11354
J. Phys. Chem. B 2000, 104, 11354-11359
B-DNA’s BII Conformer Substate Population Increases with Decreasing Water Activity. 2. A Fourier Transform Infrared Spectroscopic Study of Nonoriented d(CGCGAATTCGCG)2 Arthur Pichler, Andreas Hallbrucker, Rudolf H. Winger, Klaus R. Liedl, and Erwin Mayer* Institute of General, Inorganic and Theoretical Chemistry, UniVersity of Innsbruck, A-6020 Innsbruck, Austria ReceiVed: May 18, 2000; In Final Form: August 24, 2000
Nonoriented hydrated films of the sodium salt of the d(CGCGAATTCGCG)2 dodecamer, with Γ (water molecules per nucleotide) ) 20, 14, 8, and 6, were investigated by Fourier transform infrared spectroscopy. Curve resolution of the spectral region containing the symmetric stretching vibration of the ionic phosphate at two temperatures allows estimation of B-DNA’s BI/BII conformer substate population ratio. The BI/BII population ratio decreases at 290 K with decreasing water activity from 1.4 for Γ ) 20 to 1.2, 1.0, and 0.72 for Γ ) 14, 8, and 6. Increasing BII population with decreasing water activity is attributed to differences in hydration as the driving force for the BI f BII transition. An enhanced BII population could be significant for the interaction of proteins with B-DNA in chromatin. We surmize that the BII substate could be an intermediate in the transition of canonical B- to A-DNA.
Introduction In the preceding paper, we showed by molecular dynamics (MD) simulations of the Drew-Dickerson dodecamer (DDD), d(CGCGAATTCGCG)2, at selected hydration levels, that BI f BII transitions occur more frequently as the water activity decreases and that the BII conformer substate population increases.1 This increase in BII population occurs at water activities similar to those in the cell nucleus. Thus, we surmized that the populated BII substate could be of biological importance for the protein-DNA recognition process.1-5 Here, we report experimental evidence for an increasing BII conformer substate population with decreasing water activity by a Fourier transform infrared (FTIR) spectroscopic study of nonoriented films of DDD. Our approach has been described previously3,4 and is outlined briefly as follows. At ambient temperature, the BI and BII conformer substates (CSs) of B-DNA interconvert in aqueous solution or in nonoriented hydrated films on a subnanosecond time scale (for literature on BI and BII CSs and their properties, see ref 1). On cooling into the glassy state, the rate of CSs interconversion first slows down and eventually approaches zero. Since the equilibrium constant for BI S BII interconversion varies with temperature, a nonequilibrium distribution of CSs is generated by rapid quenching into the glassy state. On heating to an appropriate temperature in the glass f liquid transition region, isothermal relaxation toward equilibrium can be followed either by calorimetry6,7 or by FTIR spectroscopy.3,4 FTIR difference spectra allow us to distinguish between the spectral features of interconverting conformers, and we have tested our approach with spectra of chlorocyclohexane’s axial and equatorial conformers.8 Recently, we have shown by FTIR spectroscopy that in nonoriented hydrated films of DDD and of native B-DNA from salmon testes, the same BI and BII CSs are involved in relaxation toward equilibrium and that at 200 K, BI interconverts into BII.4 The spectral changes become observable in the form of IR difference spectra, in which positive peaks indicate the formation of a CS and negative peaks its disappearance. * Corresponding author.
In a next step, we compared these spectral changes on isothermal relaxation with the changes observable on cooling a film slowly in steps from ambient temperature, that is, from 290 to 270 K.4 From the mirror image-like appearance of difference spectra, we concluded that on slow cooling, BII interconverts into BI for both the hydrated films of DDD and native B-DNA from salmon testes. Once the characteristic IR spectral features of the BI and BII CS have been allocated from difference spectra, it is possible to obtain via curve resolution an estimation of the BI/BII CS population ratio at ambient temperature. The spectral region containing the symmetrical stretching vibration of the ionic phosphate, νs, of the BI and BII CS was found most suitable for curve resolution. For a hydrated nonoriented film of DDD containing 14 water molecules per nucleotide (i.e., Γ ) 14), estimation of the BI/BII CS population ratio gave 1.3 to 1 between 290 and 270 K.4 This is much lower than the 10 to 1 ratio obtained from X-ray diffraction studies of single crystals.9-11 Therefore, the BII substate is not a crystal packing artifact, as has been suggested.12 On the contrary, its population is enhanced in nonoriented films. The pronounced increase of BII CS population with decreasing water activity at a given temperature is surprising and reminds of the canonical B- to A-DNA transition13 where the “economics in the hydration of phosphate groups” is one of the interpretations.14 This dependence of the BI to BII population ratio on water activity is discussed with respect to differences in hydration as the driving force for the BI f BII transition, and it could be significant for the interaction of proteins with B-DNA in chromatin. The significance of enhanced BII population for interaction with a protein was recently discussed by Tisne´ et al.15 We note that Clark et al.16 recently reported in a single crystal study of dehydrated DDD by X-ray diffraction that “the fully dehydrated structure contains an unusually high number of BII backbone conformations”. Becker and Wang17 reported that the B f A transition is highly sequence-specific and “may actually proceed though the formation of a previously unidentified intermediate”.17,18 Here, we surmize that the BII substate could be this intermediate in the transition of canonical B- to A-DNA.
10.1021/jp001843f CCC: $19.00 © 2000 American Chemical Society Published on Web 11/01/2000
FTIR Spectroscopy of Nonoriented d(CGCGAATTCGCG)2
J. Phys. Chem. B, Vol. 104, No. 47, 2000 11355
Experimental Section Materials. Lyophilized DDD was obtained as a sodium salt from MWG Biotech and purified by HPLC or HPSF. The triethylammonium acetate buffer was removed with a NAP column. Quantitative removal of the buffer was controlled by the absence of the intense IR buffer bands at 1558 and 1414 cm-1. Films of hydrated nonoriented dodecamer with Γ ) 6, 8, 14, and 20 were obtained by keeping the aqueous solutions on AgCl disks at relative humidities of 65.2, 80.3, 84.3, and 92.5% for several days (obtainable with saturated NaNO2, (NH4)2SO4, KCl, and KNO3 solution). The hydrated DDD films were quickly covered with a second AgCl disk, and the two disks were taped and positioned in a selfmade copper holder for the cryostat. Special care was taken to avoid orientation of the film, and the method of preparation ensures nonoriented films. To generate a nonequilibrium distribution of conformer substates, sample and sample holder were quenched to ≈170 K into the glassy state while inside the cryostat by forcing liquid N2 through the cooling tubes of the sample holder. The cooling rate was ≈90 K min-1. A pressure of ≈600 mbar of N2 was maintained during the whole experiment in order to avoid dehydration of the films. FTIR Spectroscopy. IR spectra were recorded in transmission mode on Biorad’s FTS-45 model at 4 cm-1 resolution by coadding 64 scans (UDR1, DTGS detector; zero-filling factor 2; low pass filter at 1.12 kHz; triangular apodization). First, IR spectra of the films were recorded at 290, 280, 270, and 260 K. The Γ values of the films were determined from band-area ratios of the OH stretching vibration and the antisymmetric stretching vibration of the ionic PO2- group.19 No ice was formed at 260 K in films with Γ ) 14 and 20. Rapid equilibration in films recorded between 260 and 290 K was tested by recording spectra at a given temperature as a function of time. Their difference spectra contained only minute signals, of less than 10-3 absorbance units, from slight baseline instability. IR spectra of the quenched films were recorded thereafter isothermally at 200 K, and spectral changes caused by CS interconversion were followed isothermally in the form of difference spectra. This way, changes in band profiles with temperature are avoided. The absence of dehydration or of irreversible spectral changes was confirmed by comparing IR spectra recorded at 290 K before and after the low-temperature experiment; this way, changes in concentration and/or thickness of the films were ruled out. This is an important aspect of the estimation of BI/BII population ratios, which requires a constant (BI + BII) concentration and film thickness. Curve Resolution. Curve resolution was applied with a sum of Gaussian and Lorentzian peak shapes (GRAMS/32 software, from Galactic Industries Corp.).20 The slightly sloping background in the original spectra was corrected by two-point baseline subtraction, with break points set at 1155 and 995 cm-1. Second derivatives are shown inverted in Figures 3 and 4. Peak shapes of the curve-fitted component bands were in all cases pure Gaussian, although the band shape had not been fixed at the beginning of the fit. Results DDD was selected for this study because it is the best characterized oligonucleotide and it persists in the B form even at low water activity.21 Therefore, problems with formation of A-DNA with decreasing water activity are avoided. The absence of A-DNA even at low hydration was ascertained by the absence of the A-DNA IR marker bands.21,22
Figure 1. IR spectra of nonoriented films of DDD, from 950 to 1175 cm-1, recorded at 290 K. Hydration decreases from Γ ) 14 (dashed) to 8 and 6 (dash-dotted, solid). (A) shows the experimental spectra (scaled with respect to the peak maximum at 1086 cm-1); (B) shows the spectra after two-point baseline subtraction. Note the increasing intensity of the shoulder at ≈1109 cm-1 with decreasing hydration.
Figure 2. The comparison of the difference curve obtained at 200 K on isothermal annealing (A) with that obtained from spectra recorded at 290 and 260 K (B): (A) the difference curve obtained by subtracting the spectrum of a quenched DDD film (Γ ) 14) recorded at 200 K after 4 min from that recorded after 25 min; and (B) the difference curve of the same film obtained by subtracting the spectrum recorded at 290 K from that at 260 K. Note the mirror image between A and B.
Figure 1 shows the spectral region of hydrated DDD selected for curve resolution. (A) shows the experimental spectra recorded at 290 K, with hydration decreasing from Γ ) 14 (dotted line) to 8 (dash-dotted) and 6 (solid), and (B) shows the spectra after two-point baseline correction. Pronounced changes in relative areas of component bands with decreasing hydration are observable by increasing intensity of the shoulder at ≈1109 and at ≈1070 cm-1. Comparison of Difference Spectra. At ambient temperature, the hydrated films are equilibrated, whereas rapid quenching into the glassy state generates a nonequilibrium distribution of BI/BII CSs. Subsequent isothermal relaxation in the glass f liquid transition region occurs via interconversion of CSs.3,4,6,7 In Figure 2, we compare the isothermal difference curve of the nonequilibrated film with the difference curve obtained from spectra recorded with an equilibrated film at two different temperatures. Curve A is the difference curve of the hydrated film, with Γ ) 14, obtained by subtracting the spectrum recorded at 200 K after 4 min from that after 25 min. Curve B is the
11356 J. Phys. Chem. B, Vol. 104, No. 47, 2000
Hallbrucker et al. TABLE 1: Curve-Fitting Analysis of the IR Spectra of a Nonoriented d(CGCGAATTCGCG)2 Film, with Γ ) 14, Recorded at 290 and 260 Ka band number
Figure 3. Curve resolution of spectra of nonoriented DDD film recorded at 290 and 260 K (Γ ) 14, from 1175 to 975 cm-1): (A) the experimental composite spectrum recorded at 290 K (solid) and the eight curve-fitted component bands and their sum (broken); (B) the comparison of the second derivative curve of the experimental composite band profile (solid) with that of the sum of the curve-fitted component bands (broken); (C) the experimental composite spectrum recorded at 260 K (solid) and the eight curve-fitted component bands and their sum (broken); and (D) the comparison of the experimental difference curve (solid, C - A) with that of the sum of the curvefitted component bands (broken, C - A).
Figure 4. Curve resolution of spectra of nonoriented DDD film recorded at 290 and 260 K (Γ ) 6, from 1175 to 975 cm-1): (A) the experimental composite spectrum recorded at 290 K (solid) and the eight curve-fitted component bands and their sum (broken); (B) the comparison of the second derivative curve of the experimental composite band profile (solid) with that of the sum of the curve-fitted component bands (broken); (C) the experimental composite spectrum recorded at 260 K (solid) and the eight curve-fitted component bands and their sum (broken); and (D) the comparison of the experimental difference curve (solid, C - A) with that of the sum of the curvefitted component bands (broken, C - A).
difference curve obtained by subtracting the spectrum recorded at 260 K from that at 290 K. Positive peaks indicate formation of a CS, negative peaks its disappearance. Pronounced spectral changes occur for the intense bands in the antisymmetric (νas) and symmetric (νs) stretching band region of the ionic phosphate: 22-26 in curve A, ν decreases from 1237 to 1209 cm-1 on B as I to BII CS interconversion, whereas νs increases from 1083 to 1100 cm-1. Curve A is, on the whole, the mirror image of curve B. This indicates that on cooling from 290 to 260 K, an opposite CS interconversion occurs from that on isothermal relaxation at 200 K, that is, that BII interconverts into BI. BI to BII Conformer Substate Population Ratio at Ambient Temperature, with Γ ) 14. Estimation of the BI to BII CS population ratio at ambient temperature, with equilibrated
νmax (cm-1)
height
fwhh (cm-1)
area
1
290 K 260 K
1141.4 1142.1
0.01408 0.01290
13.7 14.1
0.206 0.194
2
290 K 260 K
1107.9 1109.8
0.2133 0.2089
30.7 27.5
6.973 6.119
3
290 K 260 K
1087.3b 1087.3b
0.5138 0.5681
20.2b 20.2b
11.048 12.216
4
290 K 260 K
1083.6b 1083.6b
0.0244 0.0407
6.8b 6.8b
0.177 0.295
5
290 K 260 K
1068.9 1069.4
0.3093 0.2966
18.0 16.3
5.934 5.132
6
290 K 260 K
1052.4 1052.5
0.3849 0.4466
16.2 16.9
6.621 8.035
7
290 K 260 K
1036.9 1035.9
0.1041 0.0732
18.1 13.4
2.001 1.047
8
290 K 260 K
1018.0 1019.6
0.1409 0.1567
22.2 22.1
3.327 3.692
a For Tables 1 to 4: ν -1 max (in cm ) are the peak frequencies; fwhh is the full width at half-height of the curve-fitted component bands. The spectral region from 1175 to 975 cm-1 was used for curve resolution. b Band parameters were fixed in the final optimization; all other parameters were varied.
hydrated films, was done as described previously,4 by curve resolution of the spectral region containing νs of the PO2- group. Figure 3A shows the experimental composite band of the spectrum recorded at 290 K (solid, Γ ) 14, two-point baseline corrected), the eight curve-fitted component bands that were necessary for optimizing the curve fit, and their sum (broken), which is indistinguishable from the experimental composite band. Six of the component bands are seen already in the experimental composite band in the form of peaks and shoulders. In Figure 3B, the second derivative of the experimental composite band (solid) shows these six component bands as distinct peaks. The seventh and eighth component bands were necessary to reproduce the shoulder at ≈1091 cm-1 in the second derivative and the weak feature at 1031 cm-1. We have shown recently that reliable curve resolution of highly overlapping composite bands, such as those of Figure 3A, is possible by comparison of the second or fourth derivative of the experimental composite band profile with that of the sum of the curve-fitted component bands.20 Figure 3B shows this comparison between the second derivative of the experimental composite band (solid) and the sum of the curve-fitted component bands from Figure 3A (broken). In the same manner, curve resolution of the spectrum of the same film recorded at 260 K was achieved. Figure 3C shows the experimental composite band of the spectrum recorded at 260 K (solid) and the eight curve-fitted component bands and their sum (broken, almost congruent with the solid line). Further minor refinement of the curve fit was achieved by comparison of the experimental difference curve with that obtained from the sum of the two curve fits. This optimization is in our experience a very useful additional criterion for the quality of the curve fits. In Figure 3D, the experimental difference curve obtained by subtracting the baseline-corrected experimental spectrum recorded at 290 K from that at 260 K (solid) is compared with the difference curve obtained from the sum of the corresponding curve fits (broken, C - A). The band parameters of both curve fits, optimized as described above, are listed in Table 1. The component bands centered at 1087 and 1109 cm-1 are assigned to νs of the PO2- group in BI and BII (see ref 4 for a
FTIR Spectroscopy of Nonoriented d(CGCGAATTCGCG)2 detailed discussion). The component band centered at 1109 cm-1 is much broader than that at 1087 cm-1 (Table 1, 31 vs 20 cm-1), and because of that, it is only a weak feature in the second derivative curve, in comparison to the intense peak centered at 1085 cm-1 (Figure 3B). Changes in areas of these two component bands centered at 1087 and 1109 cm-1 on cooling from 290 to 260 K are used in our following estimation of the conformer substate population because these curve-fitted component bands are the most reliable ones. For two interconverting conformers A and B, the band intensities for each conformer may be given by IA ) ACAl and IB ) BCBl, where IA and IB are the observable integrated intensities of the absorption bands of conformers A and B, A and B their molar absorptivites, CA and CB the conformer populations, and l the thickness of the absorbing film.27,28 Accordingly, for the BI and BII bands centered at 1087 and 1109 cm-1, IBI ) BICBIl, IBII ) BIICBIIl, and IBI/IBII is the ratio of the band intensities. In the two spectra recorded at 290 and 260 K, the BII CS population decreases on cooling and the BI population increases, but their sum is constant since l is constant. Thus, the BI/BII ratio can be calculated from the ratio of the intensity changes (that is, ∆IBI/∆IBII). The BI/BII ratio determined this way, together with the ratio of band intensities, IBI/ IBII, allow to calculate, for constant l, CBI/CBII which is the ratio of BI/BII CS population. The area of the BI band centered at 1087 cm-1 increases on cooling from 290 to 260 K, from 11.048 to 12.216, whereas the area of the BII band at 1109 cm-1 decreases from 6.973 to 6.119 (see Table 1). Therefore, ∆IBI/∆IBII (that is, 1.168/0.854) is 1.37. This is equivalent to saying that the area of this BI band increases by 10.6%, whereas the area of the BII band at 1109 cm-1 decreases by 7.73% (of the band centered at 1087 cm-1 (see Table 1); this % notation will be used in the following). Accordingly, the ratio of their molar absorptivities, (1087)/(1109), is 1.37. At 290 K, the ratio of band areas of the two bands centered at 1087 and 1109 cm-1 is 1.58. Therefore, the BI/BII population ratio is calculated as 1.2. In the same manner, for 260 K the BI/BII population ratio of 1.5 is calculated from the ratio of band areas (Table 1, 2.00 to 1), by using the same (1087)/ (1109) ratio. BI to BII Conformer Substate Population Ratio at Ambient Temperature, with Γ ) 6. In Figure 4, estimation of the BI/ BII population ratio is shown for a DDD film with Γ ) 6. The curves are labeled in the same way as those of Figure 3, that is, A and B contain the experimental composite band and the curvefitted component bands of the spectra recorded at 290 and 260 K, and C and D contain the comparison of second derivative curves and difference curves. The band parameters are listed in Table 2. The component band centered at 1087 cm-1 increases on cooling from 290 to 260 K by 8.96%, the band at 1109 cm-1 decreases by 7.82% (of the band centered at 1087 cm-1), and the (1087)/(1109) ratio is 1.15. At 290 K, I(1087)/I(1109) is 0.825, and the BI/BII population ratio is 0.72. At 260 K, I(1087) /I(1109) is 0.961, and the BI/BII population ratio is 0.84. BI to BII Conformer Substate Population Ratio as a Function of Hydration. Two further hydration levels (with Γ ) 20 and 8) were evaluated in the same manner, and the band parameters of the curve fits are listed in Tables 3 and 4. For the film with Γ ) 20, our estimation of the BI/BII CS population ratio gives 1.4 at 290 K (for (1087)/(1109) ) 0.88) and 1.6 at 260 K. For the film with Γ ) 8, the BI/BII CS population ratio is 1.0 at 290 K (for (1087)/(1109) ) 1.35) and 1.2 at 260 K. We further determined for each of the films five additional (1087)/(1109) ratios by using spectra recorded at 280 and 270 K
J. Phys. Chem. B, Vol. 104, No. 47, 2000 11357 TABLE 2: Curve-Fitting Analysis of the IR Spectra of a Nonoriented d(CGCGAATTCGCG)2 Film, with Γ ) 6, Recorded at 290 and 260 K band number
νmax (cm-1)
height
FWHH (cm-1)
area
1
290 K 260 K
1140.2 1140.6
0.00696 0.00774
11.4 12.2
0.0842 0.1005
2
290 K 260 K
1106.2 1107.1
0.1819 0.1777
36.1 34.5
6.984 6.533
3
290 K 260 K
1086.0a 1086.0a
0.2471 0.2693
21.9a 21.9a
5.763 6.279
4
290 K 260 K
1082.7a 1082.7a
0.0135 0.01731
7.6a 7.6a
0.109 0.140
5
290 K 260 K
1067.5 1067.7
0.1838 0.1839
17.6 16.9
3.445 3.314
6
290 K 260 K
1052.4 1052.6
0.2016 0.2167
17.5 17.4
3.746 4.019
7
290 K 260 K
1035.5 1035.6
0.0812 0.0776
31.4 30.8
2.714 2.547
8
290 K 260 K
1013.1 1013.9
0.0613 0.0631
22.2 22.3
1.447 1.496
a Band parameters were fixed in the final optimization; all other parameters were varied.
TABLE 3: Curve-Fitting Analysis of the IR Spectra of a Nonoriented d(CGCGAATTCGCG)2 Film, with Γ ) 20, Recorded at 290 and 260 K band number
νmax (cm-1)
height
FWHH (cm-1)
area
1
290 K 260 K
1142.0 1143.7
0.00977 0.00892
11.5 9.46
0.120 0.0898
2
290 K 260 K
1108.2 1109.8
0.1909 0.1879
31.5 28.7
6.393 5.738
3
290 K 260 K
1087.3a 1087.3a
0.3657 0.3918
20.2a 20.2a
7.863 8.424
4
290 K 260 K
1083.6a 1083.6a
0.02238 0.0375
7.8a 7.8a
0.198 0.3113
5
290 K 260 K
1069.2 1069.8
0.2602 0.2412
19.2 17.4
5.307 4.462
6
290 K 260 K
1051.8 1052.1
0.3245 0.3594
17.2 18.0
5.924 6.879
7
290 K 260 K
1036.0 1035.2
0.0940 0.0668
16.8 13.5
1.679 0.963
8
290 K 260 K
1018.0 1019.3
0.1399 0.1455
22.5 22.0
3.347 3.407
a Band parameters were fixed in the final optimization; all other parameters were varied.
and by evaluating curve fits at two temperatures (e.g., 290/280 K, 280/270 K) for the changes in band areas in the same manner outlined above for 290/260 K. The (1087)/(1109) ratios were constant for each hydration from 290 to 260 K. Standard deviation was (0.1 for the films with Γ ) 14, 8, and 6 and (0.2 for Γ ) 20. In Figure 5, the BI/BII population ratio is plotted versus Γ. At constant hydration, the BI/BII population ratio increases with increasing temperature, as shown by the four vertical columns. Thermodynamic parameters calculated from these temperature dependencies will be reported separately. At a given temperature, however, the BI/BII population ratio decreases with decreasing Γ values for each of the four temperatures investigated. Discussion Our MD simulations reported in the preceding paper1 and this FTIR spectroscopic study agree with respect to a pro-
11358 J. Phys. Chem. B, Vol. 104, No. 47, 2000
Hallbrucker et al.
TABLE 4: Curve-Fitting Analysis of the IR Spectra of a Nonoriented d(CGCGAATTCGCG)2 Film, with Γ ) 8, Recorded at 290 and 260 K band number
νmax (cm-1)
height
FWHH (cm-1)
area
1
290 K 260 K
1137.9 1137.9
0.0111 0.0110
17.8 17.8
0.211 0.210
2
290 K 260 K
1108.6 1109.2
0.1599 0.1572
30.4 28.8
5.177 4.819
3
290 K 260 K
1086.5a 1086.5a
0.3029 0.3235
21.9a 21.9a
7.064 7.544
4
290 K 260 K
1082.7a 1082.7a
0.0170 0.0233
7.6a 7.6a
0.138 0.189
5
290 K 260 K
1068.0 1068.3
0.1733 0.1684
16.8 15.9
3.107 2.845
6
290 K 260 K
1052.6 1052.7
0.2125 0.2517
16.8 17.3
3.804 4.630
7
290 K 260 K
1036.8 1036.0
0.0754 0.0597
30.5 21.8
2.449 1.284
8
290 K 260 K
1014.5 1016.7
0.0614 0.0765
22.1 23.1
1.447 1.878
a Band parameters were fixed in the final optimization; all other parameters were varied.
Figure 5. The BI/BII conformer substate population ratio plotted versus hydration in Γ values: [ data points for 290 K, b 280 K, 2 270 K, 1 260 K.
nounced increase in BII substate population with decreasing water activity. The MD simulation shows that this increase in BII population is achieved not only by base steps remaining longer in the BII substate but also by additional base steps interconverting into BII as the water activity is decreased. The BI/BII population ratios estimated from MD simulations by integrating over the number of base steps in BII from 500 to 10.000 ps and from FTIR spectroscopy by curve resolution differ somewhat, but the trend is evident. We emphasize that FTIR spectroscopy can give only a global view of BI/BII population ratios. Thus, contrary to the MD simulation, it is not possible to differentiate whether the experimentally observed increase in the BII population with decreasing water activity is caused by a few base steps remaining for longer times in the BII substate, by the increase in the number of base steps in the BII conformation, or by a combination of both. Effect of Water Activity on the BI/BII Population Ratio. The pronounced dependence of the BI/BII conformer substate population ratio on water activity is surprising because concentrations cancel in the equilibrium constant (eq 1) and thus, change of concentration should have little effect on the BI/BII conformer population ratio. However, medium effects on conformational equilibria in the condensed phase are wellknown, although in these cases the equilibrium distribution of conformers changes with the change of solvent, that is, in going from a nonpolar to a polar solvent.29 The thermodynamic equilibrium constant, Ka, for the BI S BII equilibrium is
Ka ) [BII]/[BI] ) [c(BII)/c(BI)][R(BII)/R(BI)]
(1)
where c(BII) and c(BI) are the BII and BI concentrations and R(BII) and R(BI) the activity coefficients. Formally speaking, the shift of equilibrium toward BII with increasing concentration of B-DNA requires that, for eq 1, the ratio of activity coefficients, [R(BII)/R(BI)], decrease with increasing concentration of B-DNA. This occurs when the decrease of R(BII) with increasing concentration is more pronounced than that of R(BI). This demonstrates that water is involved in the BI S BII equilibrium, and it is consistent with results of our FTIR spectroscopic and MD simulation studies.1,2,4,5 The dependence of the BISBII CS equilibrium on water activity is reminiscent of the B-DNASA-DNA transformation, which is explained in terms of water activity,13 where the “economics in the hydration of phosphate groups” is one of the interpretations.14 It is well-known that water is not just a medium to keep DNA dissolved; it interacts with the solute, and the distribution of water molecules in the first hydration shell is sensitive to the A- or B-type conformation of DNA.30 In a similar manner, we consider differences in hydration as the driving force for the BI f BII transition. Pronounced IR spectral changes upon BI f BII interconversion are consistent with migration of water from ionic phosphate toward the phosphodiester and sugar moieties.4 This is consistent with our recent MD simulation of the dynamics of the BI and BII substates in DDD, where analysis of radial distribution functions revealed that water migrates on BI f BII transition from ionic phosphate toward the sugar ring oxygen.2 Thus, water exchange is coupled to the conformational BI f BII transition. Our IR spectroscopic study further indicates that the BII substate is stabilized in comparison to BI by enhanced hydrogen-bond interaction with the migrating water.4 Is BII the intermediate in the Canonical B- to A-DNA transition? A central question in structural molecular biology is whether A- and B-DNA can coexist in vivo and how a conversion is achieved.31 Becker and Wang17 reported that the B f A transition is highly sequence-specific and “may actually proceed through the formation of a previously unidentified intermediate”.17,18 Since both the population of A-DNA and of the BII substate increase with decreasing water activity, we surmize that the canonical B (or BI) f A transition could occur via BII as the intermediate. In this case, BII would be a stable intermediate that is in equilibrium with A- and canonical B-DNA.32 The sugar conformation in BII is the B type, but the destacking observed with the BI f BII transition11,33 could be a hint toward an A-type conformation. Destacking is observed both with BI f BII and with the canonical B f A-DNA transition.11,30,31,33,34 Thus, destacking could induce changes in the rise that is necessary for the B f A transition. We note that Haworth et al.35 suggested BII as the intermediate for the canonical B- to Z-DNA transition. Acknowledgment. We are grateful for financial support from the Austrian Science Foundation (Project No. P12319-PHY). References and Notes (1) Winger, R. H.; Liedl, K. R.; Pichler, A.; Hallbrucker, A.; Mayer, E. J. Phys. Chem. B 2000, 104, 11349. (2) Winger, R. H.; Liedl, K. R.; Ru¨disser, S.; Pichler, A.; Hallbrucker, A.; Mayer, E. J. Phys. Chem. B 1998, 102, 8934-8940. (3) Ru¨disser, S.; Hallbrucker, A.; Mayer, E. J. Am. Chem. Soc. 1997, 119, 12251-12256. (4) Pichler, A.; Ru¨disser, S.; Mitterbo¨ck, M.; Huber, C. G.; Winger, R. H.; Liedl, K. R.; Hallbrucker, A.; Mayer, E. Biophys. J. 1999, 77, 398409. (5) Winger, R. H.; Liedl, K. R.; Pichler, A.; Hallbrucker, A.; Mayer, E. J. Biomol. Struct. Dyn. 1999, 17, 223-235.
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