Volume 55, Number 1, 2001 APPLIED SPECTROSCOPY 90003-7028 / 01 / 5501-0009$2.00 / 0q 2001 Society for Applied Spectroscopy

Estimation of the BII Conformer Substate Population inNonoriented Hydrated B-DNA via Curve Resolution ofInfrared Spectra

ARTHUR PICHLER, SIMON RUDISSER, RUDOLF H. WINGER,BERND WELLENZOHN, WOLFGANG FLADER, KLAUS R. LIEDL,ANDREAS HALLBRUCKER, and ERWIN MAYER*Institut fur Allgemeine, Anorganische und Theoretische Chemie, Universitat Innsbruck, A-6020 Innsbruck, Austria

Hydrated � lms of the self-complementary B-type d(CGCGAATT-CGCG)2 dodecamer, with 13 or 14 water molecules per nucleotide,were studied by Fourier transform infrared (FT-IR) spectroscopyeither as equilibrated samples between 290 and 260 K or, afterquenching into the glassy state, as nonequilibrated � lms isother-mally at 200 K. IR spectral changes on isothermal relaxation at 200K revealed that difference spectra are caused by interconversion ofB-DNA’s conformer substates, BI converting into BII. Curve reso-lution of IR spectra in the spectral region containing the symmetricstretching vibration of the ionic phosphate group, with a compositeband centered at 1087 cm21 for B I and at 1109 cm 21 for B II, givesthe BI/BII conformer substate population ratio and its dependenceon temperature between 290 and 260 K, and on time for isothermalrelaxation at 200 K. Curve resolution of the overlapping compositeband pro� le was optimized by comparison of second-derivative andof difference curves, aiming thereby for optimal correspondencebetween experimental and curve-� tted bands as a criterion forgoodness-of-� t. The spectroscopic aspects relevant for reliable curveresolution of the overlapping composite band pro� le are reportedand discussed in detail.

Index Headings: FT-IR spectroscopy; Conformer substates of B-DNA; Nonoriented d(CGCGAATTCGCG)2; BII conformer substatepopulation; Glass-to-liquid transition; Relaxation.

INTRODUCTION

Since 1980 single-crystal studies of synthetic B-typeoligonucleotides have shown that the dominant phospho-diester backbone conformation (B I ) is the same as thatproposed by modeling studies of oriented � bers of nativecanonical B-DNA.1–6 A minor backbone conformation(B II ) observed so far only in oligonucleotides was attri-buted to crystal packing effects.7 The B I and B II conform-er substates (CSs) involve changes in the phosphate back-bone conformation about the C3 9-O3 9-P segment of thebackbone chain, with the phosphate group rotating to-ward the minor groove in the B II conformation.2,4–6 TheseCSs are frozen-in in the crystal but interconvert in so-lution at ambient temperature on a (sub)nanosecond timescale.8–11 CSs of biologically active B-DNA could play adecisive role in nonspeci� c interaction of proteins withthe DNA backbone: in this highly dynamic system, which� uctuates between B I and B II substates, a protein ap-proaching B-DNA could select the B I or B II substate, de-pending on which one gives optimal interaction with thebases and the sugar–phosphate backbone.2,10,12

We have recently reported a new approach for inves-

Received 15 May 2000; accepted 18 September 2000.* Author to whom correspondence should be sent.

tigating experimentally the dynamics and interconversionof CSs of B-DNA,12–17 and this method is outlined brie� yas follows. At ambient temperature the B I and B II con-former substates of B-DNA interconvert rapidly in aque-ous solution or in nonoriented hydrated � lms.8–11 Oncooling into the glassy state, the rate of CSs interconver-sion � rst slows down and eventually approaches zero.Since the equilibrium constant for B I Û B II interconver-sion varies with temperature, a nonequilibrium distribu-tion of CSs is generated by rapid quenching into theglassy state. On heating to a convenient temperature inthe glass-to-liquid transition region, isothermal relaxationtoward equilibrium can be followed either by differentialscanning calorimetry (DSC)13,14,16 or by Fourier transforminfrared (FT-IR) spectroscopy.12,15–17 This is the basis ofour approach for investigating the onset of molecular mo-tions by calorimetric enthalpy relaxation and its recovery,and by distinguishing between the infrared spectral fea-tures of interconverting conformers. We have further test-ed this approach with chlorocyclohexane where equili-bration via axial ® equatorial conformer interconversioncould be observed isothermally in the glass ® liquid tran-sition region.18 Recently we have shown by FT-IR spec-troscopy that in nonoriented hydrated � lms of thed(CGCGAATTCGCG)2 dodecamer and of native B-DNAfrom salmon testes, the same B I and B II CSs are involvedin relaxation toward equilibrium, and that at 200 K B I

interconverts into B II.12,16,17 The spectral changes becomeobservable in the form of IR difference spectra wherepositive peaks indicate the formation of B II and negativepeaks the disappearance of B I. The advantage of follow-ing relaxation via conformer substate interconversion iso-thermally is that changes in band shape and position withtemperature are avoided. With the selection of an exper-imentally convenient temperature, where relaxation timeis between minutes and hours, interconversion of con-formers can be followed by conventional FT-IR spec-troscopy.12,15–17

Here we describe the IR spectroscopic details of theestimation of B I-to-B II conformer substate population ra-tios via curve resolution of IR spectra recorded eitherisothermally at 200 K or at ambient temperatures. Theself-complementary Drew–Dickerson dodecamer (DDD),d(CGCGAATTCGCG)2, was selected for this study be-cause it is the best characterized oligonucleotide,19–23 andit is in the B-form even at low water activity.24 Therefore,problems with the formation of A-DNA with decreasingwater activity are avoided. DDD was the � rst B-type ol-

10 Volume 55, Number 1, 2001

igonucleotide for which the single-crystal structure wasdetermined by X-ray diffraction.19–23 It contains the rec-ognition site of the EcoRI restriction enzyme, and be-cause of its biological importance it has remained at thefocus of biophysical studies. It has been studied, amongother methods, by NMR,10,11,25–29 Raman,30–32 and IR spec-troscopy,33,34 and by molecular dynamics (MD) simula-tion.25,35–39 In the single crystal of DDD, B II occurs at theG10-C11 and G22-C23 positions.2,5,6 Therefore, the B I-to-B II population ratio is 10 to 1. In nonoriented hydrated� lms of DDD, the B II population is higher than in thesingle crystal.12,17 Estimation of B I to B II CSs ratios con-centrates in this study on hydration with 13 or 14 watermolecules per nucleotide (i.e., G 5 13 or 5 14). Thedependence of B I-to-B II CSs ratios on a wider range ofhydration will be reported separately.40

DDD has also been intensely studied by NMR10,11,25–29

and Raman spectroscopy.30–32 Estimation of the B I-to-B II

population ratio has been made by NMR spectroscopy,and 31P NMR data in aqueous solution of DDD have beeninterpreted in terms of populations of the two thermo-dynamically stable B I and B II states, where the phosphateis assumed to make rapid jumps between the two stateson the time scale of the NMR experiment. These analysesof time-averaged NMR spectra are hindered by the factthat the proposed changes of dihedral angles e and z in-volved in the B I-to-B II transition cannot be determinedunambiguously.41 A new NMR spectroscopic approachfor distinguishing between B I and B II conformations in-volves measuring scalar couplings in 13C, 15N-labeled B-type oligonucleotides.42

Our approach of using difference spectroscopy for fol-lowing isothermal relaxation is related to the so-calledreaction-induced difference spectroscopy (RIDS) wherestructural perturbation of a protein by, for example, ab-sorption of a photon or an electrochemical event is fol-lowed by difference spectroscopy.43–48 The ratio of pro-tein spectra in the nonperturbed and perturbed state thencorresponds to the difference between the two states. Inour approach, differences in the B I-to-B II conformer sub-state population obtained either on thermally activatedisothermal relaxation or at different temperatures gener-ate the difference spectrum.12,15–17

Curve resolution of IR spectra of B-DNA resemblesthat of the amide I region of proteins with respect to bandoverlap and number of component bands.44,49–54 The ad-vantage for the B-DNA system is that only two structuralstates, B I and B II, have to be considered and that theirinterconversion as a function of time or temperature canbe followed in a systematic manner in terms of changesin B I /BII population ratios. This capability can provide aninternal control of the goodness-of-� ts.

EXPERIMENTAL

Materials. Lyophilized DDD was obtained as sodiumsalt from MWG BIOTECH, after puri� cation by HPLC.12

The triethyl ammonium acetate buffer was removed byprecipitation of DDD with cold ethanol. Quantitative re-moval of the buffer was ascertained by the absence ofthe intense IR buffer bands at 1558 and 1414 cm21. Asuitable � lm of hydrated nonoriented DDD with G 5 13was obtained by dissolving ø 0.7 mg of dry DDD in

ø 60–70 mg water, transferring the aqueous solution ona AgCl disk (the diameter of the aqueous solution on thedisk is ø 13 mm), and keeping the disk in a desiccatorfor several days over saturated KCl solution at relativehumidity of 84.3%. The hydrated DDD � lm was there-after quickly covered with a second AgCl disk, the twodisks taped, and the sample positioned in a self-madecopper holder for the cryostat. Special care was taken toavoid orientation of the � lm, and the method of prepa-ration ensures nonoriented � lms. In order to generate anonequilibrium distribution of conformer substates, sam-ple and sample holder were quenched to ø 170 K into theglassy state while inside the cryostat, by forcing liquidN2 through the cooling tubes of the sample holder, re-sulting in a cooling rate of ø 160 K min21. Thereafter theAgCl disks with the DDD � lm were stored in the des-iccator containing the saturated KCl solution for furtherstudies at ambient temperatures. During this storage, hy-dration increased slightly to G 5 14.

FT-IR Spectroscopy. IR spectra were recorded intransmission on BioRad’s FTS-45 model at 4 cm21 res-olution by coadding 64 scans [UDR (undersampling ra-tio) 5 1, DTGS detector; zero-� lling factor of 2; low-pass � lter at 1.12 kHz; triangular apodization]. The col-lection time of 150 s constitutes the time resolution. TheG values of the DDD � lms were determined from bandarea ratios of the OH stretching vibration and the anti-symmetric stre tch ing vibration of the ionic PO 2

2

group.55,56 The spectra are displayed in the � gures on thesame ordinate scale. Vertical bars indicate the ordinatescale in absorbance units. Second-derivative curves areshown inverted.

First, IR spectra of the quenched � lm were recordedisothermally at 200 K (constant to 60.18), and spectralchanges caused by CS interconversion were followed iso-thermally in the form of difference spectra. This way,changes in band pro� les with temperature are avoided.Differences in spectra were not observable at 200 K inslow-cooled � lms, or in quenched � lms after relaxationat 200 K, cooling to 180 K, and subsequent reheating.This proves that these effects are real, and not caused bybaseline instabilities. We had studied, by DSC, B-DNA’slow-temperature dynamics and its broad glass ® liquidtransition only with native DNA from salmon testes,13,14

and not with DDD. The comparison of the kinetics ofDDD and of B-DNA from salmon testes on isothermalrelaxation at low temperatures is consistent with similarglass ® liquid transition behavior.16,17

Thereafter, IR spectra of the same DDD � lm were re-corded at selected temperatures between 290 and 260 K.Rapid equilibration in � lms recorded between 260 and290 K was tested by recording spectra after a rapid tem-perature change as a function of time. Their differencespectra contained only minute signals, of less than 1023

absorbance units, from slight baseline instability.The absence of dehydration or irreversible spectral

changes was con� rmed by comparing IR spectra recordedat 290 K before and after the low-temperature experi-ment, and in this way changes in concentration and/orthickness of the � lms were ruled out. This is an importantaspect for estimation of B I /BII population ratios, whichrequires constant (B I 1 B II ) concentration and � lm thick-ness.

APPLIED SPECTROSCOPY 11

FIG. 1. Schematic representation of the d(CGCGAATTCGCG)2 do-decamer. The phosphate residues are numbered according to Kopka etal.3 The two arrows mark the B II substate positions in the single crystal(after Kopka et al.3).

Curve Resolution. Curve resolution was applied witha sum of Gaussian and Lorentzian peak shapes (GRAMS/32 software, from Galactic Industries Corp.). Reliablecurve resolution of the spectral region from 1155 to 995cm21 was accomplished as described by Fleissner et al.57

by comparison of the second derivative of the experi-mental band pro� le with that of the sum of the curve-� tted component bands, and by aiming for optimal cor-respondence between these second derivatives. As a fur-ther criterion for goodness-of-� t, we used the comparisonof the experimental difference curves with those of thedifference curves generated by ratioing sums of curve-� tted component bands, aiming again for optimal corre-spondence between the two difference curves. The slight-ly sloping background in the original spectra was cor-rected by two-point baseline subtraction, with breakpoints set at the absorbance minima at 1155 and 995cm21. Peak shapes of the curve-� tted component bandswere in all cases nearly pure Gaussian, although the bandshape had not been � xed at the beginning of the � t.

RESULTS AND DISCUSSION

Figure 1 shows a schematic representation of the self-complementary d(CGCGAATTCGCG)2 dodecamer.3,19–23

In nonoriented hydrated � lms DDD is solely in the Bform, independent of its hydration,24 and problems withthe formation of A-DNA with decreasing water activityare thus avoided. Arrows mark the two positions (at G10-

C11 and G22-C23) where in the single-crystal DDD isin the B II substate.

B-DNA’s conformer substates interconvert in solution atambient temperature on a (sub)nanosecond time scale;8–11

therefore, IR spectra recorded between 290 and 260 K arefor equilibrated DDD � lms. However, DDD � lmsquenched rapidly to low temperatures into the glassy stateare nonequilibrated, and IR spectra recorded at 200 K asa function of time monitor relaxation towards equilibriumby interconversion of B I ® B II. Our arguments for thisassignment are given in Ref. 12.

Isothermal B I ® B II Conformer Substate Intercon-version in the Dodecamer’s Glass ® Liquid Transi-tion Region. To detect the IR spectral features of thedistinct B I and B II substates of DDD, we � rst generateda nonequilibrium distribution of conformer population byquenching a hydrated nonoriented � lm into the glassystate.12–17 For the selected hydration level of G 5 13, for-mation of ice can be excluded.56 Figure 2 (top) shows IRspectra of a quenched DDD � lm recorded at 200 K after4 and 25 min (curves a and b). Below, four differencecurves are shown, which were obtained from spectra re-corded at 200 K. Curves 1 to 3 were obtained by sub-tracting the spectrum recorded after 4 min from thoseafter 10 (1), 25 (2), and 63 (3) min, respectively, wherecurve 2 is the difference b 2 a of the two spectra shownon top. These difference curves reveal the spectral chang-es on isothermal relaxation, positive peaks indicating theformation of B II and negative peaks the disappearance ofB I.

Difference curve 4 was also obtained at 200 K but withthe DDD � lm equilibrated at 220 K before spectra wererecorded at 200 K. In curve 4 the difference bands causedby B I ® B II interconversion are absent, and only minutefeatures caused by the subtraction and by slight instabilityof the instrument are observable. Thus the comparison ofcurves 1 to 3 with curve 4, which are drawn on the sameordinate scale, clearly demonstrates that the spectral fea-tures observable on isothermal relaxation and caused byB I ® B II interconversion are much more intense thanthose caused by the baseline instability and the subtrac-tion procedure.58

The decrease in intensity from disappearing B I is clear-ly observable for several peaks; this is marked by arrows(Fig. 2, curves a and b). The increase in intensity fromthe newly formed B II, however, is barely recognizable incurve b. It becomes observable only in form of increasedintensity on the wings of the main peaks. This is the � rstindication that the conformer substate population of B I ishigher than that of B II.

Pronounced IR spectral changes upon B I ® B II inter-conversion are consistent with migration of water fromionic phosphate towards the phosphodiester and/or sugarmoieties.12,16,17 This result agrees with our molecular dy-namics simulation of the dynamics of the B I and B II sub-states in DDD solution where analysis of radial distri-bution functions revealed that water migrates on the B I

® B II transition from ionic phosphate towards the sugarring oxygen.59

It would be helpful to have a zero line in the differencespectra for differentiating between positive and negativefeatures. In spite of that, we have refrained from addinga zero line because the apparent background changes

12 Volume 55, Number 1, 2001

FIG. 2. (Top) Structural relaxation by B I ® B II conformer substate interconversion as seen in infrared spectra of a quenched nonorientedd(CGCGAATTCGCG)2 dodecamer � lm with G 5 13 on isothermal annealing at 200 K. (a) Recorded after 4 min ( ), (b) after 25 min(– – –). Arrows indicate decrease of band intensity. (Bottom) Difference spectra numbered 1 to 4. Difference curves 1 to 3 were obtained bysubtracting the spectrum recorded after 4 min from those after 10 (1), 25 (2), and 63 (3) min. Difference curve 4 was obtained after heating the� lm to 220 K, keeping it at 220 K for 40 min for ensuring equilibration, cooling it to 200 K, and subtracting the spectrum recorded after 1 minfrom that after 16 min. The spectra are drawn on the same ordinate scale, with the enlargement factors given in the � gure. Difference curves areshifted for clarity (from Pichler17).

drastically on B I ® B II transition. As elaborated below,this result is attributed to the migrating water and itswavelength-dependent changes in absorptivity over thewhole spectral region. However, a zero line can be in-serted for narrow spectral regions when reliable subtrac-tion of the background is possible (cf. Figs. 9–11).

Difference Spectra Correspond to Differences be-tween Two Structural States Only. The familiar formsof difference spectra are bipolar peaks that have some‘‘zero-crossing’’ point, where the spectrum has both pos-itive and negative peaks. Such a difference spectrum ei-ther corresponds to the difference between two structuralstates or it is caused by a shift of peak frequency of thesame molecular vibration.44,60 We concentrate in Fig. 2 onthe antisymmetric stretching vibration of the ionic phos-phate group, nas PO 2

2,34,61–64 with a positive peak at 1236cm21 and a negative one at 1208 cm21 in the differencespectra, for demonstrating that these spectral features arecaused by increasing and decreasing intensity of the con-former substate bands, and not simply by changes in peakfrequency of the same molecular vibration.60 Figure 3Ashows two IR spectra of a quenched DDD � lm recordedat 200 K after 1 min and 80 min, and Fig. 3B and 3Ctheir Fourier deconvoluted spectra and their second-de-rivative curves. In both 3B and 3C, peak maxima of themain component bands are at similar positions. Sinceboth band narrowing procedures give peak maxima atsimilar positions, we conclude that these peaks are realand are not caused by artifacts due to data processing.44

In both band-narrowed spectra, peak frequencies do notchange on isothermal relaxation; only relative intensitieschange: band intensities decrease for the two bands cen-tered at 1217 and 1208 cm21, and intensity of the bandat 1238 (1239) cm21 increases.

Figure 3D shows the difference curve obtained by sub-tracting the spectrum recorded after 1 min from that after80 min, and Fig. 3E and 3F the Fourier deconvoluteddifference spectrum and the second derivative. Negativepeaks centered at 1216 and 1206 cm21 from disappearingB I have their counterparts in the band-narrowed spectraat nearly identical peak positions (Fig. 3B and 3C). Pos-itive peaks from the formed B II and centered in the band-narrowed difference spectra at 1234 and 1245 (1244)cm21 have their counterparts in Fig. 3B and 3C at similarpositions; namely, at 1238 (1239) and 1253 (1255) cm21.These band positions are not as close as those from thedisappearing B I, but since these bands are from compar-atively weak bands close to an intense composite band,their peak maximum is expected to be shifted. We thusconclude that the features of the difference spectra arecaused by variations in the relative intensities of com-ponent bands and correspond to the difference betweentwo structural states, and not by frequency shifts of com-ponent bands only.44,60

Nonexponential Kinetics of BI ® BII Interconver-sion at 200 K. Figure 4 (left) shows 11 different (in-verted) second derivatives of difference spectra of thenas PO 2

2 spectral region which were obtained from spec-

APPLIED SPECTROSCOPY 13

F IG . 3. (A) Infrared spectra of a quenched nonorientedd(CGCGAATTCGCG)2 dodecamer � lm with G 5 13, showing the spec-tral region of the antisymmetric vibration of the ionic phosphate, re-corded on isothermal annealing at 200 K after 1 min ( ) and after80 min (········); (B) the deconvoluted spectra; and (C ) the second-de-rivative curves. (D ) The difference curve obtained by subtracting thespectrum recorded after 1 min from that after 80 min; (E and F ) thedeconvoluted difference spectrum and the second derivative of the dif-ference spectrum. Deconvolution parameters are fwhh 5 34 cm21 and70% smoothing, and 19-point convolution was applied for second-de-rivative curves. Note in B and C the appearance of band maxima atsimilar positions, and in E and F the occurrence of band maxima andminima at positions similar to those in B and C .

FIG. 4. (Left) Second derivatives of difference spectra, showing thespectral region of the antisymmetric stretching vibration of the ionicphosphate, obtained from spectra of a � lm of quenched hydrated non-oriented d(CGCGAATTCGCG)2 dodecamer, with G 5 13, on isothermalannealing at 200 K. The positive feature centered at 1233 cm21 is fromthe B II substate, and the negative feature at 1214 cm21 from B I. Thespectrum recorded after 1 min had been subtracted from those after nmin (n 5 4, 7, 10, 17, 25, 49, and 63 min, respectively). (Middle) Plotsof the intensities of the positive peak centered at 1233 cm21, and of thenegative peak centered at 1214 cm21, vs. D (annealing time). (The or-dinate is in d 2DA /dn2 3 104 [cm 2] units.) The solid lines reproduce � tsobtained for nonexponential kinetics (b 5 0.58 6 0.04, t 5 961 min).(Right) for comparison the B II /BI (B I /BII ) conformer substate populationratio vs. time calculated from the ‘‘best’’ curve � ts of spectra recordedat 200 K (from Pichler17).

tra recorded at 200 K. The positive feature centered at1233 cm21 from the B II substate increases with increasingannealing time, whereas the negative feature at 1214cm21 from the B I substate decreases. These curves wereobtained from spectra of another DDD � lm, and peakpositions and relative intensities differ slightly from thesecond-derivative difference curve shown in Fig. 3F. InFig. 4 (middle) the height of the features at 1233 and1214 cm21 is plotted against D (annealing time) [D(ta)].Both plots show similar dependence on annealing timein that there is a rapid increase (decrease) for short D(ta)and a leveling off for long D(ta). But, even after 63 min,the longest time used in this experiment, equilibrium hadnot been obtained.

The continuous lines through the data points were cal-culated for nonexponential kinetics via Eq. 1:14,16,65

DI (ta) 5 I (ta) 2 I (0) 5 DI (`)[1 2 exp(2(Dta /ta)b)] (1)

DI (ta) is the difference in ordinate value at annealing timeta, I (ta) and I (0) are the ordinate values at annealing timeta and zero, and DI (`) is the difference in ordinate valueas ta ® `. ta is the characteristic relaxation time for B I

® B II interconversion at 200 K and for G 5 13, and ban empirical parameter with a value between zero and 1,which is independent of ta. DI (ta) is zero at D(ta) 5 zero.The solid lines were obtained for ta 5 9.3 6 1.0 min andb 5 0.58 6 0.04. This analysis requires that the full width

at half-height (fwhh) of the two bands be constant withtime, because evaluation of changes in band areas viatheir second derivatives would otherwise be incorrect.The two � ts of the data points are consistent with ourassumption of constant fwhh.

We emphasize that this data analysis and several othersobtained for other spectral regions15 are consistent withinterconversion of only one structural state into anotherone, and that we saw no spectroscopic evidence for athird structural state involved in isothermal equilibrationvia conformer substate interconversion.

In Fig. 4 (right), we plot for several annealing timesthe B I /B II (B II /BI ) conformer substate population ratiosthat were obtained via curve resolution as elaborated be-low. Both sets of plots show similar dependence on an-nealing times. This is strong support for the reliability ofour estimation of B I /BII population ratios.

Comparison with Conformer Substate Interconver-sion at Ambient Temperatures. In Fig. 5 we comparethe isothermal difference curve with a difference curveobtained from spectra recorded at two different temper-atures.17 Curve A is a typical difference curve obtainedon isothermal CS interconversion at 200 K (25 2 4 min);curve B is the difference curve obtained by subtractingthe spectrum of the same hydrated DDD � lm recorded at290 K from that at 270 K. Curve A is approximately amirror image of curve B. Therefore, on cooling from 290to 270 K, CS interconversion occurs in the opposite di-rection to that on isothermal annealing at 200 K, and B II

converts into B I. This is rather surprising; we will showin a separate report that reversal in the interconversion ofCSs is caused by restructuring of hydration shells, whichrules on rapid quenching the dynamics of conformer sub-states interconversion.

Subtraction of Baseline and Selection of SpectralRegion for Curve Resolution. Fairly artifact-free sub-

14 Volume 55, Number 1, 2001

FIG. 5. Comparison of a difference curve obtained on isothermal an-nealing (A) with that obtained from spectra recorded at two differenttemperatures (B). (A) The difference curve was obtained by subtractingthe spectrum of a quenched DDD � lm (G 5 13) recorded at 200 K after4 min from that recorded after 25 min. (B) The difference curve froma DDD � lm (G 5 14) was obtained by subtracting the spectrum recordedat 290 K from that at 260 K. Note the mirror image between A and B(from Pichler17).

FIG. 6. Subtraction of baseline, demonstrated for the spectrum of aDDD � lm recorded at 290 K (G 5 14, from 1470 to 800 cm21). (A)The experimental spectrum with three baselines: a and b are splinefunctions, with six break points set slightly below the spectrum; c is amultiple point baseline, with six break points set onto the spectrum at1468.9, 1335.8, 1158.3, 913.4, 815.9 and 801.5 cm21. (B) The threebaseline-corrected spectra, after subtraction of baselines a, b , and c. InA the spectral regions investigated for possible curve resolution aremarked by arrows (from Pichler17).

traction of a baseline is necessary before curve resolutionbecomes meaningful. This requirement is hampered bythe fact that, even for spectra recorded on isothermal re-laxation at a given temperature, pronounced changes ofthe apparent background do occur in several spectral re-gions; for example, in the stretching, deformation, andlibrational (below ø 1000 cm21) regions of water. Theseare caused by changes in molar absorptivity of waterbands of the water migrating on CSs interconver-sion.12,16,17

In Fig. 6 we demonstrate for a DDD � lm recorded at290 K the effects of baseline subtraction for three base-lines. Figure 6A shows the experimental spectrum, twobaselines obtained with different spline functions (a andb), and a multiple point baseline obtained with six breakpoints set onto the experimental spectrum (c). Figure 6Bshows the three baseline-corrected spectra, after subtrac-tion of baselines a to c.

The most obvious spectral region for curve resolutionis that from 1315 to 1155 cm21, because subtraction ofdifferent baselines has a minor effect only, and the inten-sity of the composite band pro� le is high. This spectralregion is shown expanded in Fig. 3A, and it contains theantisymmetric stretching vibration of the ionic phosphategroup, nas PO 2

2. However, subsequent curve � tting of thisspectral region turned out to be demanding. The band-

narrowed spectra shown in Fig. 3B and 3C show that atleast 12 component bands have to be included in thecurve � t, and four of these are necessary for the spectralrange from 1240 to 1190 cm21 containing the most in-tense feature. Curve � tting of this particular spectral re-gion turned out to be very problematic because relativeintensities of the strongly overlapping component bandsdiffer strongly.57,66,67

We had also attempted curve resolution of the confor-mation sensitive region containing sugar–phosphatebackbone vibrations, from 800 to 920 cm21. The intentionwas to compare curve � ts of nonoriented DDD with thatof the single crystal reported by Taillandier,33 where theB I-to-B II population is known to be 10 to 1.2,5,6 In ourexperience, however, reliable subtraction of water’s broadlibrational band was not possible. Figure 6B shows that,depending on the selected baseline, band shapes are sig-ni� cantly altered. For this spectral region not only wasthe low intensity of the bands problematic, but also

APPLIED SPECTROSCOPY 15

FIG. 7. Curve resolution approach demonstrated for the spectrum of anonoriented DDD � lm recorded at 290 K (G 5 14, from 1175 to 975cm21). (A) With seven component bands and all parameters varied; (C )with eight component bands, and peak position and fwhh of the twocomponent bands at 1091 and 1085 cm21 kept � xed; (E ) with eightcomponent bands, and peak position and fwhh of the two componentbands at 1083.6 and 1087.3 cm21 kept � xed. (B , D, and F ) The com-parison of second derivatives of the experimental spectrum (solid) withthose of the sum of the curve-� tted component bands of curve � ts A,C, and E (broken) (from Pichler17).

changes in apparent background of spectra recorded as afunction of time or temperature.

The only spectral region amenable to reliable curveresolution turned out to be the region from 1155 to 992cm21. This spectral region contains the symmetric stretch-ing vibration of the ionic phosphate group, ns PO 2

2,which is centered at ø 1092 cm21 in canonical B-DNA.61,64,68 Figure 6A and 6B show that subtraction ofthe three baselines has an in� uence mainly on the low-frequency side of the composite band. We preferred sub-traction of a linear two-point baseline to that of a splinefunction for the following reasons: (1) The molar ab-sorptivity at the two minima at 1155 and 992 cm21 isnearly constant in spectra recorded during an experimentas a function of time or temperature, and their minimumpositions vary at most by 61 cm21. Therefore, nearlyidentical break points can be used for a set of spectra onbaseline subtraction. (2) The error introduced by subtrac-tion of a linear baseline is expected to be small becauseintensity of the composite band is high. This error issmaller on the high-frequency side—where, according toFig. 6B, subtraction of three different baselines givesnearly identical band shapes—than at low frequency.Since analysis of the results of curve � tting concentrateson component bands at high frequency—i.e., the sym-metric stretching vibration of the ionic phosphate groupof B I (at 1087 cm21) and B II (at 1109 cm21)—the errorintroduced by baseline subtraction is expected to be mi-nor. (3) We have also considered subtraction of a splinefunction but, as shown in Fig. 6A for two spline func-tions, there is no unique method for choosing a splinefunction. We thus conclude that, for the spectral regionextending from 1155 to 992 cm21, subtraction of a two-point baseline is meaningful and gives reproducible re-sults.

Gaussian vs. Lorentzian Band Shape and the Effectof the ‘‘Offset Routine’’. We have further investigatedthe effect of the ‘‘offset routine’’ in the curve � t programof the GRAMS/32 software on the band shape parametersof a curve � t, and Ref. 17 can be consulted for the details.Brie� y, the ‘‘offset routine’’ simulates a baseline, and itallows the basis of the curve-� tted component bands tobe below that of the baseline-corrected experimentalspectrum. Curve � tting with pure Lorentzian band shapesproduced a large offset. This band shape is an artifact:when the offset is reduced, the band shape becomes in-creasingly Gaussian. Without offset we obtained Gauss-ian or nearly pure Gaussian band shapes in all curve � tsof B-DNA.12,17 Because of this offset effect we did notuse this routine and subtracted a baseline from the ex-perimental spectrum before attempting curve resolution.

Gaussian band shapes seem to contradict theory, be-cause IR band shapes are Lorentzian according to a sim-ple model in which only pressure broadening is consid-ered. However, in real samples a further line broadeningmechanism exists, and ‘‘the true line shape then is somecombination of Lorentzian and Gaussian shapes’’.69 Ses-hadri and Jones70 argue that on solute–solvent interaction,‘‘one might expect that the bands associated with suchsystems would be more pronouncedly of Gauss form be-cause the number of participating species will be verylarge’’. In our previous curve resolution studies of contact

ion pairing in glassy aqueous solution, the band shapesalso were nearly pure Gaussian.57, 71

How Many Synthetic Component Bands? Figure 7demonstrates our approach for selecting the number andpositions of component bands for the baseline-correctedspectrum of a DDD � lm recorded at 290 K. In the curve� t of Fig. 7A, seven component bands were entered inthe curve � t and all parameters varied. Figure 7B is thecomparison of the second derivative of the experimentalspectrum (solid) with that of the sum of the curve-� ttedcomponent bands (broken). It shows that the shoulder atø 1091 cm21 is not reproduced in the curve � t and, thus,that an eighth component band has to be entered. Figure7C shows the curve � t with eight component bands,where the position and fwhh of an intense componentband at 1085 cm21 and of a small band at 1091 cm21 hadbeen � xed. The comparison of second-derivative curvesin Fig. 7D shows that the shoulder in the experimentalspectrum (solid) becomes the dominant peak in the com-putational spectrum (broken). Figure 7E shows a furthercurve � t with eight component bands, where the positionand fwhh of an intense band at 1087.3 cm21 and of aweak band at 1083.6 cm21 had been � xed. The compar-ison of second-derivative curves in Fig. 7F shows thatnow the peak maximum at 1085 cm21 and the shoulderat ø 1091 cm21 are nicely reproduced by the � t. Thismeans that the weak synthetic band centered at 1083.6cm21 generates the maximum at 1085 cm21 in the second-derivative curve because of its small fwhh, and that the

16 Volume 55, Number 1, 2001

TABLE I. Curve-� tting analysis of the IR spectra (from 1155 to995 cm21) of a d(CGCGAATTCGCG)2 dodecamer � lm, with G 514, recorded at 290 and 260 K.a

Band number nmax (cm21) Heightfwhh

(cm21) Area

1

2

3

290 K260 K290 K260 K290 K260 K

1141.41142.11107.91109.81087.3*1087.3*

0.01410.01290.21330.20890.51380.5681

13.714.130.727.520.2*20.2*

0.2060.1946.9736.119

11.04812.216

4

5

6

290 K260 K290 K260 K290 K260 K

1083.6*1083.6*1068.91069.41052.41052.5

0.02440.04070.30930.29660.38490.4466

6.8*6.8*

18.015.316.216.9

0.1770.2955.9345.1326.6208.035

7

8

290 K260 K290 K260 K

1036.91035.91018.01019.6

0.10410.07320.14090.1567

18.113.422.222.1

2.0011.0473.3273.692

a For Tables I–IV, nmax (in cm 21) are the peak frequencies; fwhh is thefull width at half-height of the curve-� tted component bands; * marksband parameters which were kept � xed during optimization; and allother band parameters were varied.

FIG. 8. Difference spectra as further criterion for goodness-of-� t. Dif-ference curves were generated from two spectra of a DDD � lm recordedat 290 and 280 K (280 2 290 K, G 5 14, from 1175 to 975 cm 21). Theexperimental difference curve ( ) is compared with that obtainedfrom the sums of the curve-� tted component bands (– – –). The curve� tting parameters of the ‘‘best’’ curve � t of the spectrum recorded at290 K were inserted in the curve � t of the 280 K spectrum as follows.Peak position and fwhh of all component bands are (A) � xed and (B)varied. Peak position and fwhh are � xed for bands centered at 1107.9,1087.3, and 1083.6 cm21 (C ); for the band at 1087.3 cm21 (D ); and forthe bands centered at 1087.3 and 1083.6 cm21 (E ). The differencecurves are drawn on the same scale (from Pichler17).

intense synthetic band at 1087.3 cm21 is broader and gen-erates the shoulder at ø 1091 cm21. The peak position andfwhh of the two bands kept � xed in Fig. 7E were deter-mined by varying these parameters in small incrementsuntil optimal correspondence between the second-deriv-ative curves was achieved (according to Fleissner et al.57).The curve-� tting parameters shown in Fig. 7E are listedin Table I (290 K spectrum). These parameters were usedas starting parameters in further curve � ts of spectra re-corded at ambient temperatures. By � xing the band po-sition and fwhh of the two synthetic component bandscentered at 1087.3 and 1083.6 cm21, we inserted a sortof barrier between the left and the right side of the curve-� tted spectral region. We thus prevented additional localminima which otherwise could be caused in the curve� ts by shift of peak position, fwhh, and intensity of thesynthetic component bands.

Difference Spectra as a Further Criterion for Good-ness-of-Fit. We next show how the comparison of dif-ference spectra generated from experimental spectra withthose obtained by curve � tting from synthetic spectra canbe used as a further criterion for the goodness-of-� t. Thecurve-� tting parameters of the optimized curve � t shownin Fig. 7E, which is for a spectrum of nonoriented DDDrecorded at 290 K, were used as starting parameters forthe curve � t of the same � lm but recorded at 280 K. Theexperimental difference curve obtained by subtracting thebaseline-corrected spectrum recorded at 290 K from thatat 280 K (280 2 290 K, solid) is compared with differ-ence curves obtained by ratioing the corresponding sumsof the curve-� tted component bands (280 2 290 K, bro-ken). Figure 8A is for the case where the peak positionand fwhh of the eight component bands were � xed; forFig. 8B all parameters were varied. Correspondence be-tween solid and broken curves increases in going from8A to 8B, but it is not satisfactory at high frequency. ForFig. 8C, 8D, and 8E the peak position and fwhh were� xed for three computational component bands centeredat 1107.9, 1087.3, and 1083.6 cm21 (8C), for the com-

putational band at 1087.3 cm21 (8D), and for the twocomputational bands at 1087.3 and 1083.6 cm21 (8E).The correspondence between experimental and compu-tational difference curves increases in going from Fig.8C to 8D and 8E. Figure 8C clearly shows that � xing ofthe two band parameters of the component band at 1107.9cm21 introduces a large error. Apparently the band param-eters of this component band are altered on cooling from290 to 280 K. Optimal correspondence is achieved in Fig.8E, where even the weak feature at 1071 cm21 is correctlyreproduced. This result indicates that peak position andfwhh of these two component bands change very littlewith temperature. In the following curve � ts we will ap-ply this procedure by � rst optimizing a curve � t, and thenby keeping peak position and fwhh of these two com-ponent bands � xed in subsequent curve � ts of the same� lm recorded either at different temperatures or times.

BI-to-B II Conformer Substate Population Ratios atAmbient Temperatures. Figure 9A shows the experi-mental spectrum of a nonoriented DDD � lm recorded at290 K (solid, G 5 14, two-point baseline corrected), theeight curve-� tted component bands which were neces-sary for optimizing the curve � t, and their sum (broken),which is indistinguishable from the experimental com-posite band. Figure 9B shows the comparison betweenthe second derivative of the experimental spectrum (sol-

APPLIED SPECTROSCOPY 17

FIG. 9. Optimized curve resolution of spectra of the nonoriented DDD � lm recorded at 290 and 260 K (G 5 14, from 1175 to 975 cm21). (A)The experimental spectrum recorded at 290 K ( ), the eight curve-� tted component bands, and their sum (– – –). (B) The comparison of thesecond-derivative curve of the experimental band pro� le ( ) with that of the sum of the curve-� tted component bands (– – –). (C ) Theexperimental spectrum recorded at 260 K ( ), the eight curve-� tted component bands, and their sum (– – –). (D ) The comparison of theexperimental difference curve [( ) C 2 A] with that obtained from the sums of the curve-� tted component bands [(– – –) C 2 A]. (········)Indicates the zero-line (from Pichler17).

id) and the sum of the curve-� tted component bands from9A (broken). Figure 9C shows the experimental spectrumrecorded at 260 K (solid), the eight curve-� tted compo-nent bands, and their sum (broken, almost congruent withthe solid line). Further minor re� nement of this curve � twas achieved by comparison of difference curves: Fig.9D shows the comparison between the experimental dif-ference curve (260 2 290 K, solid) with the differencecurve obtained from the sums of the curve-� tted com-ponent bands (broken, C 2 A). As pointed out above,the band parameters of the curve � t of the spectrum re-corded at 290 K were used as starting parameters for the� t of the spectrum recorded at 260 K, and the band po-sition and fwhh of the two component bands centered at1087.3 and 1083.6 cm21 were kept constant. The bandparameters of both curve � ts are listed in Table I.

The component bands centered at 1087 and 1109 cm21

are assigned to ns of the ionic PO 22 group in B I and B II

(see Ref. 12 for detailed discussion). The componentband centered at 1109 cm21 is much broader than that at1087 cm21 (Table I, 31 vs. 20 cm21), and because of that,it is only a weak feature in the second-derivative curvein comparison to the intense peak centered at 1085 cm21

(Fig. 9B). Enhanced fwhh of the B II band at 1109 cm21

indicates a larger distribution of oscillators for ns of B II

than that of B I. This result could be caused by a broaderdistribution of backbone torsion angles in B II than in B I,which means that the B II conformation is less constrainedthan that of B I. Changes in band areas of these two com-ponent bands centered at 1087 and 1109 cm21 on cooling

from 290 to 260 K are used in our following estimationof the conformer substate population ratio, because thesecurve-� tted component bands are the most reliable ones.

For two interconverting conformers A and B, the bandintensities for each conformer may be given by

IA 5 eA · CA · l and IB 5 eB · CB · l (2)

where IA and IB are the observable integrated intensitiesof the absorption bands of conformers A and B, eA andeB are their molar absorptivites, CA and CB are the con-former populations, and l is the thickness of the absorbing� lm.72,73 For constant l the ratio of intensities, IA /IB, isequal to

I e CA A A5 (3)I e CB B B

The ratio of intensity differences for two conformers Aand B, DIA /DIB, at temperatures T 0 and T 9 is given by

DI I 0 2 I9 (e 0 C 0 2 e9 C9 )lA A A A A A A5 5 (4)DI I 0 2 I9 (e 0 C 0 2 e9 C9 )lB B B B B B B

where e 0A and C 0A are the molar absorptivity and con-centration of conformer A at T 0, e9A and C 9A are the valuesat T 9, and the same notation applies for conformer B.

For a small temperature region, e 0A 5 e9A 5 eA (ande0B 5 e9B 5 eB), and Eq. 4 can be rewritten as

DI e (C 0 2 C9 )lA A A A5 (5)DI e (C 0 2 C9 )lB B B B

18 Volume 55, Number 1, 2001

TABLE II. Curve-� tting analysis of the IR spectra (from 1155 to995 cm21) of a d(CGCGAATTCGCG)2 dodecamer � lm, with G 514, recorded at 280 and 270 K.

Band number nmax (cm21) Heightfwhh

(cm21) Area

2

3

280 K270 K280 K270 K

1108.61109.21087.3*1087.3*

0.21100.20950.53250.5505

29.628.620.2*20.2*

6.6556.368

11.44911.838

Furthermore, since C 9A 1 C 9B (C 0A 1 C 0B) is constant,and C 9A 1 C 9B 5 C 0A 1 C 0B, the above ratio in Eq. 5can be simpli� ed to

DI eA A5 2 (6)DI eB B

and the ratio of the two molar absorptivities can be cal-culated from the intensity shifts at two temperatures.Once the (temperature-independent) ratio eAB 5 eA /eB isknown, the concentration of conformers A and B can beobtained from the ratio of the observed intensities ac-cording to Eq. 3.

This concept is applied in the following to the B I andB II bands centered at 1087 and 1109 cm21, with IBI 5eBI · CBI · l, IBII 5 eBII · CBII · l, and IBI /IBII as the ratio of theband intensities. For the two spectra recorded at 290 and260 K, the B II CS population decreases on cooling andthe BI population increases, but their sum is constantsince l is constant. Thus, the eBI /eBII ratio can be calcu-lated from the ratio of the intensity changes (that is, DIBI /DIBII ). The eBI /eBII ratio determined this way together withthe ratio of band intensities, IBI /IBII, allows one to calcu-late, for constant l, CBI /CBII, which is the ratio of the B I /B II CS population.

The area of the B I band centered at 1087 cm21 increas-es on cooling from 290 to 260 K from 11.048 to 12.216,whereas the area of the B II band at 1109 cm21 decreasesfrom 6.973 to 6.119 (see Table I). Therefore, DIBI /DIBII

(that is, 1.168/0.854) is 1.37. This is equivalent to sayingthat the area of this B I band increases by 10.6%, whereasthe area of the B II band at 1109 cm21 decreases by 7.73%(of the band centered at 1087 cm21, see Table I; this %notation will be used in the following). Accordingly, theratio of their molar absorptivities, e(1087) /e(1109), is 1.37. At290 K the ratio of band areas of the two bands centeredat 1087 and 1109 cm21 is 1.58. Therefore, the B I /B II pop-ulation ratio is calculated as 1.2. In the same manner, for260 K the B I /BII population ratio of 1.5 is calculated fromthe ratio of band areas (Table I, 2.00 to 1), by using thesame e(1087) /e(1109) ratio.

Spectra of the same DDD � lm were also recorded at280 and 270 K, and their curve � ts were optimized inthe same manner described above. Band parameters ofthe two component bands centered at 1087 and 1109cm21 are listed in Table II. These data allowed us to cal-culate changes of band areas and their ratios for � ve ad-ditional temperature steps (in addition to 260–290 K:280–290 K, 270–290 K, 270–280 K, 260–270 K, 260–280 K). The mean value of the ratio of molar absorptiv-ities, e(1087) /e(1109), is 1.37 6 0.09. The small standard de-viation indicates that, between 290 and 260 K, changes

in the e(1087) /e(1109) ratio with temperature can be only mi-nor.

The B I /BII population ratios calculated from the bandarea ratios, and for e(1087) /e(1109), 5 1.37, are 1.2 at 290 K,1.3 at 280 K, 1.4 at 270 K, and 1.5 at 260 K, and thesevalues are estimated to be accurate to 60.1.

We note that different molar absorptivities were alsoobserved in recent studies of the secondary structuretypes in the amide I region of globular proteins.54

BI-to-B II Conformer Substate Population Ratios at200 K. The same � lm of nonoriented DDD that had beenused for recording spectra between 290 and 260 K wasquenched from 290 to ø 170 K into the glassy state at arate of ø 160 K min21, and this way a nonequilibriumpopulation ratio of B I /BII conformer substates was gen-erated. On heating to 200 K, isothermal relaxation to-wards equilibrium in terms of B I ® B II interconversionwas followed by FT-IR spectroscopy.

Figure 10A shows the experimental spectrum of a non-oriented DDD � lm recorded after 4 min at 200 K (solid,G 5 13, two-point baseline corrected), the eight curve-� tted component bands which were necessary for opti-mizing the curve � t, and their sum (broken, almost con-gruent with the solid line). The optimized parameters ofthe curve � t shown in Fig. 9A (Table I) were inserted asstarting parameters. Figure 10B shows the comparisonbetween the second derivative of the experimental spec-trum (solid) and the sum of the curve-� tted componentbands from 10A (broken). Figure 10C shows the exper-imental spectrum recorded after 25 min at 200 K (solid),the eight curve-� tted component bands, and their sum(broken, again congruent with the solid line). Further mi-nor re� nement of this curve � t was achieved by com-parison of difference curves: Fig. 10D shows the com-parison between the experimental difference curve (25 24 min, solid) with the difference curve obtained from thesums of the curve-� tted component bands (broken, C 2A). For the curve � t shown in Fig. 10C, the band positionand fwhh of the two component bands centered at 1087.4and 1084.0 cm21 were kept constant. The band parame-ters of both curve � ts are listed in Table III.

In the same manner changes in band areas of the twocomponent bands centered at 1087 and 1109 cm21 areused in our estimation of the B I /BII population ratio, andof the changes of this ratio on relaxation in terms ofconformer substate interconversion as a function of time.The ratio of band areas of these components is 2.02 (cf.Fig. 10A and Table III). The area of the component bandcentered at 1087 cm21 decreases by 2.62%, whereas thearea of the component band centered at 1109 cm21 in-creases by 3.15% (of the band at 1087 cm21). Therefore,the ratio of their molar absorptivities, e(1087) /e(1109), is0.831. From these values we calculate our estimation ofthe B I /BII population ratio after 4 min at 200 K as 2.4:1,and after 25 min at 200 K as 2.2:1.

Spectra of the same DDD � lm were also recorded at200 K after 10 and 63 min, and their curve � ts wereoptimized in the same manner described above. Band pa-rameters of the two component bands centered at 1087and 1109 cm21 are listed in Table IV. These data allowedus to calculate changes of band areas and their ratios for� ve additional time steps (in addition to 25 2 4 min: 102 4 min, 25 2 10 min, 63 2 4 min, 63 2 25 min, and

APPLIED SPECTROSCOPY 19

FIG. 10. Optimized curve resolution of spectra of the quenched nonoriented DDD � lm recorded at 200 K (G 5 13, from 1175 to 975 cm21). (A)The experimental spectrum at 200 K after 4 min ( ), the eight curve-� tted component bands, and their sum (– – –). (B) The comparison ofthe second-derivative curve of the experimental band pro� le ( ) with that of the sum of the curve-� tted component bands (– – –). (C ) Theexperimental spectrum recorded at 200 K after 25 min ( ), the eight curve-� tted component bands, and their sum (– – –). (D ) The comparisonof the experimental difference curve [( ) C 2 A] with that of the sum of the curve-� tted component bands [( ) C 2 A]. (········) Indicatesthe zero-line (from Ref. 12).

TABLE IV. Curve-� tting analysis of the IR spectra (from 1175 to975 cm21) of a quenched d(CGCGAATTCGCG)2 dodecamer � lm,with G 5 13, recorded at 200 K after 10 min and 63 min.

Band number nmax (cm21) Heightfwhh

(cm21) Area

2

3

10 min63 min10 min63 min

1109.51109.01087.4*1087.4*

0.19940.20220.51980.5042

26.327.119.4*19.4*

5.5745.838

10.74410.423

TABLE III. Curve-� tting analysis of the IR spectra (from 1175 to975 cm21) of a quenched d(CGCGAATTCGCG)2 dodecamer � lm,with G 5 13, recorded at 200 K after 4 min and 25 min.

Band number nmax (cm21) Heightfwhh

(cm21) Area

1

2

3

4 min25 min4 min

25 min4 min

25 min

1144.41144.21109.91109.21087.4*1087.4*

0.01420.01410.19690.20120.52580.5121

10.810.825.726.719.4*19.4*

0.1640.1635.3845.727

10.87010.585

4

5

6

4 min25 min4 min

25 min4 min

25 min

1084.0*1084.0*1069.41069.31052.31052.2

0.05820.04930.27760.27570.44650.4337

8.0*8.0*

16.116.116.416.6

0.4970.4204.7484.7297.7787.676

7

8

4 min25 min4 min

25 min

1035.41035.21020.51020.4

0.05410.05280.15000.1451

10.310.219.920.0

0.5940.5723.1693.092

63 2 10 min). Ratios obtained with data from spectrarecorded after 63 min deviated from those obtained be-tween 4 and 25 min, because changes in band areas be-tween 25 and 63 min are small and instrument instabilitystarts to become increasingly troublesome. Therefore,only band area ratios from spectra recorded after 4, 10,and 25 min were used for estimation of the mean valueof the ratio of molar absorptivities of B II /BI, e(1109) /e(1087),which is 1.22 6 0.27. The B I /BII population ratios cal-culated with this value from the band area ratios are 2.46

after 4 min at 200 K, 2.35 after 10 min, 2.26 after 25min, and 2.18 after 63 min. These ratios are estimated tobe accurate to 60.5. These B I /BII (B II /B I ) population ra-tios were plotted in Fig. 4 (right) vs. annealing time forcomparison with changes of second-derivative differencecurves plotted in Fig. 4 (middle) vs. D (annealing time).The latter values are expected to be much more accurateand, therefore, we consider their close correspondencewith changes of B I /BII (B II /BI ) population ratios with timeas strong support for the reliability of our curve-� ttingprocedure.

At 200 K the ratio of molar absorptivities of the twocomponent bands at 1087 and 1109 cm21, e(1087) /e(1109), is0.82 and therefore smaller than the ratio of 1.37 obtainedfor the temperature range 290–260 K. Changes in molarabsorptivities with temperature have often been observed,and the assumption of temperature independence of mo-lar absorptivity ratios for evaluating changes in conform-er population ratios is known to be a simpli� cation.74

The standard deviation of the e(1087) /e(1109) ratio is much

20 Volume 55, Number 1, 2001

FIG. 11. Analysis of the difference curve obtained from curve � ts ofspectra recorded at 200 K (see Fig. 10D and Table III). (A) The sumof two curve-� tted component bands, numbered 2 and 3 and centeredat 1109.9 (1109.2) and 1087.4 cm21 after 4 min ( ) and 25 min(– – –); (B) their difference (25 2 4 min). (C ) The sum of two curve-� tted component bands, numbered 5 and 6 and centered at 1069.4(1069.3) and 1052.3 (1052.2) cm 21 after 4 min ( ) and 25 min(– – –); (D ) their difference (25 2 4 min).

less for the 290–260 K temperature range than for 200K. We attribute this observation to the much smallerchanges in B I /BII band area ratios on isothermal relaxa-tion at 200 K, rather than on cooling from 290 to 260 K.

‘‘Understanding’’ the Difference Spectra. The dif-ference spectra shown in Figs. 2, 5, and 8–10 are notsimply a series of bipolar features. More complicated pat-terns emerge from changes in the peak position and fwhhof component bands on the B I ® B II transition, even onisothermal relaxation, and/or in ratios of molar absorptiv-ities. The analysis by curve resolution allows us to tracethese effects, and two examples are given in the follow-ing.

Figure 10D shows a characteristic bipolar feature ofthe difference curves containing ns of B I and B II, with amaximum at 1101 cm21 and a minimum at 1084 cm21.The minimum value is close to the peak value of theintense computational component band centered at 1084cm21, but the maximum value of 1101 cm21 is shifted by9 cm21 from the peak maximum value of the componentband centered at 1110 cm21 (Table III). A further weakminimum is observed at 1118 cm21 and a weak maximumat 1131 cm21. These features can be generated by ratioingof the two computational component bands numbered 2and 3 in Table III. Figure 11A shows the sum of the twocurve-� tted component bands centered at 1109.9 (1109.2)and 1087.4 cm21 after 4 and 25 min (from Table III).Their difference (25 2 4 min) shown in Fig. 11B containsall the features observable in Fig. 10D above 1075 cm21.This means that, on isothermal annealing at 200 K, thechange in the peak position of component band 2 from1109.9 to 1109.2 cm21 and of the fwhh from 25.7 to 26.7cm21 generates this complicated difference spectrum.

A further strange feature of the difference curve of Fig.10D is that the intense component bands centered at 1069and 1052 cm21 contribute only to the negative part of thedifference curve. These two bands are observable as dis-tinct, intense features in the second-derivative curve ofthe experimental band pro� le (Fig. 10B) and of the Fou-rier deconvoluted spectrum (not shown), and they can beassigned to C–O stretching modes coupled strongly toother normal modes.64,75,76 Figure 11C shows the sum ofthe two curve-� tted component bands after 4 and 25 min,numbered 5 and 6 in Table III and centered at 1069.4(1069.3) and 1052.3 (1052.2) cm21. Their difference (252 4 min) depicted in Fig. 11D contains most of the fea-tures observable in the difference spectrum of Fig. 10Dbelow 1080 cm21. Since the band area of both componentbands decreases on isothermal relaxation at 200 K in go-ing from 4 to 25 min (Table III), these two componentbands cannot be assigned simply to B I and B II involvedin that B I ® B II transition. The decrease in the band areaof both component bands becomes understandable by as-suming that both B I and B II have intense bands at nearlyidentical peak positions, at ø 1069 and ø 1052 cm21, andthat the decreasing band areas re� ect changes in the ratiosof molar absorptivities of the B I and B II band, eBI /eBII, andthat this ratio is .1 for both bands. This considerationthen leads to negative features only in the differencecurves.

We have further tried to separate the IR spectral fea-tures of the B I and B II conformer substate by the ‘‘ratiomethod’’,77–79 in the same manner described for chloro-

cyclohexane’s axial and equatorial conformer IR spec-trum.18 The scaling factors necessary for separation areobtainable in principle from changes in band areas of thecurve-� tted component bands on isothermal relaxation at200 K. In the case of chlorocyclohexane, spectral artifactswere observed for the minor conformer component, andthe origin of these artifacts is discussed in Ref. 18. Inparticular, intermolecular interactions can lead to alteredband shapes as the composition of a mixture is changed,80

and a � rst-derivative feature can occur in a differencespectrum when a band slightly shifts its peak position.58

The latter situation can arise when a weak band of onecomponent is superposed by an intense band of the sec-ond component, and its band shape is altered by changesin intermolecular interactions. This situation was encoun-tered in this study for the minority component B II, andchanges in the peak position and fwhh of its componentbands, even on isothermal relaxation, shown in Table III,prevented meaningful artifact-free separation into the dis-tinct spectra of B II and B I.

Enhanced B II Population is Consistent with Com-puter Simulation. In the single crystal of DDD, B II oc-curs at the G10-C11 and G22-C23 positions,2,5,6 and thus,

APPLIED SPECTROSCOPY 21

the B I-to-B II population ratio is 10 to 1. Our estimationof enhanced B I /BII population ratios in nonoriented hy-drated DDD � lms by IR spectroscopy12,17 is consistentwith estimations obtained from MD simulations ofDDD.36,39,81 For a DDD system with G 5 158, up to 6base steps show B I ® B II substate dynamics,36,39 whereasfor a DDD system with G 5 13, this number increasesup to 16 base steps involved in B I ® B II dynamics.81 Forthe latter system the time average of the B I /BII populationratio is 1.75, which is close to our estimation of B I /BII

population ratios of between 1.2 and 1.5 obtained bycurve resolution of IR spectra recorded between 290 and260 K, with G 5 14.

B II from Raman Spectroscopic Studies? Peticolasand colleagues32 have reported that Raman spectra ofDDD, recorded ‘‘in single crystals and in aqueous solu-tion, reveal signi� cant differences in the helical confor-mation exhibited in each of these states’’. They concludethat, ‘‘the structural differences are presumably due to therelaxation of solvent and packing constraints imposed bythe crystalline environment and modulated by the se-quence of the dodecamer’’.32 We surmise that some ofthese differences could be caused by an enhanced B II

population in aqueous solution in comparison to that inthe single crystal. However, the aqueous DDD solutionstudied in the Raman spectroscopic work was much moredilute30–32 than the hydrated nonoriented DDD � lms usedin our IR spectroscopic studies, and thus the B I /BII pop-ulation ratio in dilute solution is not known. Raman spec-troscopic studies of hydrated � lms and the comparisonwith the spectrum of single crystals could clarify whetherthe differences are caused by enhanced B II population.

CONCLUSION

Reliable curve resolution and estimation of the B I /BII

conformer substate population ratios is possible for theIR spectral region containing the symmetric stretchingvibration of the ionic phosphate group. In the d(CGCG-AATTCGCG)2 dodecamer, the B II conformer populationis much higher in nonoriented hydrated � lms than in thesingle crystal. Thus crystal packing effects apparently cansuppress the B II substate conformation. The same curveresolution approach outlined in this study can be appliedto native highly polymeric forms of B-DNA. This ap-proach can reveal for the � rst time their B I /B II populationratios and the contribution of B II in canonical B-DNA.Hydration is similar to that in chromatin; therefore, a sig-ni� cant B II population is expected in vivo. This consid-eration can be of biological consequence in the proteinrecognition process.

ACKNOWLEDGMENT

We are grateful for � nancial support by the ‘‘Forschungsforderungs-fonds’’ of Austria (project No. P12319-PHY).

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