Proc. Natl. Acad. Sci. USA Vol. 74, No. 5, pp. 1785-1788, May 1977
Chemistry
Circular dichroism of adenosine dinucleotides (optical activity/reciprocal relations/transition moment coupling)
J. W. PETTEGREWt, D. W. MILESt, AND H. EYRINGt t Department of Neurology and Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110; and t Department of Chemistry, University of
Utah, Salt Lake City, Utah 84112
Contributed by H. Eyring, February 7, 1977
ABSTRACT The circular dichroism and absorption spectra of (3'-5')ApA, (5'-5')AppA, and (5'-5')ApcpA [diadenosine(5',5') pyrophosphonatel under various ionic conditions and at various temperatures are reported. Temperature studies reveal that under the same ionic strength and solvation conditions the probability of base-base interaction is ApA > AppA > ApcpA. It is found that increasing the molar refractivity of the solvent decreases the base-base interaction for both ApA and AppA and that both compounds proceed to a monomeric state in solvents of high molar refractivity. Solvent effect studies also indicate that a major percentage of the driving force for the base-base interaction can be accounted for by London dispersion forces. Low ionic strength appears to favor the base-base interacted conformation. All compounds tend toward the same noninteracted form at high temperatures. Plots of [O]X against X (where X is an experimental parameter) were constructed in order to obtain information concerning which transitions might be undergoing oscillator coupling. To a first approximation, transitions coupled to each other should have very similar A[OJx/AX for the experimental range of X. Applying this technique to the experimental data yielded information concerning possible transition coupling.
In order to understand the function of chromatin (1, 2), a thorough understanding of its structure and the forces involved in maintaining that structure is needed. Of the experimental spectroscopic methods available to investigate this problem, circular dichroism remains one of the most widely used (3-26), although its usefulness here in actually defining structural details is yet to be fully realized. One of the main obstacles is the lack of reliable information about the optical properties of the nucleic acid bases and their dinucleotide derivatives, although considerable progress has been achieved (26-40). This paper contains a thorough study of the circular dichroism of the dinucleotide dimers (3'-5')ApA, diadenosine(5',5') pyrophosphate (AppA), and diadenosine(5',5') pyrophosphonate (ApcpA), which was undertaken so as to provide a model basis for understanding circular dichroism spectra of chromatin. In addition, the concept of reciprocal relations (20-23), which provides useful information concerning the details of simple dinucleotide structures, is used and extended. It is found that the diadenosine pyrophosphates, AppA and ApepA, like the closely related 5',5'-dinucleotide, NAD+, give reciprocal relations with positive couplet" circular dichroism. EXPERIMENTAL PROCEDURES Circular dichroism measurements were obtained on a Cary model 60 spectropolarimeter equipped with the Model 6001 circular dichroism attachment. Scan speeds and time constants were chosen to allow sufficient response time and achieve favorable signal-to-noise ratios. ApA was obtained commercially; AppA and ApcpA were obtained from Dr. Terrell Myers. Abbreviations: AppA, diadenosine(5',5') pyrophosphate; ApcpA, diadenosine(5',5') pyrophosphonate.
RESULTS AND DISCUSSION To understand absorption and circular dichroism spectroscopy, one needs some knowledge of the electric and magnetic transition moments in question. For this discussion, the transition moments of Hug and Tinoco (34) will be considered with the assignments as follows: B2u = 256 nm, Blu = 243-nm, Elua = 222 nm, and Elub = 185 nm. The polarization angle defined by the orientation of the double-headed transition moment vector relative to the C4-C5 bond is taken as positive for rotations that take the C5 head into the pyrimidine ring. These angles are 45°, -45°, 29°, and -69°, respectively, for the B2u, Biu, Elua, and Elub transitions. The circular dichroism curves for the mononucleotides AMP, ADP, and adenosine-5'-methylene diphosphonate shown in Fig. 1 reveal negative bands at 265 nm and 218 nm for all three monomers. Since the absorption spectra for the three monomers are the same, then any change in the rotational strength represents a change in the magnetic moment contribution to the transition o - a. This may relate to a folding of the phosphate chain over the adenine ring and therefore perturbing the adenine ir orbitals and altering the excited eigenstate. A folded conformation was first proposed by Szent-Gyorgyi (41) and has recently been supported by molecular orbital (PCILO) calculations by Perahia, Pullman, and Saran (42). Fig. 2 contains the circular dichroism for the dinucleotides (3'-5')ApA, (5'-5')AppA, and (5'-5')ApcpA. The B2u transition crossover point appears at 260 nm for the 3'-5' dimer conformation of ApA but at about 265 nm for the 5'-5' dimer conformations of AppA and ApcpA. The monomer frequency is split by the interaction between transition moments, and is also shifted by the difference in interaction between ground state permanent moments and excited state permanent moments. Differences in crossover points reflect differences in the relative orientation of the coupled transition moments. The rotational strength of the B2u couplets has greatly diminished in AppA and ApcpA, as has the rotation strength of the Eiua couplet. The 282-nm negative band in ApcpA is not present under conditions that more fully bring the two adenine chromophoric groups into juxtaposition..The negative 282-nm band likely arises from an n-1r* transition; the B2u couplet is the positive-negative pair mentioned above with crossover at 265 nm. Temperature studies were undertaken to observe the effect of thermal energy on the base-base association process, and the results are given in Figs. 3-5. Fig. 3 indicates that as the temperature is increased both (5'-5')ApcpA and (5'-5')AppA in 25% LiCl tend toward a more unstacked-like circular dichroism spectra, but the fully unstacked conformation is never achieved. Also the presence of an isoelliptic point at t268 nm for both compounds gives evidence for an equilibrium between two 1785
Chemistry: Pettegrew et al.
1786
Proc. Natl. Acad. Sci. USA 74 (1977)
.5 -
~~~~. ..v ~
5r.200
~
~
*
.
S- -
A i
220
Appcc
R
I^
240 X
(nm)
260
280
300
x
FIG. 1. Circular dichroism spectra for compounds AMP, ADP, and adenosine-5'-methylene diphosphonate (Appcp) in aqueous solution at pH 7.0.
predominant conformations, but is not proof for such an equilibrium. Similar data for (5'-5')AppA in 2 M NaCi and 0.1 M Tris (pH 7.4) are depicted in Fig. 4. The temperature data as summarized in Fig. 5 indicate: (i) that under the same ionic strength and solvation conditions the probability of base-base interactions is ApA > AppA > ApcpA; (ii) that at the same temperature,
high-ionic-strength solvents disrupt base-base
interactions more than low-ionic-strength solvents; and (Wi) that
all three compounds tend towards the same base-base interaction probability state at high temperatures but never attain a state with no base-base interaction. The high-ionic-strength conditions may increase the solvation of the phosphate groups and therefore increase steric factors that would decrease base-base interactions. Fig. 5 (right) may be explained by the fact that replacing the P-O-P group in (5'-5')AppA with the P-CH2-P- group in (5'-5')ApcpA eliminates the possibility of Pd-Op bonding and therefore increases the freedom of rotation about the P-C-P bonds and therefore decreases the probability of base-base interaction. -
4.0
A (nm)
(nm) FIG. 3. Effect of temperature on the circular dichroism of (left) AppA in 25% LiCl, (right) ApcpA in 25% LiCl. A
The influence of London dispersion forces on the adenineadenine interactions was studied by observing the effect of solvents with different polar refractivities on these interactions. Fig. 6 shows the effect of increasing the percent of dioxane in a water/dioxane mixture on the circular dichroism of (3'-5')ApA and reveals a steady regression to an unstacked circular dichroism spectra almost identical to that of the monomer AMP. There is also a well defined isoelliptic point at -260 nm. The comparable data for (5'-5')AppA shown in Fig. 7 again reveal a steady progression to an unstacked-type circular dichroism spectra but with less well defined isoelliptic points, indicating a multiconformation equilibrium as opposed to a simple stacked-unstacked equilibrium. Therefore these data would indicate that the adenine bases in (5'-5')AppA are in several thermodynamically allowed conformations under the experimental conditions but that (3'-5')ApA would appear to exist in only two allowed conformations. That a major driving force for the adenine-adenine interactions is London dispersion forces can be appreciated by referring to Fig. 8, which reveals a linear relationship between the solvent molar refractivity and [0lmax of the long-wavelength band. One of the major difficulties in interpreting circular di-
2.0' 5
2.4-
+290
1.6-
0 0~~~~~~~~~~~
O PC~~~~~I
i
t "'~~Apc
0.8
+800
-0.8 -1.6-
-2.4
220
240
260 (nm)
280
300
X
FIG. 2. Circular dichroism spectra of ApA, AppA, and ApcpA in 0.1 M NaCl/0.01 M Tris buffer at pH 7.4.
FIG. 4. Effects of temperature on the circular dichroism curves of AppA in 2 M NaCl/0.1 M Tris at pH 7.4.
Chemistry: Pettegrew et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
1787
x
!Z .I
b x -40
Temperature
Temperature
FIG. 5. (Left) Temperature dependence of circular dichroism of AppA (A), ApcpA (A) (both in 25% LiCl), and ApA (0) in 25.2% LiCl, all buffered with 0.1 M Tris at pH 7.4, and of (0) ApA in 0.1 M NaCl/0.1 M Tris at pH 7.4. (Right) Temperature dependence of circular dichroism of ApcpA (0) and AppA (@) in 1.0 M NaCl at pH 6.8.
chroism data in which one has coupled oscillator interaction of the transition moments is ascertaining which transition moments are undergoing interaction. If one can ascertain which transition moments are interacting, then one can use this information to postulate possible conformations of the molecule in question under the given experimental conditions. In an effort to accomplish this, the following approach has been adopted. If two electronic transition moments interact to give rise to reciprocal extrema, then experimental conditions that would alter this interaction would seem, at first approximation, to affect both extrema equally, with the resultant same rate of change for the two extrema, if each extremum is composed of one or only one predominant transition. Then plots of an experimental parameter, X, against [OImax for the two extrema should give rise to mirror image curves. Fig. 9 (left) plots the data in Fig. 6 for (3'-5')ApA and reveals
A (nm)
FIG. 7. Circular dichroism of AppA in water/dioxane mixtures (water:dioxane, % dioxane): (a) 10:0, 0%; (b) 13:1, 7.6%; (c) 6:1, 16.7%; (d) 5:2, 40%; (e) 1:1, 50%.
that the A % dioxane/z[O]max for the 270 nm and 255 nm peaks are almost identical. This is therefore strong evidence that the 270 nm and 255 nm peaks are the result of the coupled oscillator interaction of the 256 nm B2,1 moment. Likewise, Fig. 9 (right) is the similar plot for the data of (5'-5')AppA contained in Fig. 7. Notice, however, that this situation is not so simple or conclusive, but indicates that the 275 nm and 217 nm peaks may represent a coupled oscillator interaction between the 256 nm B2u moment and the 222 nm Eiua moment, which geometrically is plausible because the two moments have very similar geometry according to the Hug and Tinoco (34) representation. However, one could just as plausibly interpret the data as indicating interaction between the two Eiua moments and interaction between the two B2u moments with the negative 255 nm peak overlapping with the positive 235 nm peak and therefore decreasing the apparent rotational strength of the negative 255 nm peak. Therefore the plots of X against [Olmax will be useful only if there is little or no overlap of the peaks.
3
0
2
a''all\ X
Wt "I
x
0i
"
@
C
"aS /",a
-I
AppA 10
15
20
25
30
Molar refractivity
X (nm) FIG. 6. Circular dichroism of ApA in water/dioxane mixtures.
FIG. 8. Molar ellipticity of the first extremum of ApA and AppA plotted as a function of the molar refraction of the nonaqueous component of the water/organic mixtures. Concentrations of the nonaqueous component were 3.0 M. (1) Water; (2) methanol; (3) formate; (4) formamide; (5) acetate; (6) ethanol; (7) urea; (8) acetonitrile; (9) propanol; (10) dimethylformamide (11) imidazole; (12) dioxane; (13) dimethylurea, (14) trichloracetate.
1788
Proc. Nati. Acad. Sci. USA 74 (1977)
Chemistry: Pettegrew et al.
80
60
60
40
40
20
20 /l I -E
-6.0 -4.0 -2.0
0
[S1 x 10-4
2.0
4.0
-3.0 -2.0 -1.0 [lo
0
x
10
2.0
lo-'
FIG. 9. Effects of increasing percent dioxane on the molar ellipticity (left) for the 270 nm (0) and 255 nm (0) extrema of ApA; (right) for each extremum of AppA: (0) 275 nm, (-) 255 nm, (0) 217 nm, (0) 235 nm.
We thank Dr. Terrell Myers at the University of Illinois College of Medicine for providing samples of AppA and ApcpA. J.W.P. thanks Philip R. Dodge at the School of Medicine, Washington University, for his encouragement during the completion of this work. The work was supported by National Institutes of Health Grant GM12862 and National Science Foundation Grant CHE 75-10082. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
1. Heitz, E. (1928) Jahrb. Wiss. Bot. 69, 762-767. 2. Elgin, S. C. R. & Weintraub, H. (1975) Annu. Rev. Biochem. 44, 725-774. 3. Bush, C. A. & Scheraga, H. A. (1969) Biopolymers 7,395-409. 4. Powell, J. T., Richards, E. G. & Gratzer, W. B. (1972) Blopolymers
11,235-250. 5. Lowe, M. J. & Schellman, J. A. (1972) J. Mol. Biol. 65, 91109. 6. Brunner, W. C. & Maestre, M. F. (1974) Biopolymers 13, 345-357. 7. Allen, F. S. & Daub, G. W. (1974) Biopolymers 13,241-255. 8. Bush, C. A. & Tinoco, J. (1965) Biochem. Biophys. Res. Commun. 18,633-638. 9. Van Holde, K. E., Brahms, J. & Michelson, A. M. (1965) J. Mol. Biol. 12, 726-739. 10. Miles, D. W., Robins, R. K. & Eyring, H. (1967) 57, 11381145. 11. Ivanov, V. I., Minchenkova, L. E., Schyolkina, A. K. & Poletayev, A. I. (1973) Biopolymers 12, 89-110. 12. Lee, C. H., Chang, C.-T. & Wetmur, J. G. (1973) Biopolymers 12, 1099-1122. 13. Shih, T. Y. & Lake, R. S. (1972) Biochemistry 11, 4811-4817.
14. Mandel, R. & Fasman, G. D. (1974) Biochem. Biophys. Res. Commun. 59, 672-679. 15. Frisman, E. V., Sibileva, M. A., D'lakova, E. B., Voroblev, V. I. & Turoverova, L. V. (1974) Biopolymers 13,863-877. 16. Jordan, C. F., Lermon, L. S. & Venable, J. H., Jr. (1972) Nature New Biol. 236,67-70. 17. Yu, S. S. & Li, H. J. (1973) Biopolymers 12, 2777-2788. 18. Yang, J. T. (1969) Prog. Nucleic Acid Res. Mol. Biol. 9, 223300. 19. Bush, C. A. (1973) Phys. Chem. Prop. Nucleic Acids 2, 147186. 20. Urry, D. W. (1965) Proc. Natl. Acad. Sci. USA 54,640-648. 21. Miles, D. W. & Urry, D. W. (1967) J. Phys. Chem. 71, 44484454. 22. Miles, D. W. & Urry, D. W. (1968) J. Blol. Chem. 243, 41814188. 23. Miles, D. W. & Urry, D. W. (1968) Biochemistry 7, 27912799. 24. Richard, H., Schreiber, J. P. & Daune, M. (1973) Biopolymers 12, 1-10. 25. Shin, Y. A. (1973) Biopolymers 12,2459-2475. 26. Baba, Y. & Kagemoto, A. (1974) Biopolymers 13,339-344. 27. Moore, D. A. & Wagner, T. E. (1973) Biopolymers 12, 201221. 28. Moore, D. S. & Wagner, T. E. (1974) Biopolymers 13, 977986. 29. Bush, C. A. & Tinoco, I., Jr. (1967) J. Mol. Blol. 23,601-614. 30. Johnson, W. C., Itzkowitz, M. S. & Tinoco, I., Jr. (1972) Biopolymers 11,225-234. 31. Johnson, W. C. & Tinoco, I., Jr. (1969) Blopolymers 7, 727749. 32. Perahia, D., Pullman, B. & Saran, A. (1974) Biochim. Blophys. Acta 340, 229-313. 33. Jordan, F. (1974) Biopolymers 13,289-306. 34. Hug, W. & Tinoco, I., Jr. (1973) J. Am. Chem. Soc. 95,28032813. 35. Hug, W. & Tinoco, I., Jr. (1974) J. Am. Chem. Soc. 96, 665673. 36. Studdert, D. S. & Davis, R. C. (1974) Blopolymers 13, 13771389. 37. Studdert, D. S. & Davis, R. C. (1974) Blopolymers 13, 13911403. 38. Studdert, D. S. & Davis, R. C. (1974) Blopolymers 13, 14051416. 39. Pullman, B. & Pullman, A. (1969) Prog. Nucleic Acid Res. Mol. Biol. 9, 327-402. 40. Chen, H. H. & Clark, L. B. (1969) J. Chem. Phys. 51, 18621871. 41. Szent-Gyorgyi, A. (1957) Bioenergetics (Academic Press, New
York). 42. Perahia, D., Pullman, B. & Saran, A. (1972) Blochem. Biophys. Res. Commun. 47, 1284-1289.
Chemistry
Circular dichroism of adenosine dinucleotides (optical activity/reciprocal relations/transition moment coupling)
J. W. PETTEGREWt, D. W. MILESt, AND H. EYRINGt t Department of Neurology and Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110; and t Department of Chemistry, University of
Utah, Salt Lake City, Utah 84112
Contributed by H. Eyring, February 7, 1977
ABSTRACT The circular dichroism and absorption spectra of (3'-5')ApA, (5'-5')AppA, and (5'-5')ApcpA [diadenosine(5',5') pyrophosphonatel under various ionic conditions and at various temperatures are reported. Temperature studies reveal that under the same ionic strength and solvation conditions the probability of base-base interaction is ApA > AppA > ApcpA. It is found that increasing the molar refractivity of the solvent decreases the base-base interaction for both ApA and AppA and that both compounds proceed to a monomeric state in solvents of high molar refractivity. Solvent effect studies also indicate that a major percentage of the driving force for the base-base interaction can be accounted for by London dispersion forces. Low ionic strength appears to favor the base-base interacted conformation. All compounds tend toward the same noninteracted form at high temperatures. Plots of [O]X against X (where X is an experimental parameter) were constructed in order to obtain information concerning which transitions might be undergoing oscillator coupling. To a first approximation, transitions coupled to each other should have very similar A[OJx/AX for the experimental range of X. Applying this technique to the experimental data yielded information concerning possible transition coupling.
In order to understand the function of chromatin (1, 2), a thorough understanding of its structure and the forces involved in maintaining that structure is needed. Of the experimental spectroscopic methods available to investigate this problem, circular dichroism remains one of the most widely used (3-26), although its usefulness here in actually defining structural details is yet to be fully realized. One of the main obstacles is the lack of reliable information about the optical properties of the nucleic acid bases and their dinucleotide derivatives, although considerable progress has been achieved (26-40). This paper contains a thorough study of the circular dichroism of the dinucleotide dimers (3'-5')ApA, diadenosine(5',5') pyrophosphate (AppA), and diadenosine(5',5') pyrophosphonate (ApcpA), which was undertaken so as to provide a model basis for understanding circular dichroism spectra of chromatin. In addition, the concept of reciprocal relations (20-23), which provides useful information concerning the details of simple dinucleotide structures, is used and extended. It is found that the diadenosine pyrophosphates, AppA and ApepA, like the closely related 5',5'-dinucleotide, NAD+, give reciprocal relations with positive couplet" circular dichroism. EXPERIMENTAL PROCEDURES Circular dichroism measurements were obtained on a Cary model 60 spectropolarimeter equipped with the Model 6001 circular dichroism attachment. Scan speeds and time constants were chosen to allow sufficient response time and achieve favorable signal-to-noise ratios. ApA was obtained commercially; AppA and ApcpA were obtained from Dr. Terrell Myers. Abbreviations: AppA, diadenosine(5',5') pyrophosphate; ApcpA, diadenosine(5',5') pyrophosphonate.
RESULTS AND DISCUSSION To understand absorption and circular dichroism spectroscopy, one needs some knowledge of the electric and magnetic transition moments in question. For this discussion, the transition moments of Hug and Tinoco (34) will be considered with the assignments as follows: B2u = 256 nm, Blu = 243-nm, Elua = 222 nm, and Elub = 185 nm. The polarization angle defined by the orientation of the double-headed transition moment vector relative to the C4-C5 bond is taken as positive for rotations that take the C5 head into the pyrimidine ring. These angles are 45°, -45°, 29°, and -69°, respectively, for the B2u, Biu, Elua, and Elub transitions. The circular dichroism curves for the mononucleotides AMP, ADP, and adenosine-5'-methylene diphosphonate shown in Fig. 1 reveal negative bands at 265 nm and 218 nm for all three monomers. Since the absorption spectra for the three monomers are the same, then any change in the rotational strength represents a change in the magnetic moment contribution to the transition o - a. This may relate to a folding of the phosphate chain over the adenine ring and therefore perturbing the adenine ir orbitals and altering the excited eigenstate. A folded conformation was first proposed by Szent-Gyorgyi (41) and has recently been supported by molecular orbital (PCILO) calculations by Perahia, Pullman, and Saran (42). Fig. 2 contains the circular dichroism for the dinucleotides (3'-5')ApA, (5'-5')AppA, and (5'-5')ApcpA. The B2u transition crossover point appears at 260 nm for the 3'-5' dimer conformation of ApA but at about 265 nm for the 5'-5' dimer conformations of AppA and ApcpA. The monomer frequency is split by the interaction between transition moments, and is also shifted by the difference in interaction between ground state permanent moments and excited state permanent moments. Differences in crossover points reflect differences in the relative orientation of the coupled transition moments. The rotational strength of the B2u couplets has greatly diminished in AppA and ApcpA, as has the rotation strength of the Eiua couplet. The 282-nm negative band in ApcpA is not present under conditions that more fully bring the two adenine chromophoric groups into juxtaposition..The negative 282-nm band likely arises from an n-1r* transition; the B2u couplet is the positive-negative pair mentioned above with crossover at 265 nm. Temperature studies were undertaken to observe the effect of thermal energy on the base-base association process, and the results are given in Figs. 3-5. Fig. 3 indicates that as the temperature is increased both (5'-5')ApcpA and (5'-5')AppA in 25% LiCl tend toward a more unstacked-like circular dichroism spectra, but the fully unstacked conformation is never achieved. Also the presence of an isoelliptic point at t268 nm for both compounds gives evidence for an equilibrium between two 1785
Chemistry: Pettegrew et al.
1786
Proc. Natl. Acad. Sci. USA 74 (1977)
.5 -
~~~~. ..v ~
5r.200
~
~
*
.
S- -
A i
220
Appcc
R
I^
240 X
(nm)
260
280
300
x
FIG. 1. Circular dichroism spectra for compounds AMP, ADP, and adenosine-5'-methylene diphosphonate (Appcp) in aqueous solution at pH 7.0.
predominant conformations, but is not proof for such an equilibrium. Similar data for (5'-5')AppA in 2 M NaCi and 0.1 M Tris (pH 7.4) are depicted in Fig. 4. The temperature data as summarized in Fig. 5 indicate: (i) that under the same ionic strength and solvation conditions the probability of base-base interactions is ApA > AppA > ApcpA; (ii) that at the same temperature,
high-ionic-strength solvents disrupt base-base
interactions more than low-ionic-strength solvents; and (Wi) that
all three compounds tend towards the same base-base interaction probability state at high temperatures but never attain a state with no base-base interaction. The high-ionic-strength conditions may increase the solvation of the phosphate groups and therefore increase steric factors that would decrease base-base interactions. Fig. 5 (right) may be explained by the fact that replacing the P-O-P group in (5'-5')AppA with the P-CH2-P- group in (5'-5')ApcpA eliminates the possibility of Pd-Op bonding and therefore increases the freedom of rotation about the P-C-P bonds and therefore decreases the probability of base-base interaction. -
4.0
A (nm)
(nm) FIG. 3. Effect of temperature on the circular dichroism of (left) AppA in 25% LiCl, (right) ApcpA in 25% LiCl. A
The influence of London dispersion forces on the adenineadenine interactions was studied by observing the effect of solvents with different polar refractivities on these interactions. Fig. 6 shows the effect of increasing the percent of dioxane in a water/dioxane mixture on the circular dichroism of (3'-5')ApA and reveals a steady regression to an unstacked circular dichroism spectra almost identical to that of the monomer AMP. There is also a well defined isoelliptic point at -260 nm. The comparable data for (5'-5')AppA shown in Fig. 7 again reveal a steady progression to an unstacked-type circular dichroism spectra but with less well defined isoelliptic points, indicating a multiconformation equilibrium as opposed to a simple stacked-unstacked equilibrium. Therefore these data would indicate that the adenine bases in (5'-5')AppA are in several thermodynamically allowed conformations under the experimental conditions but that (3'-5')ApA would appear to exist in only two allowed conformations. That a major driving force for the adenine-adenine interactions is London dispersion forces can be appreciated by referring to Fig. 8, which reveals a linear relationship between the solvent molar refractivity and [0lmax of the long-wavelength band. One of the major difficulties in interpreting circular di-
2.0' 5
2.4-
+290
1.6-
0 0~~~~~~~~~~~
O PC~~~~~I
i
t "'~~Apc
0.8
+800
-0.8 -1.6-
-2.4
220
240
260 (nm)
280
300
X
FIG. 2. Circular dichroism spectra of ApA, AppA, and ApcpA in 0.1 M NaCl/0.01 M Tris buffer at pH 7.4.
FIG. 4. Effects of temperature on the circular dichroism curves of AppA in 2 M NaCl/0.1 M Tris at pH 7.4.
Chemistry: Pettegrew et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
1787
x
!Z .I
b x -40
Temperature
Temperature
FIG. 5. (Left) Temperature dependence of circular dichroism of AppA (A), ApcpA (A) (both in 25% LiCl), and ApA (0) in 25.2% LiCl, all buffered with 0.1 M Tris at pH 7.4, and of (0) ApA in 0.1 M NaCl/0.1 M Tris at pH 7.4. (Right) Temperature dependence of circular dichroism of ApcpA (0) and AppA (@) in 1.0 M NaCl at pH 6.8.
chroism data in which one has coupled oscillator interaction of the transition moments is ascertaining which transition moments are undergoing interaction. If one can ascertain which transition moments are interacting, then one can use this information to postulate possible conformations of the molecule in question under the given experimental conditions. In an effort to accomplish this, the following approach has been adopted. If two electronic transition moments interact to give rise to reciprocal extrema, then experimental conditions that would alter this interaction would seem, at first approximation, to affect both extrema equally, with the resultant same rate of change for the two extrema, if each extremum is composed of one or only one predominant transition. Then plots of an experimental parameter, X, against [OImax for the two extrema should give rise to mirror image curves. Fig. 9 (left) plots the data in Fig. 6 for (3'-5')ApA and reveals
A (nm)
FIG. 7. Circular dichroism of AppA in water/dioxane mixtures (water:dioxane, % dioxane): (a) 10:0, 0%; (b) 13:1, 7.6%; (c) 6:1, 16.7%; (d) 5:2, 40%; (e) 1:1, 50%.
that the A % dioxane/z[O]max for the 270 nm and 255 nm peaks are almost identical. This is therefore strong evidence that the 270 nm and 255 nm peaks are the result of the coupled oscillator interaction of the 256 nm B2,1 moment. Likewise, Fig. 9 (right) is the similar plot for the data of (5'-5')AppA contained in Fig. 7. Notice, however, that this situation is not so simple or conclusive, but indicates that the 275 nm and 217 nm peaks may represent a coupled oscillator interaction between the 256 nm B2u moment and the 222 nm Eiua moment, which geometrically is plausible because the two moments have very similar geometry according to the Hug and Tinoco (34) representation. However, one could just as plausibly interpret the data as indicating interaction between the two Eiua moments and interaction between the two B2u moments with the negative 255 nm peak overlapping with the positive 235 nm peak and therefore decreasing the apparent rotational strength of the negative 255 nm peak. Therefore the plots of X against [Olmax will be useful only if there is little or no overlap of the peaks.
3
0
2
a''all\ X
Wt "I
x
0i
"
@
C
"aS /",a
-I
AppA 10
15
20
25
30
Molar refractivity
X (nm) FIG. 6. Circular dichroism of ApA in water/dioxane mixtures.
FIG. 8. Molar ellipticity of the first extremum of ApA and AppA plotted as a function of the molar refraction of the nonaqueous component of the water/organic mixtures. Concentrations of the nonaqueous component were 3.0 M. (1) Water; (2) methanol; (3) formate; (4) formamide; (5) acetate; (6) ethanol; (7) urea; (8) acetonitrile; (9) propanol; (10) dimethylformamide (11) imidazole; (12) dioxane; (13) dimethylurea, (14) trichloracetate.
1788
Proc. Nati. Acad. Sci. USA 74 (1977)
Chemistry: Pettegrew et al.
80
60
60
40
40
20
20 /l I -E
-6.0 -4.0 -2.0
0
[S1 x 10-4
2.0
4.0
-3.0 -2.0 -1.0 [lo
0
x
10
2.0
lo-'
FIG. 9. Effects of increasing percent dioxane on the molar ellipticity (left) for the 270 nm (0) and 255 nm (0) extrema of ApA; (right) for each extremum of AppA: (0) 275 nm, (-) 255 nm, (0) 217 nm, (0) 235 nm.
We thank Dr. Terrell Myers at the University of Illinois College of Medicine for providing samples of AppA and ApcpA. J.W.P. thanks Philip R. Dodge at the School of Medicine, Washington University, for his encouragement during the completion of this work. The work was supported by National Institutes of Health Grant GM12862 and National Science Foundation Grant CHE 75-10082. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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