J. Phys. Chem. 1985,89, 583-588
583
show that the T I gets shorter in going from 23 OC (Figure 20a) been used to describe side-chain motions, to understand backbone motions, to unravel the effects of stereochemistry, and to examine to 96 O C (Figure 20b). This result indicates that the phenyl ring motions are on the slow correlation time side of the T I minimum the effects of mechanical deformation in the solid state. Much (see Figure 2). In addition, the data at 96 O C show evidence of of this progress can be attributed to the development of physical several components that invert at different times. models to describe N M R relaxation. If the only motion of the phenyl rings were pure 180' ring flips, The advent of high-resolution solid-state N M R spectroscopy the correlation times determined from the line shape. analyses (see and its availability on a routine basis have also done much to above) and from the T l should be identical. Analysis of the T I increase the current level of understanding of polymer dynamics. data according to the two-site hop model of Torchia and S ~ a b o ~ ~In particular, definitive work has been done to establish the reprovides a correlation time of 1.6 X lo-' s at 70 O C , approximately lationships between polymer structure and polymer dynamics in 1 order of magnitude shorter than that obtained from the line the solid state. It is likely that many future advances in the field will be made shapes. This result, in conjunction with the multicomponent nature on the basis of solid-state deuterium N M R experiments. This of the T 1 data, is taken as evidence for heterogeneity in the environments of the amorphous phenyl rings. In addition, the technique provides exceptionally detailed information about both discrepancy in correlation times suggests that the phenyl motions the frequency and the angular range of molecular motions and are best described by low-angle, high-frequency librations at the furthermore provides ready assessment of motional heterogeneity. bottom of the conformational potential energy well, in addition In all of these approaches it is necessary to couple theory with to the 180' ring flips. experiment. There is still a need for more complete theory, particularly for describing motional heterogeneity in polymers. IV. Summary Taken as a whole, all of the results described in this review underlie During the past decade major advances have been made in the conclusion that synthetic polymers, whether in solution or in understanding the dynamics of polymer chains, both in solution the solid state, exhibit a considerable degree of motional heteand in the solid state. The results from N M R experiments have rogeneity. Adequate methods for measuring this heterogeneity and physically insightful models for describing it are the challenge (63) Torchia, D. A.; Szabo, A. J . Mugn. Reson. 1982, 49, 107. for the near future.
ARTICLES ''P Magnetization-Transfer NMR Studies of the Interchange between Structures of Self-Assembled Disodium Guanosine 5'-Monophosphate In Solution Jens J. Led* and Henrik Gesmar Department of Chemical Physics, University of Copenhagen, The H.C. 0rsted Institute, 5, Universitetsparken, DK-2100 Copenhagen 0 , Denmark (Received: July 2, 1984)
Mutual exchange between the ordered forms of disodium guanosine 5'-monophosphate in aqueous media at pD 7.5 and 1 NMR spectroscopy using the magnetization-transfer technique. At 1 O C four 31PNMR signals OC has been studied by (a,0, y, 6) are observed. On the basis of their relative intensities and their appearances as function of temperature and by comparison with previous IH studies, these signals are assigned to four nonequivalent positions in the ordered structures, one of the signals (y) receiving a further contribution from disordered 5'-GMP. By applying the magnetization-transfer technique, exchange is found to take place only between the fl and y sites ( k , = 5.7 0.5 s-'), whereas exchange between any other pair of sites is considerably smaller or negligible (10.20h 0.08 s-I). On the basis of these results, the fl signal and part of the y signal are assigned to two previously suggested isomeric octamers (Bouhoutsos-Brown, E.; Marshall, C. L.; Pinnavaia, T. J. J. Am. Chem. Soc. 1982,104,6576-6584) with D4symmetry, consisting of two coaxially reversed stacked tetramers in a head-to-head and tail-to-tail arrangement, respectively, while the interchange between the two signals is attributed to a relative twisting of the two tetramers about the D4axis. The nonexchanging a and S 31Psignals are assigned to different environments in two diastereomericoctamers with C4symmetry formed by coaxial stacking of tetramer plates in a head-to-tail arrangement.
Introduction Guanosine 5'-monophosphate dianions (5'-GMP) are unique among the nucleic acids in their ability to form regular, ordered structures in aqueous solution at neutral or slightly basic pH, when Na+, K', or Rb' are counter ion^.^-^ Numerous studies1-" con(1) Miles, H. T.; Frazier, J. Biochem. Biophys. Res. Commun. 1972, 49, 199-204.
0022-3654/85/2089-0583$01.50/0
cerning the nature of these structures have been made using IR,I Raman,637and several types of N M R spectroscopy (1H,2-4v8-1 (2) Pinnavaia, T. J.; Miles, H. T.; Becker, E. D. J. Am. Chem. SOC.1975, 97, 7198-7200. (3) Pinnavaia, T. J.; Marshall, C. L.;Mettler, C. M.; Fisk, C. L.; Miles, H. T.; Becker, E. D. J. Am. Chem. SOC.1978, 100, 3625-3627. (4) Borzo, M.; Detellier, C.; Laszlo, P.; Paris, A. J. Am. Chem. Soc. 1980, 102, 1124-1 134.
0 1985 American Chemical Society
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The Journal of Physical Chemistry, Vol. 89, No. 4, 1985
23Na,513C,839and 31P438). In particular N M R spectroscopy has provided detailed information, one of the reasons being that the individual aggregates are sufficiently stable on an N M R time scale to produce separate signals. Different models for the ordered species have been proposed, including structures with tetramers as their basic unit'-5 and a structure consisting of stacked d i m e s 8 Recent 13C N M R relaxation studies9 of the size of the ordered aggregates as well as IH N M R studies of their stoichiometry9J0 and the number of exchangeable H-bonded protons" per ordered nucleotide unit favor isomeric octamers formed by stacking of tetramers, and stabilized by alkali ion chelations. The present study provides further support of the isomeric octamers in the case of Na2(5'-GMP), through an investigation of the mutual exchange between the individual ordered species of this nucleotide, using high-field 31PN M R spectroscopy and the magnetization-transfer (MT) technique previously developed.I2 This technique allows a detailed unraveling of complicated exchange and relaxation schemesl2-I4 if cross-relaxation can be neglected.12 As shown here, this condition holds for the Na2(5'-GMP) system when 31P is the observed nuclei.
Experimental Section Preparation of Solutions. Na2(5'-GMP) (Sigma), twice lyophilized from 99.8% D 2 0 (Norsk Hydro), was used without further purification. Solutions were prepared by dissolving the appropriate amount of the salt in 99.8% D 2 0 and adjusting the pD to 7.5 (pD = meter reading + 0.415)with diminutive amounts of 12 M hydrochloric acid. EDTA (1 mg/ 1 mL of solution) was added to bind possible paramagnetic impurities. The 5'-GMP concentrations were determined spectrophotometrically. ''P NMR Measurements. 31PN M R spectra were obtained at 109.3 MHz on a Bruker H X 270 spectrometer. Broad-band proton noise decoupling was performed continuously with the center of the decoupling frequency band close to the resonance of the H(5') protons of the ribose ring. This allowed a decoupling power of only 0.6 W to be applied, thereby avoiding any appreciable heating of the samples due to decoupling.I6 Temperatures were measured by using an alcohol thermometer coaxially mounted in an N M R tube containing the applied solution. In all cases a 3-kHz spectral range defined by 8192 data points was employed. The chemical shifts were measured in parts per million relative to 85% H3PO4 as external reference. In the magnetization-transfer experiments the selective inversion of the individual signals was accomplished by means of a DANTE pulse sequence" consisting of 36 consecutive pulses, each 2.3-ps long and separated by 0.68-0.92 ms. Either the center band or the first sideband generated by the pulse sequence was used for excitation. The 90' nonselective analyzing pulse was 36 ~ s . Data Analysis. The model used in the analysis of the magnetization-transfer data is basically the McConnellI8 and For~&n-Hoffman'~ equations for chemical exchange, modified as (5) Detellier, C.; Laszlo, P. J . Am. Chem. SOC.1980, 102, 1135-1141. (6) Faurskov Nielsen, 0.; Lund, P.-A.; Petersen, S.B. J.Rumun Spectrosc. 1981, 1 1 , 493-495. (7) Faurskov Nielsen, 0.;Lund, P.-A,; Petersen S. B. J. Am. Chem. SOC. 1982, 104, 1991-1995. ( 8 ) Petersen, S.B.; Led, J. J.; Johnston, E. R.; Grant, D. M. J . Am. Chem. SOC.1982, 104, 5007-5015. (9) Fisk, C. L.; Becker, E. D.; Miles, H. T.; Pinnavaia, T. J. J. Am. Chem. SOC.1982, 104, 3307-3314. (10) Bouhoutsos-Brown, E.; Marshall, C. L.; Pinnavaia, T. J. J. Am. Chem. SOC.1982, 104, 6576-6584. (1 1) Walmsley, J. A.; Barr, R. G.; Bouhoutsos-Brown, E.; Pinnavaia, T. J. J . Phys. Chem. 1984, 88, 2599-2605. (12) Led,J. J.; Gesmar, H. J . Magn. Reson. 1982, 49, 444-463. (13) Led, J. J.; Neesgaard, E.; Johansen, J. T. FEBS Lerf. 1982, 147, 74-80. (14) Hvidt, Aa.; Gesmar, H.; Led, J. J. Acta Chem. Scand., Ser. B 1983, 837, 227-234. (15) Glasoe, P. K.; Long, F. A. J . Phys. Chem. 1960, 64, 188-190. (16) Led, J. J.; Petersen, S. B. J . Muan. Reson. 1978, 32, 1-17. (17) Bodenhausen, G.; Freeman, R.; Morris, G. A. J . Magn. Reson. 1976, 23, 171-175. Morris, G. A.; Freeman, R. J . Magn. Reson. 2978, 29,433-462. (18) McConnell, H. M. J . Chem. Phys. 1958, 28, 430-431
Led and Gesmar
,-------dL
2
h 11
60
50
LC
30
20
9
10 ppm
Figure 1. Temperature variation of the 109.3-MHz31PNMR spectrum of a 0.49 M solution of Na2(5'-GMP) in D 2 0 at pD 7.5 (25 "C). The shifts are relative to 85% H3P04as external standard.
previously describedI2and extended to the general multisite (n-site) case. The exchange and relaxation rates were obtained by a simultaneous nonlinear least-squares analysis of the n X n signal recoveries measured after the selective inversion of each one of the signals corresponding to the n sites among which exchange may occur. Also the signal recoveries obtained in a normal nonselective inversion recovery experiment were included in the analysis, which was based on peak heights as previously described.12 The relative concentrations of the individual species were obtained from the signal intensities of the equilibrium spectra, evaluated by a nonlinear least-squares Lorentzian curve fitting.
Results and Discussion Temperature Studies. Figure 1 shows the 31P N M R spectrum of a 0.49 M solution of Na2(5'-GMP) at pD 7.5, obtained at different temperatures. At 29 OC the spectrum is dominated by a single narrow line at 4.0 ppm which is assigned to disordered 5'-GMP, in rapid chemical exchange with minor amounts of tetramers and aggregates of this species (see below). As the temperature is lowered, however, three major changes occur. First, two new peaks ( a and 6) of equal intensities appear, one on each side of the original resonance. While the intensities of both of these peaks increase with decreasing temperature at the expense of the original signal, their intensities relative to one another, as well as their mutual separation of 2.35 ppm, remain unchanged over the entire experimental temperature range. Similar observations have been made by Laszlo et al.4 On the basis of these results, together with line-width measurements of the 23NaNMR signals, and a joint consideration of the 31Pand H(8) proton spectra, these authors suggested that the new 31P resonances correspond to 31Pnuclei belonging to the same chemical species as the lowest field ( a ) and the highest field (6) H(8) protons, respectively. Here, additional support of this suggestion is provided by the observation that the appearance of the a and 6 31Psignals is concomitant with the appearance of the a and 6 H(8) signals in the corresponding proton spectrum (Figure 2). Furthermore, the normalized intensities of the two pairs of signals, as evaluated by the nonlinear least-squares Lorentzian curve fitting, are equal at the same temperature, within the experimental uncertainty. Thus, (19) Forscrl, S.; Hoffman, R. A. J . Chem. Phys. 1963, 39, 2892-2901.
Magnetization-Transfer N M R Studies of Naz(5'-GMP)
The Journal of Physical Chemistry, Vol. 89, No. 4, 1985 585
I CL 1*30"1
111 DLICW, CCW, *30"1
I1 CLi-30")
IV D'ICW, CCW. +60"1
I
75
70
65
60
55ppm
Figure 2. Temperature variation of the 270-MHz 'H NMR spectrum of Na2(5'-GMP) obtained from the same sample as the slP spectra in Figure 1. The shifts are relative to Me4Si as external standard.
at 1 "C the normalized intensity of the a and 6 jlP signals together is 0.48 f 0.04 while that of the two outer H(8) signals amounts to 0.46 f 0.04. It seems therefore incontestable that the a and 6 31Psignals (at 1.62 and 3.98 ppm at 1 "C,respectively) belong to the same regular, ordered structures as the a and 6 H(8) resonances in the proton spectrum. In terms of the octamer models, recently proposed by Pinnavaia et al.,IOthese structures are two isomeric octamers with C, symmetry (structures I and I1 in Figure 3) consisting of two coaxially stacked tetramers with the same H-bonding directionality (head-to-tail arrangementz0) and stabilized by Na+ chelations. Further, the two tetramers are twisted either +30° or -30' relative to each other. The latter two arrangements correspond to two diastereoisomers with the D-ribophosphate bound either to a left- or right-handed form of the chiral core of stacked guanine tetramers. A second major change in the spectra in Figure 1 is the substantial shift of the original y jlP signal toward higher field as the temperature is lowered. This is in contrast to the previously reported 31Pspectra4 where a small (-0.15 ppm) and apparently temperature-independent difference between the shifts of the a and y 31P signals was observed. This inconsistency might well be due to a small, but important, difference in pD in the two cases. Thus a pD of about 8.3, obtained when dissolving about 0.5 M Na2(5'-GMP) in DzO, may apply in ref 4 where no adjustment or measurement of the pD of the sample used in the 31Pexperiments is reported. Whereas this pD is well above the highest pK value of the phosphate group in 5'-GMP (6.lZ1 0.415at 25 "C), the pD value of 7.5 used here is sufficiently close to this pK value to allow a significant increase of the ratio of monoprotonated to nonprotonated species when the temperature is lowered and may thereby give rise to a change in the 3iPchemical shift toward higher field.zz The third important feature in Figure 1 is the appearance of a fourth 31Psignal (6) close to the y resonance at the lowest temperatures. N o report was made of this signal in previous 31P
+
(20) Here the same definition of the stacking arrangements as given by Pinnavaia et al. (ref 9 footnote 37) was adopted; that is, "head" and ''tail" of 5'-GMP are defined as follows: the side of the base facing up from the page when 5'-GMP is drawn with the five-membered ring to the right is called the head; the side facing down is called the tail. (21) 'Handbook of Biochemistry", 2nd ed.; Sober,H. A,, Ed.; CRC Press: Cleveland, OH, 1973; p 5210. (22).Gardian, D. G.; Radda, G . K.; Richards, R. E.; Seeley, P. J. In 'Biological Applications of Magnetic Resonance"; Shulman, R. G., Ed.; Academic Press: New York, 1979; pp 463-535.
V D'ICCW. CW, -60") VI D'(CCW. CW, -3O"I Figure 3. The six possible diastereomeric octamers of 5'-GMP proposed by Pinnavaia et a1.I0 In the C4isomers the two tetramers are stacked in a normal head-to-tail arrangement with the same H-bonding directionality for the two tetramers while in the D4isomers the stacking is inverted (tail-to-tail or head-to-head);that is, the two tetramers have opposite H-bonding directionality. The heavier lines represent the upper tetramer unit, the lighter lines the lower tetramers. R represents the chiral ribophosphate group. The CW and CCW notation for the D4isomers designate the clockwise or counterclockwise sense of the hydrogen bonding for the upper and lower tetramer units. The twist angles are f30° or &60° as indicated.
N M R s t ~ d i e s . ~ JAgain, l most likely this is due to a higher pD value in ref 4 and 11, and the appearance of the /3 signal in the present study is a result of the temperaturedependent shift of the y signal caused by the lower pD value, as discussed above. As to the assignment of the 6 signal, it must be attributed to a third ordered structure of Na2(5'-GMP). Furhtermore, it seems reasonable to assign it to the same structure as the c signal of the proton spectrum (Figure 2) since the normalized intensities of both signals at 1 'C, as evaluated by a nonlinear least-squares Lorentzian curve fitting, are almost identical (0.16 0.05 and 0.1 1 f 0.01, respectively). A similar agreement between the normalized intensities of the y 31Psignal and the y p signals of the 'H spectrum (0.37 f 0.05 vs. 0.42 f 0.01) indicates that the former, in addition to the 31Psignal of disordered 5'-GMP, contains the 31Psignal of a fourth ordered species identical with the one giving rise to the H(8) signal. According to Pinnavaia et a1.I0these two new ordered structures should be identified as a second and a third pair of isomeric octamers (D4symmetry) consisting of two reversed-stacked tetramers (opposite H-bonding directionality) in a head-to-head and a tail-to-tail arrangement, respectively, and twisted either +30° or +60°, and -30" or -60" relative to one another (structures 111-VI in Figure 3). The attachment of the D-ribophosphate to each of these structures results in four diastereomeric species. Finally, the fact that the p 31Psignal coalesces and moves with the y signal at the higher temperatures (>8 "C) suggests that it belongs to an ordered structure which is in relatively fast chemical exchange with the species giving rise to the y signal and, therefore, less stable than those associated with the a and 6 signals. This suggestion is amply confirmed by the following magnetization-transfer studies.
*
+
Led and Gesmar
586 The Journal of Physical Chemistry, Vol. 89, No. 4, 1985
z s 15
TABLE I: Exchange Rates,' kXy,in 8,at 1 "C for 0.49 M Na,(S'-GMP) at DD 7.Sb exchange from x exchange to v C Y l3 Y 0.21 f 0.10 B Y -0.2' f 0.1 5.1 f 0.5 6 0.002 f 0.010 0.015' f 0.16 -0.003' f 0.08 a Including 1u standard deviations. Signal assignments are as given in Figure 1. 'The negative signs (key and kt6) and large uncertainties are due to high correlations (see text).
TABLE II: 31PRelaxation Rates," in 8, at 1 "C for 0.49 M NaZ(S'-GMP) at pD 7.Sb R.. Rtl R, RA selective 0.16 f 0.03 0.66c k 0.02 0.10 f 0.03 nonselective 0.13 f 0.01 0.25 f 0.01 0.24 f 0.01 0.10 f 0.01 ~~
50
LO
30
20
10
ppm
Figure 4. magnetization-transferNMR spectra of a 0.49 M solution of Naz(5'-GMP) in DzO at pD 7 . 5 and 1 "C. Only one-third of the spectra monitoring the time dependence of the signal intensities after a selective inversion of the signal are shown. The shifts are relative to 85%
HsP04as external standard.
Magnetization-Transfer Studies. Further insight into the ordered structures of Na2(5'-GMP) and their mutual relations was achieved through an unraveling of the pattern of exchanges between the individual species. This, in turn, was accomplished by complementary sets of M T experiments.I2 Figure 4 illustrates the essence of these experiments while plots of the measured signal intensities and their time dependencies are shown in Figure 5 together with the best fit resulting from a simultaneous nonlinear least-squares analysis of the total amount of data. Besides the data of the four M T experiments, the signal intensities measured in a nonselective inversion recovery experiment were included in the analysis. The excellent agreement between these data and those of the M T experiment emphasizes the reliability of the procedureI2 and the quality of the data. The variation of the signal intensities in the M T experiments depends not only on the exchange and relaxation rates of 31Pnuclei in the different chemical environments but also on cross-relaxation ~ only caused by a direct 31P-31Pdipolar i n t e r a c t i ~ n . ~However, if this interaction can compete with other relaxation paths for the 31Pnuclei will the cross-relaxation affect the data and the analysis. For the present system this is not the case. Here the relaxation of the 31Pnuclei24is controlled by chemical shift anisotropy and by their dipolar interactions with the ribose protons. Even the relaxation caused by the interaction with the two H(5') protons dominates over the relaxation due to the 31P-31Pinteraction. Thus, the shortest interphosphorus distance, which is found in the Na+ stabilized octamers, should be 4.8 8, or less for Na+ chelation to be feasible,I0 while the average 31P-H(5') distance is about 2.9 A.25 Moreover, the gyromagnetic ratio of IH is 2.5 times that of 31P.Therefore, even if the 31P-31Pdistance should be as short as -3 8, the difference in gyromagnetic ratios of 31Pand 'H assures that the 31P-31Prelaxation path is negligible compared with the 31P-H(5') path, assuming similar correlation functions for the two interactions. Experimentally this is supported by the observation that for the CY and 6 resonances, which are assigned to the 31Pnuclei of the Na+ chelated C4 octamers, the selective and nonselective relaxation rates, respectively, are identical within the experimental uncertainties (see below and Table 11), a fact only compatible with ~ ~the case of the an insignificant 31P-31Pdipolar i n t e r a ~ t i o n .In nonchelated tetramers and the disordered 5'-GMP, cross-relaxation (23) Noggle, J. H.; Schirmer, R. E. 'The Nuclear Overhauser Effect"; Academic Press: New York, 1971; Chapter 2. (24) James, T. L. Bull. Mugn. Reson. 1983, 4 (3-4), 119-159. (25) Katti, S. K.; Seshadri, T. P.; Viswamitra, M. A. Acta Crystallogr., Secr. B 1981, 837, 1825-1831.
~
a Including 1u standard deviations. Signal assignments are as given in Figure 1. CThe value of the combination 0.25R8 + 0.97R, (see text).
seems even less likely due to the electrostatic repulsion of the negatively charged phosphate groups. Consequently, the 31PM T experiments provide exchange rates that are unaffected by cross-relaxation, The exchange rates, resulting from the least-squares analysis of the experimental data, are given in Table I. The selective relaxation rates, also obtained in this analysis, are given in Table I1 together with a set of relaxation rates derived from a separate analysis of the nonselective relaxation data (last row in Figure 5). As seen from Table I a significant exchange occurs between the sites corresponding to the fl and y signals (k,, = 5.7 f 0.5 s-]), whereas the rest of the exchange rates are too small to be determined ( 5 0 . 2 f 0.1). The high relative uncertainties of k,,, k,, and k, and the negative signs of k,, and kr6 are due to strong correlations, and only combinations of these rates can be determined with higher accuracy. Thus, the combinations that can be determined with the highest accuracy are 0.76k,, + 0.65ka, and 0.25k0, 0.97kY6,which amount to 0.017 f 0.006 s-I and 0.004 f 0.003 s-l, respectively. Among the selective relaxation rates, R , and R6 are well determined whereas R , and R , are indeterminable, and only the combination 0.25R, 0.97R7 can be obtained. As previously described in detailI2 this indeterminable character stems from the fact that k,, >> R , R,, which only allows a precise determination of kD7. For R , and R6a similar prohibitive condition does not apply. As for the nonselective relaxation rates, excellent agreements with the selective rates are obtained in the case of R , and R , for reasons discussed above, whereas, again, for R , and R , only an average value results, because of the relatively fast exchange between the corresponding sites. The value of 5.7 f 0.5 S-I obtained for k,, shows that a significant direct exchange takes place between the structured species associated with the fl 31Psignal and one or both of the species (one structured and one disordered) giving rise to the y signal. While an exchange with the disordered species implies a separation of the two tetramers of an octamer, an exchange between the two structured species is compatible with the relative twisting of 30' of the two tetramers in the D4 isomers, which interconverts structures I11 and IV, or V and VI (see Figure 3), and, thereby, exchanges the phosphate groups between two different chemical environments. Formally the four D4isomers should give rise to four different signals,I0 one from each of the four diastereomeric D4 species in Figure 3. However, the fact that only two interchanging signals are observed shows that the chemical shifts of the 31Pnuclei are unaffected by whether the D-ribophosphate is bound to a tail-to-tail (structure I11 and IV) or a head-to-head (structure V and VI) stacking arrangement of the guanine tetramers. In contrast to the 0 and y signals, the a! and 6 31Presonances, assigned to the C, isomers, are not connected by direct exchange (ka6 = 0.002 f 0.010 s-l). In fact, no measurable exchange
+
+
+
31PMagnetization-Transfer N M R Studies of Na2(5'-GMP)
The Journal of Physical Chemistry, Vol. 89, No. 4, 1985 581
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