reduction of the salt con- centration. These are the general boundary conditions for Special Issue: Chemical Kinetics, Biological Mechanisms and experimental studies of the B-A transition in solution—obvi- Molecular Evolution. ously valid for studies of both the equilibrium and kinetics. Boundary conditions and the high rate of the B-A transition * Dietmar Porschke restrict the experimental potential for analysis of the kinet- dpoersc@gwdg.de ics. The dynamics of the B-A transition could only be ana- Max Planck Institute for Biophysical Chemistry, lyzed by the electric field jump technique (Jose and Porschke 37077 Göttingen, Germany Vol.:(0123456789) 1 3 326 European Biophysics Journal (2018) 47:325–332 2004, 2005). Electric field pulses induce a reaction from the barrier for the transition lies in the organization of the the A toward the B form. Experimental evidence (Jose and solvent” (Knee et al. 2008). Porschke 2004) indicates that the A → B reaction is driven by “dipolar stretching;” a dipole increase is expected upon the A → B reaction resulting from an increase of the contour Materials and methods length. A contribution to the driving force may also come from a dissociation field effect (Onsager 1934). The B  → A Poly[d(A-T)] from Sigma was dialyzed extensively, first reaction recorded at zero field strength (after electric field against a high salt buffer with 0.2 M NaCl, 1 mM caco- pulses) always showed a spectrum of time constants with a dylate pH 7.0, 1 mM EDTA and finally against 250 μM major amplitude at ~ 10 μs followed by slower components NaCl, 250 μM cacodylate pH 7.0, 50 μM EDTA. Solutions with time constants not larger than ~ 1 ms. were prepared for measurements by mixing DNA, salt and The dynamics of the B-A transition has been studied buffer first; ethanol was added in the last step, except for more extensively by molecular dynamics simulations than minor water quantities for filling up to the desired volume. by experiments in solution. Initial simulations suggested that Quantities were controlled by pipetting and weighing. the transition proceeds within ~ 1 ns in both directions. Fur- Samples exposed to electric field pulses must be bubble ther simulations with accepted force fields indicated that the free to avoid cavitation. Thus, these samples had to be transition is observed only in the direction from the A to the degassed under vacuum after filling them into the field B form in aqueous solutions with time constants of ~ 1 ns, jump cells. Because some reduction of the ethanol content whereas the opposite reaction from the B to the A form, cannot be avoided during this procedure, the ethanol con- expected to occur at reduced water activity, has not been tent was determined after completion of the field jumps by observed under these conditions yet (Cheatham and Kollman measurement of the density in a densitometer DMA 602 1996; Cheatham et al. 1997; Sprous et al. 1998; Noy et al. (Anton Paar, Graz, Austria). UV spectra of the solutions 2007; Knee et al. 2008). Meanwhile the conclusion about a in the cell were recorded directly after filling the cell and low rate for the B-A transition at, for example, 85 volume% after completion of the measurements in a Perkin-Elmer ethanol (vol% EtOH) appears to be accepted in general. Lambda 17. Conductivities were also determined. The Apparently, there is a reaction barrier for the B-A transition buffers always contained 1 mM Na-cacodylate pH 7 and at reduced water activity. Cheatham et al. (1997) observed 0.2 mM EDTA; in addition, there was 1 mM NaCl in buffer a reaction from the B to A form in 85 vol% EtOH when A, 3  mM NaCl in buffer B and 10  mM NaCl in buffer the C3′-endo sugar pucker was stabilized, and Knee et al. C. All field jump experiments were conducted at 2 °C to (2008) observed the reaction from the B to the A form, when avoid field-induced denaturation of poly[d(A-T)]. “waters were restrained in the major groove of B DNA.” In The potential effect of hydrogen/deuterium replacement summary, the simulations indicate the existence of a reac- was tested in solutions prepared in buffer A in H O ini- tion barrier under the conditions of reduced water activity. tially. Then, H O was evaporated—finally residual H O 2 2 The present investigation was executed with the goal to was removed in vacuo over phosphorus pentoxide. Sub- obtain more detailed information about contributions to the sequently, the components were dissolved in the required activation barrier. The experimental data obtained in the volume of D O, and finally EtOD was added. D O was 2 2 preceding investigations were restricted to a limited range from Sigma Aldrich, EtOD from Deutero GmbH. of low salt concentrations, which may be a serious problem Processes induced by electric field pulses were meas- in particular for a reaction of a polyelectrolyte. Thus, an ured in a device originally constructed by Grünhagen extension of the data on the B-A dynamics to higher salt (1974). The main parts of the high-voltage pulse genera- concentrations should be useful. Poly[d(AT)] was selected tor are still as described by Grünhagen (1974), whereas for these tests because its B-A transition appears in a very other components such as the light source, measuring cell narrow range of solvent conditions and thus can be char- and detector were replaced. The light source used in the acterized at a higher accuracy than for natural DNAs with present experiments was a 200-W high-stability L2423 Hg/ mixed sequences. Another favorable property of poly[d(AT)] Xe arc-lamp from Hamamatsu together with a Schoeffel for this analysis is a relatively high solubility at reduced GM250 grating monochromator and a Glan air polarizer. water activity. A homemade photomultiplier detector was used together Because the B-A transition is induced by reduction of the with a Tektronix DSA 601A digitizer for data collection. water activity and hydration of the double helix is essen- The data were evaluated by a set of previously described tial for the transition, effects upon replacement of H O by programs (Diekmann et  al. 1982; Porschke and Jung D O may provide information about the nature of interac- 1985). tions. Such analysis is also suggested by the possibility “that Densities required for conversion between the differ- ent units used to define the composition of water-ethanol 1 3 European Biophysics Journal (2018) 47:325–332 327 mixtures were taken from Haynes and Lide (2015) and from “International alcoholometric tables” ( https :// www .oiml.or g/en/f iles /pdf_r/r022-e75.pdf). Densities for D O-EtOD mixtures were measured by the DMA 602 densitometer (Anton Paar, Graz, Austria). Conditions for magic angle detection and construction of improved cell windows Separation of field-induced reaction effects from orien- tation effects requires measurements at the magic angle, which is not trivial and demands special experience. The “magic” angle refers to an orientation of polarized light at 54.8° with respect to the vector of the applied electric field (Labhart 1961). Theory and experiments demon- strate that reaction effects are observed selectively at the magic angle (Porschke 1974, 1996). In practice, ampli- tudes should not exceed ~ 10% of the total light intensity, because magic angle conditions may be violated other- wise. Changes in the light intensity resulting from orien- tation are usually much larger than those from reactions Fig. 1 Construction of the cell used for exposure of samples to elec- tric field pulses. Form and dimensions of the cell body (macrolon) such as conformation changes; thus, magic angle condi- and the electrodes (platinum) are essentially as used previously. A tions must be rigorously adhered to. Polarized light at the major improvement is based on the construction of the cell windows: magic angle can be generated relatively easily, but the explosion view (left) and assembled view (right). The windows are state of polarization may be seriously affected by strain in not in direct contact with the cell body: the quartz cylinder is held at an outer segment of larger diameter between elastic O-rings (soft the cell windows. Such strain is induced by any mechani- silicon rubber), such that the inner segment is “free” in a bore hole cal stress at the optical windows. Quartz is convenient for adjacent to the sample space measurements in the UV, but minor stress may cause a large birefringence, resulting in changes of the polarized light. In the past, birefringence of quartz windows was path lengths of 7 mm (Fig.  1) and 20 mm (not shown) were used in the present investigation. avoided by a layer of silicon grease between the cell body and the window (Porschke 1996). The viscosity of the grease had to be low enough to avoid mechanical coupling with the cell, but high enough to avoid smearing out into Results the cell and on the surface of the windows. In practice, cell windows had to be cleaned and reinserted frequently. Salt dependence These problems are avoided by a novel form of window construction and insertion into cell bodies developed Poly[d(AT)] samples in water–ethanol mixtures at speci- fied buffer concentrations were subjected to DC electric recently for extension of birefringence measurements to a particularly high sensitivity (Porschke 2011). This mode field pulses, and the absorbance changes at the magic angle were recorded. As described previously (Jose and of construction also proves to be very useful for absorb- ance measurements in field jump cells. Because arc lamps Porschke 2004, 2005), the relative absorbance changes associated with the B-A transition are particularly high are still favorable for measurements in the UV and high light intensities are required for high time resolution, the at wavelengths ≥ 290 nm. For convenient magnitudes of absolute changes in this spectral range, DNA concentra- window dimensions had to be increased. The construction used for the present measurements is shown in Fig. 1. The tions in the range of ~ 20 to ~ 280 μM (monomer units) were used. Examples of field-induced transients in the essential conditions for optimal implementation are: (1) selection of quartz windows completely free of strain; (2) range of the B-A transition are shown in Fig.  2 for the buffers A, B and C (green, blue and red line, respectively) the quartz windows are held at their outer cylinder planes by f lexible O-rings made from soft silicon rubber and are at ethanol contents close to the centers of the B-A transi- tion. The shape of the transients and the time constants are not in direct contact with the cell body. Cells with optical similar in buffers A and B. The reduction of the amplitude 1 3 328 European Biophysics Journal (2018) 47:325–332 standard spectrophotometer demonstrate that the shifts are due to a field-induced temperature jump. This effect is minimal in buffer A because of its low conductivity, but is clearly detectable in buffer B because of its increased salt concentration. A further increase of the salt concentration in buffer C leads to a clear change of the transient with respect to those found in buffers A and B: (1) the amplitude is much higher, although electrostatic shielding is increased; (2) the response time constants are much larger. These changes in the tran- sient are accompanied by the appearance of turbidity. All these observations indicate the onset of aggregation. The limiting value of the ethanol content, where aggrega- tion is initiated, decreases with increasing salt concentration. At salt concentrations up to 4 mM, the B-A transition is observed before the onset of aggregation, whereas the B-A Fig. 2 Transients induced by application of electric field pulses to solutions of poly[d(AT)] at different salt concentrations: 2.44 mM at transition and aggregation appear at closely corresponding 67.5 vol% EtOH (buffer A, green line), 4.44 mM at 66.71 vol% EtOH ethanol contents in 10 mM salt. (buffer B, blue line) and 11.4 mM at 65.96 vol% EtOH (buffer C, red The aggregation is not only reflected in the response to line). The measured changes of light intensity are converted to extinc- electric field pulses, but is also indicated by an increase of tion changes, which are then normalized to a concentration of 80 μM phosphate units. Electric field pulses start at t = 20 μs; pulse lengths the absorbance due to light scattering. This effect is clearly are 83, 63 and 83 μs in buffers A, B and C, respectively; pulse ampli- visible, for example, at long wavelengths outside the usual tudes are 39 kV/cm absorbance band, already at 63.6 vol% EtOH in the buffer with 10 mM salt, whereas a minor increase was found at 63.0 vol% EtOH. At 4 mM salt, there is no increase of the in buffer B compared to buffer A reflects the increased absorbance in the long wavelength range in the B-A transi- electrostatic shielding resulting from the increase of the tion range resulting from turbidity; a minor increase was ion concentration in buffer B. A corresponding depend- observed above the B-A transition at 68.23 vol% EtOH and ence was observed at the lower salt concentrations used a DNA concentration of 260  μM (monomer units). This in the previous investigation (Jose and Porschke 2004). effect was not visible at a DNA concentration of 72.6 μM A detailed discussion of the processes observed upon (monomer units) and 68.04 vol% EtOH content. Both EtOH application of the electric field and after pulse termination content and salt concentration are particularly important has been presented previously (Jose and Porschke 2004). parameters for aggregation. In addition, the onset of aggre- Transients measured close to the center of the B-A tran- gation is also affected by the DNA concentration. sition represent large perturbations induced by the field The field-induced change of the absorbance is maximal pulses. Clear differences are observed in the reaction pro- at the center of the B-A transition and decays to very small gress curves upon application of electric field pulses and values outside the transition range. The dependence of the after pulse termination. Here, discussions are restricted amplitudes on the ethanol content is fitted to Gaussians, to reaction curves recorded after pulse termination (i.e., providing the center and width of the transition (Fig. 3). at electric field strength zero), representing a net reaction The amplitudes were measured at different poly[d(AT)] flow from B to A DNA. These transients show a spectrum concentrations and were normalized for fitting to a stand - of times with a relatively broad range of time constants. ard concentration of 80 μM. An extreme case is included Thus, individual time constants cannot be defined as usual, in the data set at 4.44 mM salt: one of the points (at 66.707 and only approximate numerical values can be given. This vol% EtOH) was measured at a poly[d(AT)] concentration is partly because the B-A transition is a cooperative reac- of 279.6 mM and another one (at 66.775 vol% EtOH) at tion. Fitting of the transients showed a spectrum with a 23.1 μM. Although these data points were taken at widely first process at ~ 10 μs followed by slower components different poly[d(AT)] concentrations, the ∆E values normal- with time constants not exceeding ~ 1 ms. ized to 80 μM are very close to each other (Fig. 3). This is The transient in buffer B indicates a shift in the ∆E further evidence against aggregation ee ff cts in the BA transi- level after the field jump relaxation with respect to that tion range of poly[d(AT)] at 4.44 mM salt. before application of the electric field (cf. Fig.  2). Analysis There are now data for the B-A transition obtained by of the field jump data and comparison with measurements the electric field jump method at four different salt con- of the absorbance as a function of the temperature in a centrations ranging from 0.183 to 4.44 mM [data from the 1 3 European Biophysics Journal (2018) 47:325–332 329 different decamer helices together with respective changes on the trifluoroethanol volume% scale (Minchenkova et al. 1986), (vol% TFE) provides a correspondence of ~ 235 cal per decamer for 1% change on the vol% TFE scale. Each decamer helix involves nine base stacks. Thus, a change of the salt by a factor of 10 in the case of poly[d(AT)] corresponds to ~ 70 cal per mole of base stack. The change of the polyelectrolyte free energy ∆G for the B-A transition of double helical DNA was calculated by Manning (2002) as a function of the salt concentration c : −2 −3 when c is decreased from 10 to 10 M, ∆G is increased by ~ 100 cal per mole base pair. Because of the approxi- mations involved in the comparison, an exact agreement should not be expected. It may be concluded that the orders Fig. 3 Transition amplitudes induced by electric field pulses of fixed of magnitude of the experimental result and of the theoreti- amplitude and duration in poly[d(AT)] as a function of the ethanol cal prediction are consistent. content at salt concentrations of 2.44 mM (data points and ∆E scale in red, buffer A) and 4.44  mM (data points and ∆E scale in blue, buffer B). The data are fitted to Gaussians providing the transition H O/D O exchange 2 2 centers 67.99 and 67.12 together with the widths of the transition 1.59 and 1.46 for the buffers A and B, respectively. ∆E values nor - The test for any dependence of the B-A transition param- malized to a concentration of 80 μM phosphate units eters upon an exchange of the solvent from H O to D O was 2 2 performed in buffer A, because the field-induced amplitudes are still favorable under these salt concentration conditions. Field-induced amplitudes were measured as a function of the EtOH and EtOD content, respectively. In both cases, distinct maxima of the amplitudes were observed at the center of the B-A transition. Fitting of the data by Gaussians showed maxima at 67.7 vol% EtOH in H O and at 69.3 vol% EtOD in D O. The estimated accuracy is ± 0.2 vol%. The cor- responding procedure applied to the same amplitude data but using a scale based on molar concentrations of H O and D O provided maxima at 20.0 and 20.1 mol/l, respectively, with an estimated accuracy ± 0.1 mol/l. Thus, the center of the B-A transition appears at a slightly higher D O than H O value on the mol/l scale, but the difference is within the limit of accuracy. Because of the sharp maximum of the amplitudes at the transition center, this center can be determined at a relatively Fig. 4 Centers of the B-A transition of poly[d(AT)] as a function of the logarithm of the salt concentration. The slope obtained by linear high accuracy. This advantage does not exist for the time regression is − 2.57 constants. Moreover, a special problem appears because of multiexponential transients. Separation of multiexponentials is notoriously difficult; thus, exact values of individual time present investigation and from Jose and Porschke (2004)]. constants cannot be specified. Under these conditions, the The ethanol content at the center of the transition is a conclusion is limited to the statement that the time constants linear function of the logarithm of the salt concentration are very similar in H O and D O. 2 2 (Fig. 4). It has been suggested that the ethanol content can be used as a scale of the free energy at least approximately. Based on the assignments presented by Ivanov et  al., Discussion changes in the vol% EtOH scale may be translated to free energy changes. The slope obtained by linear regression As demonstrated by high-resolution structures of protein of the data in Fig. 4 is − 2.57, corresponding to a change DNA complexes, the B-A transition of DNA is induced of vol% EtOH by 2.57 upon a change of the salt by a factor upon binding of regulatory proteins to DNA in many cases of 10. Using the free energy changes evaluated for three (Jacobo-Molina et al. 1993; Kiefer et al. 1998; Cheetham 1 3 330 European Biophysics Journal (2018) 47:325–332 and Steitz 1999; Jones et al. 1999; Lu et al. 2000). Thus, the rearrangements were characterized in aqueous salt solutions. B-A transition is directly involved in the mechanism of gene Thus, the time constants observed for the B-A transition by regulation. An unambiguous assignment of the steps contrib- relaxation measurements at reduced water activity are in a uting to this biologic function requires sufficient information time range that may be expected from the time constants of about the B-A transition. usual stacking rearrangements in a usual aqueous environ- ment. From this point of view, there is no problem regarding Comparison of experimental and simulated results the nature of an activation barrier during the B-A transition. There are many publications on molecular dynamics simula- The aggregation problem tions from different authors. Because of initial problems in the early phase of simulations, some conclusions about the One of the problems associated with the experimental analy- B-A dynamics were not apparent from the beginning. Fast sis of the B-A transition has always been the decrease of conversion of A-DNA to B-DNA within ~ 1 ns in aqueous DNA solubility at reduced water activity. Precipitation has solutions has been simulated in many cases, whereas the been avoided simply by reduction of the salt concentration. opposite reaction from B- to A-DNA, expected to occur at However, the criteria for the absence of aggregation have not reduced water activity, has not been observed in unbiased always been presented clearly enough. Minchenkova et al. MD simulations (Cheatham et al. 1997; Sprous et al. 1998; (1986) reported that precipitation can be avoided by using Noy et al. 2007). Apparently, there is a considerable activa- trifluoroethanol (TFE) instead of ethanol for reduction of tion barrier at reduced water activity. This result may be the water activity. A detailed analysis based on electro-opti- considered consistent with the experimental data obtained cal measurements of rotational diffusion for natural DNA at reduced water activity by the electric field jump technique restriction fragments with mixed AT/GC sequences has been (Jose and Porschke 2004, 2005) in the sense that both experi- presented recently (Porschke 2016), showing a range with- ments and simulations show the existence of activation bar- out aggregation or condensation upon addition of ethanol at riers at reduced water activity. However, there is no explicit monovalent salt ≤ 1 mM. This range for analysis of the B-A information on the time scale of the B-A reaction at reduced transition without aggregation or condensation is extended water activity from molecular dynamics. Conversely, the to monovalent salt ≤ 4.4 mM upon addition of TFE. field jump technique does not provide information on the In the present investigation, the special effect of TFE B-A kinetics in usual aqueous solutions. It is remarkable was not used. Instead it was tested, how far the set of data that molecular dynamics simulations indicate the existence available for poly[d(AT)] in H O-EtOH mixtures could be of a large activation barrier at reduced water activity and the extended. The results show that an extension is possible absence of this barrier in usual aqueous environments. This up to a salt concentration of 4.4 mM. Strong perturbations contrast raises questions about the nature of the activation resulting from aggregation were observed at 11.4 mM salt barrier. concentration. The aggregation tendency of DNA without The B-A transition is a special case of a stacking rear- GC base pairs is expected to be reduced. The chain length rangement, which is a frequently observed reaction in may be another parameter affecting aggregation. Finally, nucleic acids. A simple example is single-strand stacking, the kinetics of aggregation should be considered, which is which has been studied for various cases by cable tem- expected to be accelerated with increasing salt and ethanol perature jump measurements (Porschke 1976, 1977). Time content. Intermediate experimental conditions of salt and constants in the range of ~ 10 ns to ~ 1 μs were observed. ethanol content are expected to exist where aggregation can- Corresponding results were found by laser temperature not be avoided at long times but remains negligible at suf- jump measurements (Dewey and Turner 1979; Freier et al. ficiently short times. Under these conditions there are time 1981). The time constant of stacking in a model compound windows that can be used for analysis of the B-A transition. with two adenine residues connected by a simple and flex- ible aliphatic –(CH ) -bridge could not be resolved by the Magnitude of the electrostatic barrier 2 3 cable temperature jump technique (Porschke 1978); thus, its stacking time constant is faster than ~ 10 ns. This result An obvious candidate for a contribution to an activation indicates that the “activation barrier” in the case of single- barrier is electrostatic repulsion. The experimental analysis stranded stacking is imposed by the ribose- or deoxyribose- by measurements of the ionic strength dependence has now phosphate backbone. Stacking is slowed down even further been extended to a wider range from 0.183 to 4.44 mM. in more complex structures. In simple nucleic acid loops, for The change of B-A transition time constants in this range example, the rearrangement of stacking was found to occur is relatively small, indicating that the electrostatic contri- with time constants ≥ 10 μs (Bujalowski et al. 1986; Menger bution to the reaction barrier is not as large as may have et al. 2000). All these time constants for simple stacking been expected. The experimental time constants have always 1 3 European Biophysics Journal (2018) 47:325–332 331 been determined close to the center of the transition. For not induce changes of the equilibrium and kinetic parameters each of the analyzed salt concentrations there is a strong of the B-A transition within the limits of experimental accu- dependence of the time constants on the EtOH content. The racy. A comparison of the experimental results for the B-A magnitude of the time constants observed under the impact transition with those for other stacking rearrangements indi- of the electric field is always reduced with decreasing EtOH cates that the B-A activation barrier found at reduced water contents (Jose and Porschke 2004). However, extrapolation activity is on the expected order of magnitude, whereas the of these dependences to the absence of EtOH is not prac- absence of a barrier indicated by MD simulations for the ticable, because the interval available for measurements B-A transition in the absence of ethanol appears to be unu- is very limited; thus, extrapolation over the wide range of sual from this point of view. EtOH content to the absence of EtOH cannot be sufficiently Acknowledgements Open access funding provided by Max Planck accurate. Moreover, the effect of the electric field must be Society. The author is indebted to Thorsten Freiberg for fine mechani- extrapolated as well. Under these conditions, the available cal construction. The facilities of the Gesellschaft für wissenschaftliche experimental data can only be used with sufficient reliability Datenverarbeitung mbH, Göttingen, were used for data processing. for the conclusion that the time constants at the center of the transition are almost independent of the salt concentration. Compliance with ethical standards Because the overall activation barrier corresponds to a factor of ~ 10 , the present results indicate that the main part of this Conflict of interest The author declares that he does not have a conflict of interest. barrier is not due to electrostatics. Open Access This article is distributed under the terms of the Crea- Solvent isotope experiment tive Commons Attribution 4.0 International License (http://creat iveco mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate The H O/D O exchange was conducted as a simple test for 2 2 credit to the original author(s) and the source, provide a link to the an influence of the hydrogen bonding and hydration network Creative Commons license, and indicate if changes were made. on the thermodynamics and kinetics of the B-A transition. The experimental data demonstrate that the B-A transition is observed at a slightly higher value on the mol/l scale for D O than H O, but the difference is within the limit of accu- 2 2 References racy. Thus, the effect of the H O/D O exchange on the B-A 2 2 transition is relatively small. 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Kinetics of the B-A transition of DNA: analysis of potential contributions to a reaction barrier

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Life Sciences; Biochemistry, general; Biological and Medical Physics, Biophysics; Cell Biology; Neurobiology; Membrane Biology; Nanotechnology
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Abstract

Because of open problems in the relation between results obtained by relaxation experiments and molecular dynamics simulations on the B-A transition of DNA, relaxation measurements of the B-A dynamics have been extended to a wider range of conditions. Field-induced reaction effects are measured selectively by the magic angle technique using a novel cell construction preventing perturbations from cell window anisotropy. The kinetics was recorded for the case of poly[d(AT)] up to the salt concentration limit of 4.4 mM, where aggregation does not yet interfere. Now experimental data on the B-A dynamics are available for poly[d(AT)] at salt concentrations of 0.18, 0.73, 2.44 and 4.4 mM. In all cases, a spectrum of time constants is found, ranging from ~ 10 μs up to components approaching ~ 1 ms. The relatively small dependence of these data on the salt concentration indicates that electrostatic effects on the kinetics are not as strong as may be expected. The ethanol content at the transition center is a linear function of the logarithm of the salt concentration, and the slope is close to that expected from polyelectrolyte theory. The B-A transition dynamics was also measured in D O at a salt concentra- tion of 2.4 mM: the center of the transition is found at 20.0 mol/l H O and at 20.1 mol/l D O with an estimated accuracy of 2 2 ± 0.1 mol/l; the spectrum of time constants at the respective transition centers is very similar. The experimental results are discussed regarding the data obtained by molecular dynamics simulations. Keywords B-A transition · Kinetics · Magic angle measurements · Electrostatics · Solvent isotope effect Introduction sugar pucker is C2′-endo for the B and C3′-endo for the A form; (4) the hydration is more extensive in the B form and Looking at molecular models of the B- and A-form DNA more “economic” in the A form (Saenger et al. 1986). Dick- suggests that the transition between these well-ordered, erson and Ng ( 2001, 2002) concluded from their analysis of right-handed double-helical structures (Saenger 1984) is a the structures that there is a smooth transition between the B relatively simple rearrangement. The molecular details of B- and A forms without major activation barriers. and A-helix structures in crystals have been analyzed at high The equilibrium conditions for the existence of the B- resolution (Egli et al. 1998; Schneider et al. 1998; Dickerson and A-form DNA in solution have been characterized in and Ng 2001; Ng and Dickerson 2002). A short summary of detail (Ivanov et al. 1974). In general, the B form is observed the major differences: (1) the helix increase per base pair is in usual aqueous solutions, whereas formation of the A form 3.4 Å in the B form and ~ 2.9 Å in the A form; (2) the planes requires reduction of the water activity, e.g., by addition of of the base pairs are inclined with respect to the helix axis at ethanol. Because addition of ethanol not only favors the A angles of ~ 90° for the B and of 74° for the A form; (3) the form but also aggregation and precipitation of DNA, such reactions had to be avoided by reduction of the salt con- centration. These are the general boundary conditions for Special Issue: Chemical Kinetics, Biological Mechanisms and experimental studies of the B-A transition in solution—obvi- Molecular Evolution. ously valid for studies of both the equilibrium and kinetics. Boundary conditions and the high rate of the B-A transition * Dietmar Porschke restrict the experimental potential for analysis of the kinet- dpoersc@gwdg.de ics. The dynamics of the B-A transition could only be ana- Max Planck Institute for Biophysical Chemistry, lyzed by the electric field jump technique (Jose and Porschke 37077 Göttingen, Germany Vol.:(0123456789) 1 3 326 European Biophysics Journal (2018) 47:325–332 2004, 2005). Electric field pulses induce a reaction from the barrier for the transition lies in the organization of the the A toward the B form. Experimental evidence (Jose and solvent” (Knee et al. 2008). Porschke 2004) indicates that the A → B reaction is driven by “dipolar stretching;” a dipole increase is expected upon the A → B reaction resulting from an increase of the contour Materials and methods length. A contribution to the driving force may also come from a dissociation field effect (Onsager 1934). The B  → A Poly[d(A-T)] from Sigma was dialyzed extensively, first reaction recorded at zero field strength (after electric field against a high salt buffer with 0.2 M NaCl, 1 mM caco- pulses) always showed a spectrum of time constants with a dylate pH 7.0, 1 mM EDTA and finally against 250 μM major amplitude at ~ 10 μs followed by slower components NaCl, 250 μM cacodylate pH 7.0, 50 μM EDTA. Solutions with time constants not larger than ~ 1 ms. were prepared for measurements by mixing DNA, salt and The dynamics of the B-A transition has been studied buffer first; ethanol was added in the last step, except for more extensively by molecular dynamics simulations than minor water quantities for filling up to the desired volume. by experiments in solution. Initial simulations suggested that Quantities were controlled by pipetting and weighing. the transition proceeds within ~ 1 ns in both directions. Fur- Samples exposed to electric field pulses must be bubble ther simulations with accepted force fields indicated that the free to avoid cavitation. Thus, these samples had to be transition is observed only in the direction from the A to the degassed under vacuum after filling them into the field B form in aqueous solutions with time constants of ~ 1 ns, jump cells. Because some reduction of the ethanol content whereas the opposite reaction from the B to the A form, cannot be avoided during this procedure, the ethanol con- expected to occur at reduced water activity, has not been tent was determined after completion of the field jumps by observed under these conditions yet (Cheatham and Kollman measurement of the density in a densitometer DMA 602 1996; Cheatham et al. 1997; Sprous et al. 1998; Noy et al. (Anton Paar, Graz, Austria). UV spectra of the solutions 2007; Knee et al. 2008). Meanwhile the conclusion about a in the cell were recorded directly after filling the cell and low rate for the B-A transition at, for example, 85 volume% after completion of the measurements in a Perkin-Elmer ethanol (vol% EtOH) appears to be accepted in general. Lambda 17. Conductivities were also determined. The Apparently, there is a reaction barrier for the B-A transition buffers always contained 1 mM Na-cacodylate pH 7 and at reduced water activity. Cheatham et al. (1997) observed 0.2 mM EDTA; in addition, there was 1 mM NaCl in buffer a reaction from the B to A form in 85 vol% EtOH when A, 3  mM NaCl in buffer B and 10  mM NaCl in buffer the C3′-endo sugar pucker was stabilized, and Knee et al. C. All field jump experiments were conducted at 2 °C to (2008) observed the reaction from the B to the A form, when avoid field-induced denaturation of poly[d(A-T)]. “waters were restrained in the major groove of B DNA.” In The potential effect of hydrogen/deuterium replacement summary, the simulations indicate the existence of a reac- was tested in solutions prepared in buffer A in H O ini- tion barrier under the conditions of reduced water activity. tially. Then, H O was evaporated—finally residual H O 2 2 The present investigation was executed with the goal to was removed in vacuo over phosphorus pentoxide. Sub- obtain more detailed information about contributions to the sequently, the components were dissolved in the required activation barrier. The experimental data obtained in the volume of D O, and finally EtOD was added. D O was 2 2 preceding investigations were restricted to a limited range from Sigma Aldrich, EtOD from Deutero GmbH. of low salt concentrations, which may be a serious problem Processes induced by electric field pulses were meas- in particular for a reaction of a polyelectrolyte. Thus, an ured in a device originally constructed by Grünhagen extension of the data on the B-A dynamics to higher salt (1974). The main parts of the high-voltage pulse genera- concentrations should be useful. Poly[d(AT)] was selected tor are still as described by Grünhagen (1974), whereas for these tests because its B-A transition appears in a very other components such as the light source, measuring cell narrow range of solvent conditions and thus can be char- and detector were replaced. The light source used in the acterized at a higher accuracy than for natural DNAs with present experiments was a 200-W high-stability L2423 Hg/ mixed sequences. Another favorable property of poly[d(AT)] Xe arc-lamp from Hamamatsu together with a Schoeffel for this analysis is a relatively high solubility at reduced GM250 grating monochromator and a Glan air polarizer. water activity. A homemade photomultiplier detector was used together Because the B-A transition is induced by reduction of the with a Tektronix DSA 601A digitizer for data collection. water activity and hydration of the double helix is essen- The data were evaluated by a set of previously described tial for the transition, effects upon replacement of H O by programs (Diekmann et  al. 1982; Porschke and Jung D O may provide information about the nature of interac- 1985). tions. Such analysis is also suggested by the possibility “that Densities required for conversion between the differ- ent units used to define the composition of water-ethanol 1 3 European Biophysics Journal (2018) 47:325–332 327 mixtures were taken from Haynes and Lide (2015) and from “International alcoholometric tables” ( https :// www .oiml.or g/en/f iles /pdf_r/r022-e75.pdf). Densities for D O-EtOD mixtures were measured by the DMA 602 densitometer (Anton Paar, Graz, Austria). Conditions for magic angle detection and construction of improved cell windows Separation of field-induced reaction effects from orien- tation effects requires measurements at the magic angle, which is not trivial and demands special experience. The “magic” angle refers to an orientation of polarized light at 54.8° with respect to the vector of the applied electric field (Labhart 1961). Theory and experiments demon- strate that reaction effects are observed selectively at the magic angle (Porschke 1974, 1996). In practice, ampli- tudes should not exceed ~ 10% of the total light intensity, because magic angle conditions may be violated other- wise. Changes in the light intensity resulting from orien- tation are usually much larger than those from reactions Fig. 1 Construction of the cell used for exposure of samples to elec- tric field pulses. Form and dimensions of the cell body (macrolon) such as conformation changes; thus, magic angle condi- and the electrodes (platinum) are essentially as used previously. A tions must be rigorously adhered to. Polarized light at the major improvement is based on the construction of the cell windows: magic angle can be generated relatively easily, but the explosion view (left) and assembled view (right). The windows are state of polarization may be seriously affected by strain in not in direct contact with the cell body: the quartz cylinder is held at an outer segment of larger diameter between elastic O-rings (soft the cell windows. Such strain is induced by any mechani- silicon rubber), such that the inner segment is “free” in a bore hole cal stress at the optical windows. Quartz is convenient for adjacent to the sample space measurements in the UV, but minor stress may cause a large birefringence, resulting in changes of the polarized light. In the past, birefringence of quartz windows was path lengths of 7 mm (Fig.  1) and 20 mm (not shown) were used in the present investigation. avoided by a layer of silicon grease between the cell body and the window (Porschke 1996). The viscosity of the grease had to be low enough to avoid mechanical coupling with the cell, but high enough to avoid smearing out into Results the cell and on the surface of the windows. In practice, cell windows had to be cleaned and reinserted frequently. Salt dependence These problems are avoided by a novel form of window construction and insertion into cell bodies developed Poly[d(AT)] samples in water–ethanol mixtures at speci- fied buffer concentrations were subjected to DC electric recently for extension of birefringence measurements to a particularly high sensitivity (Porschke 2011). This mode field pulses, and the absorbance changes at the magic angle were recorded. As described previously (Jose and of construction also proves to be very useful for absorb- ance measurements in field jump cells. Because arc lamps Porschke 2004, 2005), the relative absorbance changes associated with the B-A transition are particularly high are still favorable for measurements in the UV and high light intensities are required for high time resolution, the at wavelengths ≥ 290 nm. For convenient magnitudes of absolute changes in this spectral range, DNA concentra- window dimensions had to be increased. The construction used for the present measurements is shown in Fig. 1. The tions in the range of ~ 20 to ~ 280 μM (monomer units) were used. Examples of field-induced transients in the essential conditions for optimal implementation are: (1) selection of quartz windows completely free of strain; (2) range of the B-A transition are shown in Fig.  2 for the buffers A, B and C (green, blue and red line, respectively) the quartz windows are held at their outer cylinder planes by f lexible O-rings made from soft silicon rubber and are at ethanol contents close to the centers of the B-A transi- tion. The shape of the transients and the time constants are not in direct contact with the cell body. Cells with optical similar in buffers A and B. The reduction of the amplitude 1 3 328 European Biophysics Journal (2018) 47:325–332 standard spectrophotometer demonstrate that the shifts are due to a field-induced temperature jump. This effect is minimal in buffer A because of its low conductivity, but is clearly detectable in buffer B because of its increased salt concentration. A further increase of the salt concentration in buffer C leads to a clear change of the transient with respect to those found in buffers A and B: (1) the amplitude is much higher, although electrostatic shielding is increased; (2) the response time constants are much larger. These changes in the tran- sient are accompanied by the appearance of turbidity. All these observations indicate the onset of aggregation. The limiting value of the ethanol content, where aggrega- tion is initiated, decreases with increasing salt concentration. At salt concentrations up to 4 mM, the B-A transition is observed before the onset of aggregation, whereas the B-A Fig. 2 Transients induced by application of electric field pulses to solutions of poly[d(AT)] at different salt concentrations: 2.44 mM at transition and aggregation appear at closely corresponding 67.5 vol% EtOH (buffer A, green line), 4.44 mM at 66.71 vol% EtOH ethanol contents in 10 mM salt. (buffer B, blue line) and 11.4 mM at 65.96 vol% EtOH (buffer C, red The aggregation is not only reflected in the response to line). The measured changes of light intensity are converted to extinc- electric field pulses, but is also indicated by an increase of tion changes, which are then normalized to a concentration of 80 μM phosphate units. Electric field pulses start at t = 20 μs; pulse lengths the absorbance due to light scattering. This effect is clearly are 83, 63 and 83 μs in buffers A, B and C, respectively; pulse ampli- visible, for example, at long wavelengths outside the usual tudes are 39 kV/cm absorbance band, already at 63.6 vol% EtOH in the buffer with 10 mM salt, whereas a minor increase was found at 63.0 vol% EtOH. At 4 mM salt, there is no increase of the in buffer B compared to buffer A reflects the increased absorbance in the long wavelength range in the B-A transi- electrostatic shielding resulting from the increase of the tion range resulting from turbidity; a minor increase was ion concentration in buffer B. A corresponding depend- observed above the B-A transition at 68.23 vol% EtOH and ence was observed at the lower salt concentrations used a DNA concentration of 260  μM (monomer units). This in the previous investigation (Jose and Porschke 2004). effect was not visible at a DNA concentration of 72.6 μM A detailed discussion of the processes observed upon (monomer units) and 68.04 vol% EtOH content. Both EtOH application of the electric field and after pulse termination content and salt concentration are particularly important has been presented previously (Jose and Porschke 2004). parameters for aggregation. In addition, the onset of aggre- Transients measured close to the center of the B-A tran- gation is also affected by the DNA concentration. sition represent large perturbations induced by the field The field-induced change of the absorbance is maximal pulses. Clear differences are observed in the reaction pro- at the center of the B-A transition and decays to very small gress curves upon application of electric field pulses and values outside the transition range. The dependence of the after pulse termination. Here, discussions are restricted amplitudes on the ethanol content is fitted to Gaussians, to reaction curves recorded after pulse termination (i.e., providing the center and width of the transition (Fig. 3). at electric field strength zero), representing a net reaction The amplitudes were measured at different poly[d(AT)] flow from B to A DNA. These transients show a spectrum concentrations and were normalized for fitting to a stand - of times with a relatively broad range of time constants. ard concentration of 80 μM. An extreme case is included Thus, individual time constants cannot be defined as usual, in the data set at 4.44 mM salt: one of the points (at 66.707 and only approximate numerical values can be given. This vol% EtOH) was measured at a poly[d(AT)] concentration is partly because the B-A transition is a cooperative reac- of 279.6 mM and another one (at 66.775 vol% EtOH) at tion. Fitting of the transients showed a spectrum with a 23.1 μM. Although these data points were taken at widely first process at ~ 10 μs followed by slower components different poly[d(AT)] concentrations, the ∆E values normal- with time constants not exceeding ~ 1 ms. ized to 80 μM are very close to each other (Fig. 3). This is The transient in buffer B indicates a shift in the ∆E further evidence against aggregation ee ff cts in the BA transi- level after the field jump relaxation with respect to that tion range of poly[d(AT)] at 4.44 mM salt. before application of the electric field (cf. Fig.  2). Analysis There are now data for the B-A transition obtained by of the field jump data and comparison with measurements the electric field jump method at four different salt con- of the absorbance as a function of the temperature in a centrations ranging from 0.183 to 4.44 mM [data from the 1 3 European Biophysics Journal (2018) 47:325–332 329 different decamer helices together with respective changes on the trifluoroethanol volume% scale (Minchenkova et al. 1986), (vol% TFE) provides a correspondence of ~ 235 cal per decamer for 1% change on the vol% TFE scale. Each decamer helix involves nine base stacks. Thus, a change of the salt by a factor of 10 in the case of poly[d(AT)] corresponds to ~ 70 cal per mole of base stack. The change of the polyelectrolyte free energy ∆G for the B-A transition of double helical DNA was calculated by Manning (2002) as a function of the salt concentration c : −2 −3 when c is decreased from 10 to 10 M, ∆G is increased by ~ 100 cal per mole base pair. Because of the approxi- mations involved in the comparison, an exact agreement should not be expected. It may be concluded that the orders Fig. 3 Transition amplitudes induced by electric field pulses of fixed of magnitude of the experimental result and of the theoreti- amplitude and duration in poly[d(AT)] as a function of the ethanol cal prediction are consistent. content at salt concentrations of 2.44 mM (data points and ∆E scale in red, buffer A) and 4.44  mM (data points and ∆E scale in blue, buffer B). The data are fitted to Gaussians providing the transition H O/D O exchange 2 2 centers 67.99 and 67.12 together with the widths of the transition 1.59 and 1.46 for the buffers A and B, respectively. ∆E values nor - The test for any dependence of the B-A transition param- malized to a concentration of 80 μM phosphate units eters upon an exchange of the solvent from H O to D O was 2 2 performed in buffer A, because the field-induced amplitudes are still favorable under these salt concentration conditions. Field-induced amplitudes were measured as a function of the EtOH and EtOD content, respectively. In both cases, distinct maxima of the amplitudes were observed at the center of the B-A transition. Fitting of the data by Gaussians showed maxima at 67.7 vol% EtOH in H O and at 69.3 vol% EtOD in D O. The estimated accuracy is ± 0.2 vol%. The cor- responding procedure applied to the same amplitude data but using a scale based on molar concentrations of H O and D O provided maxima at 20.0 and 20.1 mol/l, respectively, with an estimated accuracy ± 0.1 mol/l. Thus, the center of the B-A transition appears at a slightly higher D O than H O value on the mol/l scale, but the difference is within the limit of accuracy. Because of the sharp maximum of the amplitudes at the transition center, this center can be determined at a relatively Fig. 4 Centers of the B-A transition of poly[d(AT)] as a function of the logarithm of the salt concentration. The slope obtained by linear high accuracy. This advantage does not exist for the time regression is − 2.57 constants. Moreover, a special problem appears because of multiexponential transients. Separation of multiexponentials is notoriously difficult; thus, exact values of individual time present investigation and from Jose and Porschke (2004)]. constants cannot be specified. Under these conditions, the The ethanol content at the center of the transition is a conclusion is limited to the statement that the time constants linear function of the logarithm of the salt concentration are very similar in H O and D O. 2 2 (Fig. 4). It has been suggested that the ethanol content can be used as a scale of the free energy at least approximately. Based on the assignments presented by Ivanov et  al., Discussion changes in the vol% EtOH scale may be translated to free energy changes. The slope obtained by linear regression As demonstrated by high-resolution structures of protein of the data in Fig. 4 is − 2.57, corresponding to a change DNA complexes, the B-A transition of DNA is induced of vol% EtOH by 2.57 upon a change of the salt by a factor upon binding of regulatory proteins to DNA in many cases of 10. Using the free energy changes evaluated for three (Jacobo-Molina et al. 1993; Kiefer et al. 1998; Cheetham 1 3 330 European Biophysics Journal (2018) 47:325–332 and Steitz 1999; Jones et al. 1999; Lu et al. 2000). Thus, the rearrangements were characterized in aqueous salt solutions. B-A transition is directly involved in the mechanism of gene Thus, the time constants observed for the B-A transition by regulation. An unambiguous assignment of the steps contrib- relaxation measurements at reduced water activity are in a uting to this biologic function requires sufficient information time range that may be expected from the time constants of about the B-A transition. usual stacking rearrangements in a usual aqueous environ- ment. From this point of view, there is no problem regarding Comparison of experimental and simulated results the nature of an activation barrier during the B-A transition. There are many publications on molecular dynamics simula- The aggregation problem tions from different authors. Because of initial problems in the early phase of simulations, some conclusions about the One of the problems associated with the experimental analy- B-A dynamics were not apparent from the beginning. Fast sis of the B-A transition has always been the decrease of conversion of A-DNA to B-DNA within ~ 1 ns in aqueous DNA solubility at reduced water activity. Precipitation has solutions has been simulated in many cases, whereas the been avoided simply by reduction of the salt concentration. opposite reaction from B- to A-DNA, expected to occur at However, the criteria for the absence of aggregation have not reduced water activity, has not been observed in unbiased always been presented clearly enough. Minchenkova et al. MD simulations (Cheatham et al. 1997; Sprous et al. 1998; (1986) reported that precipitation can be avoided by using Noy et al. 2007). Apparently, there is a considerable activa- trifluoroethanol (TFE) instead of ethanol for reduction of tion barrier at reduced water activity. This result may be the water activity. A detailed analysis based on electro-opti- considered consistent with the experimental data obtained cal measurements of rotational diffusion for natural DNA at reduced water activity by the electric field jump technique restriction fragments with mixed AT/GC sequences has been (Jose and Porschke 2004, 2005) in the sense that both experi- presented recently (Porschke 2016), showing a range with- ments and simulations show the existence of activation bar- out aggregation or condensation upon addition of ethanol at riers at reduced water activity. However, there is no explicit monovalent salt ≤ 1 mM. This range for analysis of the B-A information on the time scale of the B-A reaction at reduced transition without aggregation or condensation is extended water activity from molecular dynamics. Conversely, the to monovalent salt ≤ 4.4 mM upon addition of TFE. field jump technique does not provide information on the In the present investigation, the special effect of TFE B-A kinetics in usual aqueous solutions. It is remarkable was not used. Instead it was tested, how far the set of data that molecular dynamics simulations indicate the existence available for poly[d(AT)] in H O-EtOH mixtures could be of a large activation barrier at reduced water activity and the extended. The results show that an extension is possible absence of this barrier in usual aqueous environments. This up to a salt concentration of 4.4 mM. Strong perturbations contrast raises questions about the nature of the activation resulting from aggregation were observed at 11.4 mM salt barrier. concentration. The aggregation tendency of DNA without The B-A transition is a special case of a stacking rear- GC base pairs is expected to be reduced. The chain length rangement, which is a frequently observed reaction in may be another parameter affecting aggregation. Finally, nucleic acids. A simple example is single-strand stacking, the kinetics of aggregation should be considered, which is which has been studied for various cases by cable tem- expected to be accelerated with increasing salt and ethanol perature jump measurements (Porschke 1976, 1977). Time content. Intermediate experimental conditions of salt and constants in the range of ~ 10 ns to ~ 1 μs were observed. ethanol content are expected to exist where aggregation can- Corresponding results were found by laser temperature not be avoided at long times but remains negligible at suf- jump measurements (Dewey and Turner 1979; Freier et al. ficiently short times. Under these conditions there are time 1981). The time constant of stacking in a model compound windows that can be used for analysis of the B-A transition. with two adenine residues connected by a simple and flex- ible aliphatic –(CH ) -bridge could not be resolved by the Magnitude of the electrostatic barrier 2 3 cable temperature jump technique (Porschke 1978); thus, its stacking time constant is faster than ~ 10 ns. This result An obvious candidate for a contribution to an activation indicates that the “activation barrier” in the case of single- barrier is electrostatic repulsion. The experimental analysis stranded stacking is imposed by the ribose- or deoxyribose- by measurements of the ionic strength dependence has now phosphate backbone. Stacking is slowed down even further been extended to a wider range from 0.183 to 4.44 mM. in more complex structures. In simple nucleic acid loops, for The change of B-A transition time constants in this range example, the rearrangement of stacking was found to occur is relatively small, indicating that the electrostatic contri- with time constants ≥ 10 μs (Bujalowski et al. 1986; Menger bution to the reaction barrier is not as large as may have et al. 2000). All these time constants for simple stacking been expected. The experimental time constants have always 1 3 European Biophysics Journal (2018) 47:325–332 331 been determined close to the center of the transition. For not induce changes of the equilibrium and kinetic parameters each of the analyzed salt concentrations there is a strong of the B-A transition within the limits of experimental accu- dependence of the time constants on the EtOH content. The racy. A comparison of the experimental results for the B-A magnitude of the time constants observed under the impact transition with those for other stacking rearrangements indi- of the electric field is always reduced with decreasing EtOH cates that the B-A activation barrier found at reduced water contents (Jose and Porschke 2004). However, extrapolation activity is on the expected order of magnitude, whereas the of these dependences to the absence of EtOH is not prac- absence of a barrier indicated by MD simulations for the ticable, because the interval available for measurements B-A transition in the absence of ethanol appears to be unu- is very limited; thus, extrapolation over the wide range of sual from this point of view. EtOH content to the absence of EtOH cannot be sufficiently Acknowledgements Open access funding provided by Max Planck accurate. Moreover, the effect of the electric field must be Society. The author is indebted to Thorsten Freiberg for fine mechani- extrapolated as well. Under these conditions, the available cal construction. The facilities of the Gesellschaft für wissenschaftliche experimental data can only be used with sufficient reliability Datenverarbeitung mbH, Göttingen, were used for data processing. for the conclusion that the time constants at the center of the transition are almost independent of the salt concentration. Compliance with ethical standards Because the overall activation barrier corresponds to a factor of ~ 10 , the present results indicate that the main part of this Conflict of interest The author declares that he does not have a conflict of interest. barrier is not due to electrostatics. Open Access This article is distributed under the terms of the Crea- Solvent isotope experiment tive Commons Attribution 4.0 International License (http://creat iveco mmons.or g/licenses/b y/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided you give appropriate The H O/D O exchange was conducted as a simple test for 2 2 credit to the original author(s) and the source, provide a link to the an influence of the hydrogen bonding and hydration network Creative Commons license, and indicate if changes were made. on the thermodynamics and kinetics of the B-A transition. The experimental data demonstrate that the B-A transition is observed at a slightly higher value on the mol/l scale for D O than H O, but the difference is within the limit of accu- 2 2 References racy. Thus, the effect of the H O/D O exchange on the B-A 2 2 transition is relatively small. 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European Biophysics JournalSpringer Journals

Published: Feb 5, 2018

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