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J. Phys. Chem. 1996, 100, 458-461
Probing DNA’s Dynamics and Conformational Substates by Enthalpy Relaxation and Its Recovery Simon Ru1 disser, Andreas Hallbrucker, and Erwin Mayer* Institut fu¨ r Allgemeine, Anorganische und Theoretische Chemie, UniVersita¨ t Innsbruck, A-6020 Innsbruck, Austria ReceiVed: August 30, 1995; In Final Form: NoVember 15, 1995X
The A and B form of NaDNA with hydration level of between 0.15 and 0.64 (g of water)/(g of NaDNA) have been vitrified by cooling at rates of ≈80 K min-1, and their thermal behavior on reheating at 30 K min-1 was studied from ≈120 to 300 K by differential scanning calorimetry. The calorimetric effects of annealing time and temperature are characteristic of a glass, and they are attributed to enthalpy relaxation of conformational substates of B-DNA. Structural relaxation becomes observable in form of endothermic enthalpy recovery for Γ > 3-4 (water molecules per nucleotide), and the heat effects increase with hydration level linearly up to Γ ≈ 12. Molecular motions caused by the presence of water hydrogen bonded to DNA’s functional groups become unfrozen from ≈153 to ≈263 K which is attributed to a very broad distribution of relaxation times. The data suggest that structural relaxation effects as reported here can be linked to the conformational flexibility of B-DNA necessary for protein-DNA interaction.
Introduction DNA’s dynamics and conformational substates have been studied by, e.g., molecular dynamics calculations, X-ray diffraction, and NMR spectroscopy.1-7 A further technique, namely, enthalpy relaxation of a glass and its recovery on heating,8-12 is widely used for synthetic polymers but has only recently been applied to proteins.13,14 Physical aging, or annealing, of a glass below its calorimetric glass f liquid transition leads to relaxation of the structure toward a state of lower enthalpy and entropy, at a rate that decreases with decreasing temperature. This enthalpy relaxation becomes observable on reheating by differential scanning calorimetry (DSC) when the enthalpy recovery shows up as an endothermic peak.8-12 We report here a DSC study of enthalpy relaxation and its recovery of vitrified hydrated native sodium salt of DNA (NaDNA) and of the effects of hydration level, annealing temperature, and time. Experimental Section As-received NaDNA (obtained from Fluka, salmon testes, No. 31163, containing 0.08 wt % protein and 12.9 wt % water) was hydrated by keeping over saturated salt solutions for several weeks, and the water content determined by weighing to (0.01 mg, taking into account the water content of as-received NaDNA. The hydrated samples were quickly transferred into steel capsules. The accuracy of the given hydration values is estimated to be (0.02 (g of water)/(g of dry NaDNA). The samples were cooled in the DSC instrument at a rate of ≈80 K min-1. The DSC scans were recorded on heating from 103 to 298 K at a rate of 30 K min-1. For investigating the effect of annealing, the samples were heated from 103 K to the given annealing temperature, kept at this temperature for a given annealing time and cooled thereafter to 103 K at 30 K min-1 for final heating and recording the DSC scan. A differential scanning calorimeter (Model DSC-4, PerkinElmer Corp.) with a self-written computer program was used. A baseline obtained with empty sample pans was subtracted from all scans to eliminate curvature of the traces. In addition, X
Abstract published in AdVance ACS Abstracts, December 15, 1995.
0022-3654/96/20100-0458$12.00/0
a straight line was subtracted from each DSC scan, with set points placed at ≈110 and ≈130 K. This is equivalent to slope control in the original software. Because of this change of slope at low temperature, the plateau region seen in curves 4-6 of Figure 1 where the slope is almost zero between ≈240 and 290 K, might not be real but the decrease in slope certainly is. In curves 6 of Figure 1, an endothermic peak centered at 259 K is assigned to melting of ice. The weights of the samples were between ≈10 and 30 mg. The temperature scales in the figures are not corrected for thermal lag of the instrument which is 1.6° for heating at a rate of 30 K min-1. The DSC scans of each figure are normalized with respect to the samples’ weights but shifted vertically for clarity. The ordinate bar is for 1 mg of hydrated NaDNA. For further experimental details on DSC see ref 13. NaDNA was characterized further by FTIR spectra of films with two different hydration levels, one containing pure B-DNA, the other a mixture of A-DNA and B-DNA, and the comparison with those shown in Figure 2 of ref 16. Results and Discussion Figure 1 shows for the hydrated native sodium salt of DNA (NaDNA, from salmon testes) the effect of hydration level and of annealing on the DSC scans recorded on heating from 150 to 290 K at a rate of 30 K min-1 (recording of the scans was started at 103 K). The effect of hydration level is shown in Figure 1a where curves 1-6 are the DSC scans of unannealed hydrated NaDNA samples, the hydration level increasing from 0.19 (1), 0.26 (2), 0.29 (3), 0.45 (4), 0.52 (5), to 0.64 (6) (g of water)/(g of NaDNA). The hydration level of curves 1 corresponds to that of nearly pure A-DNA, those of curves 2-6 to mixtures of A- and B-DNA, the percentage of B-DNA increasing with increasing hydration.3,15-18 The effect of annealing is shown in Figure 1b where the samples used for Figure 1a were in addition annealed for 20 min at 183 K (marked by the broken line) before recording the DSC scans. The labeling of the curves is the same as that used for Figure 1a. The samples were cooled for all curves at a rate of ≈80 K min-1 in the DSC instrument. In Figure1b, curves 2-6 show that an endothermic feature develops upon annealing at a temperature © 1996 American Chemical Society
Letters
Figure 1. DSC scans of increasingly hydrated NaDNA, from curve 1 to 6, obtained during heating at 30 K min-1. Water content in (g of water)/(g of NaDNA) is 0.19 (1), 0.26 (2), 0.29 (3), 0.45 (4), 0.52 (5), and 0.64 (6). (a) DSC scans of unannealed samples, (b) samples were annealed for 20 min at 183 K (broken line) before recording the DSC scans (from ref 35 with changes).
slightly higher than that used for annealing, whose size apparently increases with the hydration level. The thermal features observable in Figure 1, e.g., the effect of hydration level and of annealing, and in the following figures are reversible. This was ascertained by repeating in some selected cases the procedure with the same sample. For curves 1-5 the hydration level is within the region of unfreezable water,19 and consequently no distinct melting endotherm of ice is observable. For curves 6, the endotherm centered at 259 K is assigned to melting of ice. For hydration level of 0.64 (g of water)/(g of NaDNA) used for curves 6, a small amount of freezable water is to be expected in addition to the unfreezable water.19 This small amount of freezable water is also expected to vitrify on cooling at ≈80 K min-1 in comparison with freezable water on a protein.20 Curves 2-6 in Figure 1 further show that a change of slope of the heat capacity (Cp) at ≈175-220 K occurs for both unannealed and annealed samples which increases with increasing hydration level and simultaneously shifts to lower temperature. In curves 1 a comparable change of slope is absent in this temperature region. We note that increasing slope of DNA’s Cp with increasing water content at similar temperatures has been reported by Mrevlishvili.21 For the water-rich samples shown in curves 4-6 decrease in slope occurs on heating above ≈240 K. The effect of annealing is seen more clearly in form of difference curves where for a given hydration level the DSC scan of the unannealed sample is subtracted from that of the same but annealed sample. The corresponding difference curves are shown in Figure 2a. Figure 2a shows more clearly than Figure 1 that the area of the endothermic feature with a peak maximum ≈20 K above that of the annealing temperature Ta of 183 K increases with increasing hydration level in curves 2-6 with an accompanied shift of its peak to lower temperature. This endothermic feature with an extrapolated peak temperature of ≈206 K is absent in difference curve 1. (In curve 1 the spike centered at 198 K is considered as noise. A further weak endothermic feature with the peak close to Ta is probably caused by the instrument.) In Figure 2b the recovered enthalpy is plotted versus the hydration level of NaDNA. The solid line is obtained by linear regression of the data points from 0.26 to 0.64 (g of water)/(g of NaDNA). Its extrapolation gives ≈0.10 (g of water)/(g of NaDNA) necessary for the onset of heat effect.
J. Phys. Chem., Vol. 100, No. 2, 1996 459
Figure 2. (a) DSC difference curves, obtained by subtracting the scan of an unannealed sample (from Figure 1a) from that of the same sample but annealed in addition for 20 min at 183 K (from Figure 1b). The labeling is the same as that used in Figure 1. The broken line is at 183 K. (b) Recovered enthalpy plotted in J/(g of sample) versus water content (from ref 35 with changes).
Figure 3. (a) DSC difference curves, indicating the influence of annealing time for annealing at 183 K. The same sample containing 0.48 (g of water)/(g of NaDNA) was used throughout. The difference curves were obtained by subtracting the scan of an unannealed sample from that of the same sample but annealed in addition for a given time. (b) Plots of recovered enthalpy in J/(g of sample) and of peak maximum versus annealing time, and versus ln(annealing time) (c).
Figure 3a shows the influence of annealing time (ta) in form of DSC difference curves (i.e., annealed - unannealed) for the same sample containing 0.48 (g of water)/(g of NaDNA). Ta was 183 K (marked by broken line), and ta was increased from 1 min in curve 1 to 58 min in curve 8. In Figure 3b the enthalpy recovered (in J/(g of sample)) and the peak temperature are plotted versus ta. Both plots show similar dependence on ta in that there is a rapid increase for short ta and a leveling off for long ta. Figure 3c contains plots of peak temperature and recovered enthalpy versus ln(ta) which shows the nearly linear relationship often reported for enthalpy relaxation of a glass and its recovery.8-12 In Figure 4 we show the influence of Ta (marked by the arrow) in form of DSC difference curves (i.e., annealed - unannealed) for the same sample containing 0.46 (g of water)/(g of NaDNA) and the same ta of 20 min. Ta was increased in 10 K steps from 153 K (curve 1) to 223 K (curve 8), and it was 263 and 278 K for curves 9 and 10. Endothermic heat effects increase in going from 153 K (curve 1) to 193 K (curve 5) but decrease in curves 6-9 on further increase in Ta. Endothermic heat effects are absent on annealing at 143 K (not shown) or at 278 K (curve 10). In curves 7-9, the endotherms are preceded by
460 J. Phys. Chem., Vol. 100, No. 2, 1996
Figure 4. DSC difference curves for demonstrating the influence of annealing temperature for constant annealing time of 20 min. The same sample containing 0.46 (g of water)/(g of NaDNA) was used throughout. The difference curves were obtained by subtracting the scan of an unannealed sample from that of the same sample but annealed in addition at the given temperature. The annealing temperatures are 153 (1), 163 (2), 173 (3), 183 (4), 193 (5), 203 (6), 213 (7), 223 (8), 243 (9), 263 (10), and 278 K (11), and these temperatures are marked by the arrow.
an exothermic feature whose peak temperature is centered near Ta. We attribute this feature to some exothermic process occurring during annealing and causing an apparent decrease of Cp. Formation of ice is unlikely because Falk et al.19 have shown for this hydration that the water does not crystallize. The area under the endothermic feature recovered on heating is a quantitative measure of the enthalpy released on annealing.9 This recovery allows us to quantitate the influence of the hydration level of Ta and of ta on the dynamics of DNA and then to relate the results to DNA’s hydration sites in terms of water molecules per nucleotide, Γ, as the increase in hydration level causes a conformer transition from A to B state. (i) Effect of Ta. Enthalpy recovery and the development of endothermic features on annealing at a given temperature is, for a given hydration level and ta, an indicator for the temperature region where atomic and segmental motions occur.8-13 In Figure 1, curves 2-6 have the appearance of a glass f liquid transition with an increase in Cp at ≈175-220 K and a decrease in slope above ≈240 K. The endothermic features shown in Figure 4 are also characteristic for enthalpy recovery in and below a glass f liquid transition region as observed in poly(vinyl chloride).12 However, the temperature region where endothermic effects are observable is much larger here than those observed for molecular and polymeric glasses. When the comparison is made on a reduced temperature scale, Tg/T, the difference will appear even more pronounced.22 The large width indicates a very broad distribution of relaxation times and is attributed to the presence of a large number of local structures of hydrated NaDNA in which atomic and molecular segments diffuse at very different time scales at a fixed temperature. Therefore instead of assigning these calorimetric features to a single glass transition, we attribute them to a superposition of glass transitions of local structures, each with its own relaxation time and its temperature dependence, as for hydrated proteins13 and a synthetic polymer.23 But there is a
Letters difference between the DNA and proteins’ behavior in that the temperature region where enthalpy recovery occurs is smaller for hydrated DNA than for hydrated proteins.13 This indicates a narrower distribution of relaxation times for hydrated DNA than for hydrated proteins and is a reflection of the number of substates. For 30 K min-1 heating rate the relaxation time at the onset temperature of glass transition (Tg) in a DSC scan is ≈70 s.24 Annealing at any given Ta would lead to decrease of enthalpy of those structures whose Tg’s are below Ta. In addition, since ta is longer than the usual relaxation time at Tg, some structures with Tg slightly above Ta also relax and contribute to the experimentally observed endothermic feature. The weak endothermic feature observable upon annealing at 153 K (Figure 4, curve 1) has to be due to enthalpy relaxation of the local structures with the shortest relaxation time which would be for the rearrangement of -O-H groups H-bonded to the macromolecules’ structure. The intense endotherms developing upon annealing between 163 and 203 K (Figure 4, curves 2-6) are attributed mainly to kinetic unfreezing of the hydrated phosphate ester moiety because experimental and theoretical studies have shown that “the phosphate ester conformation is the most variable part of the nucleic acid structure”.2 This is also consistent with single-crystal studies of a B-DNA dodecamer where at low temperature mean crystallographic temperature factors4 and mean vibrational amplitudes were much higher for the phosphate sugar moiety than for the bases (see Table 24.3 in ref 3). The absence of enthalpy recovery on annealing at 278 K (Figure 4, curve 10) indicates that at this temperature the sample is in the “fluid” state where interconversion between conformational substates is rapid.2,6 Mrevlishvili21 assumed that the anomalous increase in Cp of native hydrated DNA observed in his studies at ≈200-220 K “involves the vitrification of a certain fraction of the water” whereas Gol’danskii et al.25 preferred assignment of this feature to “a vitrification point in the polymers”. Indications of a glass transition in DNA by thermally stimulated depolarization current and dc conductivity methods were reported by Laudat and Laudat.26 (ii) Effect of ta. Figure 3 shows that recovered enthalpy and peak temperature of the endotherm both increase with increasing ta and that they are an approximately linear function of ln(ta). This is the behavior generally observed for short-time annealing of polymeric8-10,12 and molecular11 glasses below Tg: the longer a sample is annealed at Ta < Tg, the more it relaxes toward its equilibrium configuration at Ta. This state is lower in energy and more stable than the equilibrium configuration at Tg. Therefore, the more nearly the sample has annealed to equilibrium at Ta, the more is the area of its enthalpy recovery when the sample is brought back to Tg, and the higher the temperature at which the peak appears.11 At long ta and/or high Ta both recovered enthalpy and peak temperature become ultimately independent of ta. This is seen in leveling off at high ta when the sample has approached its equilibrium configuration at Ta. Such leveling off is not observable in the ln(ta) plots of Figure 3c, which indicates that the equilibrium configuration at Ta has not been attained yet. These concepts although developed for annealing of glasses with single glass transition also hold for vitrified states of hydrated DNA. (iii) Effect of Hydration Level. For annealing at 183 K for 20 min, enthalpy decrease and its recovery on reheating is not observable at Γ ≈ 3.4 (Figure 2a, curve 1, with 0.19 (g of water)/ (g of NaDNA)). It becomes observable only at higher hydration level and increases nearly linearly from Γ ≈4.7 up to ≈12 (Figure 2a, curves 2-6). Linear extrapolation to zero enthalpy
Letters recovered gives Γ ≈ 2, or 0.10 (g of water)/(g of NaDNA). The linear increase of recovered enthalpy with increasing hydration is consistent with dynamic coupling of the water with NaDNA.27 The maximal value of recovered enthalpy is ≈0.4 J/(g of sample) (Figure 2b). Much higher values of ≈30 J/(g of sample) were obtained here on heat denaturation and are reported in the literature.28 Completion of DNA’s primary hydration shell including hydration of the base atoms is achieved for Γ ≈ 12 which is also the upper hydration level studied here. These water molecules can be grouped in three classes with decreasing binding affinity for (1) phosphate, (2) phosphodiester and sugar atoms, and (3) functional groups of bases.3,15,17,18 With increasing hydration a change in the conformer population of A-DNA and B-DNA occurs,3,15-18 the amount of the B-DNA conformer increasing linearly in this hydration range from 0% at Γ ) 3-4 to 50-70% at Γ ) 12 (read from Figures 5 and 6 in ref 18). Transition between the A and B conformers is comparatively slow at ≈300 K and even slower at lower temperatures.6,16,17 So, we expect that by quenching at ≈80 K min-1 we have retained at 103 K the original A to B conformer ratio and that it does not change on annealing at low temperatures or heating at 30 K min-1. Experimental2,6,29,30 and theoretical31 studies do suggest that B-DNA contains a multiplicity of conformational substates, whereas for A-DNA only one low-energy conformation was found.6,31 Fluctuation between the conformational substates of B-DNA is highly dynamic at ambient temperature but is frozenin at low temperatures in a nonequilibrium state.29,30 Enthalpy decrease and its recovery thus must be caused by the B-DNA conformer, and it is a result of approach toward equilibrium of some of the conformational substates of B-DNA. The linear increase observed in the amount of B-DNA with hydration3,15-18 coincides with the linear increase of enthalpy recovery shown in Figure 2b. Onset of observable enthalpy recovery for Γ ≈ 4.7 shown in curve 2 of Figure 2a is consistent with this interpretation because B-DNA is formed above Γ ) 3-4. Enthalpy recovery is absent for NaDNA containing only the A-form (curve 1 of Figure 2a, 0.19 (g of water)/(g of NaDNA), corresponding to Γ ≈ 3.4). This is further evidence for the inference that for A-DNA only one low-energy conformation exists.6,31 These observations are significant because the B form of DNA is considered to be biologically active,3 and this activity is attributed to the multiplicity of its conformational substates.31 Considering the effects of temperature on the enthalpy recovery effects as shown in Figure 4, and by implication on the availability of conformational substates, B-DNA would be biological inactive in the glassy state where conformer populations are frozen-in.29,30 It is biologically active in the “fluid” state where conformational fluctuations are highly dynamic. Similar correlation of biological activity with low-temperature dynamics and a glasslike transition at ≈220 K has been proposed also for proteins.32
J. Phys. Chem., Vol. 100, No. 2, 1996 461 For protein-DNA interaction, conformational flexibility and distortion of B-DNA is a prerequisite.33,34 We surmize that enthalpy recovery effects of B-DNA as reported here can be quantified to reveal the conformational dynamics necessary for interaction with a protein. Acknowledgment. We are grateful for financial support by the Forschungsfo¨rderungsfonds of Austria (Project Number P10404-PHY), and to Prof. G. P. Johari for reading and discussing the manuscript. References and Notes (1) Reviewed by: McGammon, J. A.; Harvey, S. C. Dynamics of proteins and nucleic acids; Cambridge University Press: Cambridge, 1987. (2) Reviewed by: Gorenstein, D. G. Chem. ReV. 1994, 94, 1315. (3) Reviewed by: Jeffrey, G. A.; Saenger, W. Hydrogen Bonding in Biological Structures; Springer-Verlag: Berlin, 1994. (4) Kopka, M. L.; Fratini, A. V.; Drew, H. R.; Dickerson, R. E. J. Mol. Biol. 1983, 163, 129. (5) Holbrook, S. R.; Kim, S.-H. J. Mol. Biol. 1984, 173, 361. (6) Shindo, H.; Fujiwara, T.; Akutsu, H.; Matsumoto, U.; Shimidzu, M. J. Mol. Biol. 1984, 174, 221. (7) Kintanar, A.; Huang, W.-C.; Schindele, D. C.; Wemmer, D. E.; Drobny, G. Biochemistry 1989, 28, 282. (8) Scherer, G. W. Relaxation in Glass and Composites; Wiley: New York, 1986. (9) Hodge, I. M. J. Non-Cryst. Solids 1994, 169, 211. (10) Illers, K. H. Makromol. Chem. 1969, 127, 1. (11) Stephens, R. B. J. Non-Cryst. Solids 1976, 20, 75. (12) Hodge, I. M.; Berens, A. R. Macromolecules 1982, 15, 756. (13) Sartor, G.; Mayer, E.; Johari, G. P. Biophys. J. 1994, 66, 249. (14) Green, J. L.; Fan, J.; Angell, C. A. J. Phys. Chem. 1994, 98, 13780. (15) Falk, M.; Hartman, K. A., Jr.; Lord, R. C. J. Am. Chem. Soc. 1962, 84, 3843; 1963, 85, 387; 1963, 85, 391. (16) Taillandier, E.; Liquier, J.; Taboury, J. A. In AdVances in Infrared and Raman Spectroscopy; Clark, R. J. H., Hester, R. E., Eds.; Wiley: New York, 1985; Vol. 12, Chapter 2. (17) Lindsay, S. M.; Lee, S. A.; Powell, J. W.; Weidlich, T.; Demarco, C.; Lewen, G. D.; Tao, N. J.; Rupprecht, A. Biopolymers 1988, 27, 1015. (18) Wolf, B.; Hanlon, S. Biochemistry 1975, 14, 1661. (19) Falk, M.; Poole, A. G.; Goymour, C. G. Can. J. Chem. 1970, 48, 1536. (20) Sartor, G.; Hallbrucker, A.; Hofer, K.; Mayer, E. J. Phys. Chem. 1992, 96, 5133. (21) Mrevlishvili, G. M. SoV. Phys. Usp. 1979, 22, 433. (22) Angell, C. A. Science 1995, 267, 1924. (23) Sartor, G.; Mayer, E.; Johari, G. P. J. Polym. Sci. B: Polym. Phys. 1994, 32, 683. (24) Angell, C. A.; Torrell, L. M. J. Chem. Phys. 1983, 78, 937. (25) Gold’anskii, V. I.; Krupyanskii, Yu. F.; Fleurov, V. N. Phys. Scr. 1986, 33, 527. (26) Laudat, J.; Laudat, F. Europhys. Lett. 1992, 20, 663. (27) Tao, N. J.; Lindsay, S. M.; Rupprecht, A. Biopolymers 1988, 27, 1655. (28) Ackermann, Th. Angew. Chem. 1989, 101, 1005. (29) DiVerdi, J. A.; Opella, S. J. J. Mol. Biol. 1981, 149, 307. (30) HolbrooK, S. R.; Kim, S.-H. J. Mol. Biol. 1984, 173, 361. (31) Poncin, M.; Piazzola, D.; Lavery, R. Biopolymers 1992, 32, 1077. (32) Perutz, M. Nature 1992, 358, 548. (33) Somers, W. S.; Philipps, S. E. V. Nature 1992, 359, 387. (34) Pavletich, N. P.; Pabo, C. O. Science 1993, 261, 1701. (35) Ru¨disser, S. Diplomarbeit, Kalorimetrische Untersuchung der Dynamik von Wasser und NaDNA an hydratisierten NaDNA Fasern, Universita¨t Innsbruck, 1995.
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