- Email: [email protected]
Dynamics of B-DNA in the solid state
J. Mol.
Riol. (1981) 149, 307-311
LETTERSTO THE EDITOR
Dynamics of B-DNA in the Solid State Hydrated solid B-form DNA is shown to have large amplitude motions of the phosphodiester groups and immobile bases at temperatures above 5°C. The 31P nuclear magnetic resonance chemical shift powder pattern of DNA in the solid state is significantly averaged by motions that are rapid compared to lo4 Hz. The DNA was labelled with deuterium at the C-8 purine position by exchange. nuclear magnetic resonance spectra have complete static quadrupole patterns indicative of no motions fast compared to IO6 Hz at 50°C.
The *H powder
Structural information must always be interpreted with reference to the time-scale defined by the experimental measurements. Much of the evidence for the doublehelical structure of DNA comes from X-ray diffraction data obtained on hydrated fibers of DNA (Watson & Crick, 1953). This structure represents a long time average of hours to days because of the exposure times of the determinations. The relatively low resolution of the structure of the DNA helix may be the result of the time averaged smearing out of spatial ‘positions combined with sequence-dependent conformational heterogeneity. Nuclear magnetic resonance spectroscopy is very sensitive to motions in samples. Depending on the experimental details and the sample, the n.m.r.7 time-scale can range from a few Hz to hundreds of MHz. Solid state n.m.r. of nucleic acids is particularly valuable because it allows the study of the polymers as crystalline or amorphous samples (Terao et al., 1977; Opella et nl., 1981) as well as nucleoprotein complexes in solution (Cross et al., 1979; Opella et al., 1980; Akutsu et al., 1980: Munowitz et al., 1980; DiVerdi & Opella, 1981; J. A. DiVerdi, S. I. Opella, R. I. Ma, N. R. Kallenbach & N. C. Seeman, unpublished results). The presence of motion is clearly indicated in solid state n.m.r. spectra by the averaging of the static second rank tensor nuclear spin interactions such as dipole-dipole, chemical shift anisotropy, or nuclear quadrupole. The B-form of DNA as a hydrated solid has been studied in detail by X-ray 1970) and n.m.r. spectroscopy diffraction (Langridge et aZ., 1960; Arnott, (Migchelsen et al., 1968; Edzes et al., 1972; Shindo et al., 1980). The recent 31P n.m.r. study of Shindo et aZ. (1980) is in general agreement with the structural parameters from the diffraction work, but indicates the presence of motion in the DNA fibers and conformational heterogeneity in the DNA backbone. The results presented here are consistent with the experimental data of Shindo et aZ. (1980) but, by performing experiments that monitor the dynamics of both the bases and the chosphodiester backbone over a large temperature range, we reach different t Abbreviation
used: n.m.r., nuclear magnetic resonance.
002%2836/81/180307-05
$02.00/0
307
0 1981 Aradcmic
Press Inc. (London) Ltd.
308
,I. A. DrVEKDI
AND
S. J. OPELLA
conclusions about the motions of solid B-form DNA. The structural parameters of DNA are not addressed with these experiments. The only phosphorus atoms in DNA4 are in the backbone, therefore 31P n.m.r’. spectra of solid DNA obtained with proton decoupling display the chemical shift’ powder pattern from the phosphodiester groups. 31P n.m.r. spectra in a 3.5 T magnetic field are sensitive to motions faster than the lo* Hz anisotropg of the 31P n m.r. spectra of solid B-form phosphorus chemical shift. Figure 1 contains DKA as a function of temperature. At - 20°C the hydrated B-form DNA gives the characteristic asymmetric 31P chemical shift powder pattern for a static
*XL A h\ x. ,;;;.,
200
0
$50 oc
+40
+20
“C
oc
+5 oc
-2
oc
-200
p.p.m.
FIG. 1. “P n.m.r. (609 MHz) of B-form DNA. Chemical shifts werr measured from external %I”,, H3P04. 10’ transients were recorded at each temperature using a cross polarization time of 1 ms. aquisition time of 50 ms (with high power proton decoupling) and a recycle delay of 1 s. Both proton and phosphorus TT/~pulses welp 5 p’s,
LETTERS
TO THE
309
EDITOR
phosphodiester group. The principal values of the chemical shielding tensor measured from this spectrum are the same as those determined for dehydrated solid DNA samples (Terao et al., 1977 ; Opella et al., 1981). The presence of motions affecting the phosphates is seen at temperatures higher than about 5°C. Although the breadth of the powder pattern is reduced as the temperature is raised, it remains significantly until about 50°C. At relatively high asymmetric temperatures, the 31P resonances collapse to an isotropically averaged line. The 20°C spectrum is very similar to that reported by Shindo et al. (1980). The high molecular weight calf thymus DNA was specifically labelled at the C-8 position of the purine bases by exchange (Doppler-Bernardi & Felsenfeld, 1969). therefore ‘H n.m.r. monitors the dynamics of the bases because of the location of the deuterium in the DNA. The line shapes of 2H n.m.r. spectra of solids are determined by the nuclear quadrupole interaction of C-D bonds. Deuterium has spin I = 1 and a static quadrupole coupling constant of e2qQ/h = 180 kHz with near axial symmetry for an aromatic C-D bond (Barnes & Bloom, 1972; C. H. Gall. J. 4. DiVerdi & S. J. Opella, unpublished results). A theoretical static 2H n.m.r. powder pattern is compared to experimental spectra for purine C-8 labelled DNA at “P
eH
IL A
I ” IO
i-50
oc
t20
oc
Theory
”
I”“1 0 kHZ
-10
A A A
I ’ 200
’
’
1 ’ 0
’
’ I -200
kHz
FIG. 2. 3’P (609 MHz) and ‘H (37% MHz) n.m.r. spectra of B-form DNA. 31P n.m.r. spectra are for t,he reproduced from Fig. 1 for comparison. The theoretical “P powder pattern was calculated experimentally determined principal elements of the chemical shielding tensor for DNA with o,, = 85 p.p.m., oz2 = 25 p.p.m., and 033 = - 109 p.p.m. The ‘H n.m.r. spectra were lo4 quadrupolar spin echoes at each temperature using 2 ps n/z pulses, 50 ps inter-pulse delay, 1 ms acquisition time and 0.1 s recycle delay. The phase of the first pulse was shifted by 180” on every other transient and these data alternately added and subtracted to minimize probe artifacts. The theoretical ‘H powder pattern was calculated for the experimentally determined static quadrupole coupling constant e*qQ/h = 180 kHz and asymmetry parameter 7 = 0.05.
310
+I. A. DIVERDI
AND
S. .I. OPELLB
20°C and 50°C in Figure 2. These solid state ‘H n.m.r. spectra are nearly indistinguishable within the limits of experimental signal-to-noise, especially with respect to the splitting of the major discontinuities, which is 128 kHz in all these cases. By contrast, the 31P chemical shift powder patterns from the same sample at the same temperature show significant reductions in breadth compared to the static theoretical chemical shift anisotropy. These data show that the backbone of B-form DNA in the solid state has motions of substantial amplitude that occur more rapidly than lo4 Hz at 50°C as well as at, lower temperatures. There are no motions influencing the bases that are on the order of or faster than lo6 Hz, even at 50°C. These results are incompatible with a model of re-orientation about the long axis of DNA as proposed by Shindo et al. (1980). First of all, rotation about any axis defined in a molecular frame would average the 31P chemical shift line-shapes to axially symmetric powder patterns. Instead, at the higher temperatures the line shape becomes completely averaged to its isotropic value, indicative of motions in all directions faster than the relevant n.m.r. time-scale. Second, rotations much faster than lo4 Hz would reduce the ‘H quadrupole powder splitting of 128 kHz observed in the powder patterns from the C-8 deuterium labels. This does not occur, since the 50°C *H n.m.r. spectrum is essentially identical to the 20°C spectrum. It is unlikely that the somewhat smaller time-scale for phosphorus chemical shielding compared to the deuterium quadrupole interaction affects the conclusions because of the large extent, of motional averaging of the phosphorus line shape at 50°C’. The data presented here are too limited to provide a detailed description of the motions of solid B-form DNA. Even though the influence of specific models of rotational or jump motions on powder patterns can be calculated. the effectively isotropic averaging of the “P chemical shift pattern and the complete lack of averaging of the *H quadrupole pattern make such modelling difficult. However. the qualitative conclusions of independent re-orientations of the phosphodiester groups on a time scale faster than IO4 Hz and the rigidity of the bases are valid. Backbone motions in DSA clearly could have a significant influence on the interpretation of diffraction data. and it may be of interest to examine such data for a sample of B-form DNA at an elevated tempera~ture where these n.m.r. results would predict rigid bases and significant motions of the phosphodiester groups. It is important to keep the physical state of DNA in mind when discussing USA dynamics. At the present time, there is no evidence of motion in dehydrated DX.4 at 25°C or hydrated DNA at - 20°C. The solid B-form DNA shows large amplitude backbone but not base motions, especially at elevated temperatures. In contrast,. B-form DEA in solution behaves as a flexible polymer with phosphate motions having a rotational correlation time of about 1W6 seconds at 30°C (Opella ft al.. 1981). The phosphate motions in the solid state probably are slower or of limited amplitude compared to those observed for the polymer in solution. DNA backbone motions are significantly restricted by interaction with proteins. as seen for both prokaryotic (Cross et al., 1979; Opella et al.. 1980: Akutsu et al.. 1980; DiVerdi & Opella, 1981) and eukaryotic (J. A. DiVerdi, S. ,J. Opella. R. T. Ma. N. R. Kallenbach & 9. C. Seeman, unpublished results) nucleoprotein complexes.
LETTERS
TO THE
311
EDITOR
This research is being supported by grants from the American Cancer Society (NP-225) and the National Institutes of Health (GM-24266). One of us (S. J. 0.) is a fellow of the A. P. Sloan Foundation (1980-1982). J. A. DIVERDI
Department of Chemistry, University of Pennsylvania Philadelphia, Penn. 19104, U.S.A. Received 16 February
AND S. J. OPELLA
1981
REFERENCES Akutsu, H., Satake, H. & Franklin, R. M. (1980). Biochemistry, 19, 5264-5270. Arnott, S. (1970). Progr. Biophys. Mol. Biol. 21, 267-319. Barnes, R. G. & Bloom, J. W. (1972). J. Chem. Phys. 57, 3082-3086. Cross, T. A., DiVerdi, J. A., Wise, W. B. & Opella, S. J. (1979). In NMR and Biochemistry. pp. 67-74, Marcel Dekker, New York. DiVerdi, J. A. & Opella, S. J. (1981). Biochemistry, 20, 28&284. Doppler-Bernardi, F. & Felsenfeld, G. (1969). Biopolymers, 8, 733-741. Edzes, H. T., Rupprecht, A. & Berendsen, H. S. (1972). Biochem. Biophys. Res. Cwmmun. 46, 790-794.
Langridge,
R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. & Hamilton, L. D. (1960). J. 2, 19-37. Migchelsen, C., Berendsen, H. J. C. & Rupprecht, A. (1968). J. Mol. Biol. 37, 235-237. Munowitz, M. C., Dobson, C. M., Griffin, R. G. & Harrison, S. C. (1980). J. Mol. Biol. 141, 327-333. Opella, S. J., Cross, T. A., DiVerdi, J. A. & Sturm, C. F. (1980). Biophys. J. 32, 531-548. Opella, S. J., Wise, W. B. & DiVerdi, J. A. (1981). Biochemistry, 20, 284-290. Shindo, H., Wooten, J. B., Pheiffer, B. H. & Zimmerman, S. B. (1980). Biochemistry, 19,518526. Terao, T.. Matsui, S. & Akasaka, K. (1977). J. Amer. Chem. Sot. 99, 613-138. Watson, *J. D. & Crick, F. H. C. (1953). ,Vature (London), 171, 737-738. Mol.
Biol.
Riol. (1981) 149, 307-311
LETTERSTO THE EDITOR
Dynamics of B-DNA in the Solid State Hydrated solid B-form DNA is shown to have large amplitude motions of the phosphodiester groups and immobile bases at temperatures above 5°C. The 31P nuclear magnetic resonance chemical shift powder pattern of DNA in the solid state is significantly averaged by motions that are rapid compared to lo4 Hz. The DNA was labelled with deuterium at the C-8 purine position by exchange. nuclear magnetic resonance spectra have complete static quadrupole patterns indicative of no motions fast compared to IO6 Hz at 50°C.
The *H powder
Structural information must always be interpreted with reference to the time-scale defined by the experimental measurements. Much of the evidence for the doublehelical structure of DNA comes from X-ray diffraction data obtained on hydrated fibers of DNA (Watson & Crick, 1953). This structure represents a long time average of hours to days because of the exposure times of the determinations. The relatively low resolution of the structure of the DNA helix may be the result of the time averaged smearing out of spatial ‘positions combined with sequence-dependent conformational heterogeneity. Nuclear magnetic resonance spectroscopy is very sensitive to motions in samples. Depending on the experimental details and the sample, the n.m.r.7 time-scale can range from a few Hz to hundreds of MHz. Solid state n.m.r. of nucleic acids is particularly valuable because it allows the study of the polymers as crystalline or amorphous samples (Terao et al., 1977; Opella et nl., 1981) as well as nucleoprotein complexes in solution (Cross et al., 1979; Opella et al., 1980; Akutsu et al., 1980: Munowitz et al., 1980; DiVerdi & Opella, 1981; J. A. DiVerdi, S. I. Opella, R. I. Ma, N. R. Kallenbach & N. C. Seeman, unpublished results). The presence of motion is clearly indicated in solid state n.m.r. spectra by the averaging of the static second rank tensor nuclear spin interactions such as dipole-dipole, chemical shift anisotropy, or nuclear quadrupole. The B-form of DNA as a hydrated solid has been studied in detail by X-ray 1970) and n.m.r. spectroscopy diffraction (Langridge et aZ., 1960; Arnott, (Migchelsen et al., 1968; Edzes et al., 1972; Shindo et al., 1980). The recent 31P n.m.r. study of Shindo et aZ. (1980) is in general agreement with the structural parameters from the diffraction work, but indicates the presence of motion in the DNA fibers and conformational heterogeneity in the DNA backbone. The results presented here are consistent with the experimental data of Shindo et aZ. (1980) but, by performing experiments that monitor the dynamics of both the bases and the chosphodiester backbone over a large temperature range, we reach different t Abbreviation
used: n.m.r., nuclear magnetic resonance.
002%2836/81/180307-05
$02.00/0
307
0 1981 Aradcmic
Press Inc. (London) Ltd.
308
,I. A. DrVEKDI
AND
S. J. OPELLA
conclusions about the motions of solid B-form DNA. The structural parameters of DNA are not addressed with these experiments. The only phosphorus atoms in DNA4 are in the backbone, therefore 31P n.m.r’. spectra of solid DNA obtained with proton decoupling display the chemical shift’ powder pattern from the phosphodiester groups. 31P n.m.r. spectra in a 3.5 T magnetic field are sensitive to motions faster than the lo* Hz anisotropg of the 31P n m.r. spectra of solid B-form phosphorus chemical shift. Figure 1 contains DKA as a function of temperature. At - 20°C the hydrated B-form DNA gives the characteristic asymmetric 31P chemical shift powder pattern for a static
*XL A h\ x. ,;;;.,
200
0
$50 oc
+40
+20
“C
oc
+5 oc
-2
oc
-200
p.p.m.
FIG. 1. “P n.m.r. (609 MHz) of B-form DNA. Chemical shifts werr measured from external %I”,, H3P04. 10’ transients were recorded at each temperature using a cross polarization time of 1 ms. aquisition time of 50 ms (with high power proton decoupling) and a recycle delay of 1 s. Both proton and phosphorus TT/~pulses welp 5 p’s,
LETTERS
TO THE
309
EDITOR
phosphodiester group. The principal values of the chemical shielding tensor measured from this spectrum are the same as those determined for dehydrated solid DNA samples (Terao et al., 1977 ; Opella et al., 1981). The presence of motions affecting the phosphates is seen at temperatures higher than about 5°C. Although the breadth of the powder pattern is reduced as the temperature is raised, it remains significantly until about 50°C. At relatively high asymmetric temperatures, the 31P resonances collapse to an isotropically averaged line. The 20°C spectrum is very similar to that reported by Shindo et al. (1980). The high molecular weight calf thymus DNA was specifically labelled at the C-8 position of the purine bases by exchange (Doppler-Bernardi & Felsenfeld, 1969). therefore ‘H n.m.r. monitors the dynamics of the bases because of the location of the deuterium in the DNA. The line shapes of 2H n.m.r. spectra of solids are determined by the nuclear quadrupole interaction of C-D bonds. Deuterium has spin I = 1 and a static quadrupole coupling constant of e2qQ/h = 180 kHz with near axial symmetry for an aromatic C-D bond (Barnes & Bloom, 1972; C. H. Gall. J. 4. DiVerdi & S. J. Opella, unpublished results). A theoretical static 2H n.m.r. powder pattern is compared to experimental spectra for purine C-8 labelled DNA at “P
eH
IL A
I ” IO
i-50
oc
t20
oc
Theory
”
I”“1 0 kHZ
-10
A A A
I ’ 200
’
’
1 ’ 0
’
’ I -200
kHz
FIG. 2. 3’P (609 MHz) and ‘H (37% MHz) n.m.r. spectra of B-form DNA. 31P n.m.r. spectra are for t,he reproduced from Fig. 1 for comparison. The theoretical “P powder pattern was calculated experimentally determined principal elements of the chemical shielding tensor for DNA with o,, = 85 p.p.m., oz2 = 25 p.p.m., and 033 = - 109 p.p.m. The ‘H n.m.r. spectra were lo4 quadrupolar spin echoes at each temperature using 2 ps n/z pulses, 50 ps inter-pulse delay, 1 ms acquisition time and 0.1 s recycle delay. The phase of the first pulse was shifted by 180” on every other transient and these data alternately added and subtracted to minimize probe artifacts. The theoretical ‘H powder pattern was calculated for the experimentally determined static quadrupole coupling constant e*qQ/h = 180 kHz and asymmetry parameter 7 = 0.05.
310
+I. A. DIVERDI
AND
S. .I. OPELLB
20°C and 50°C in Figure 2. These solid state ‘H n.m.r. spectra are nearly indistinguishable within the limits of experimental signal-to-noise, especially with respect to the splitting of the major discontinuities, which is 128 kHz in all these cases. By contrast, the 31P chemical shift powder patterns from the same sample at the same temperature show significant reductions in breadth compared to the static theoretical chemical shift anisotropy. These data show that the backbone of B-form DNA in the solid state has motions of substantial amplitude that occur more rapidly than lo4 Hz at 50°C as well as at, lower temperatures. There are no motions influencing the bases that are on the order of or faster than lo6 Hz, even at 50°C. These results are incompatible with a model of re-orientation about the long axis of DNA as proposed by Shindo et al. (1980). First of all, rotation about any axis defined in a molecular frame would average the 31P chemical shift line-shapes to axially symmetric powder patterns. Instead, at the higher temperatures the line shape becomes completely averaged to its isotropic value, indicative of motions in all directions faster than the relevant n.m.r. time-scale. Second, rotations much faster than lo4 Hz would reduce the ‘H quadrupole powder splitting of 128 kHz observed in the powder patterns from the C-8 deuterium labels. This does not occur, since the 50°C *H n.m.r. spectrum is essentially identical to the 20°C spectrum. It is unlikely that the somewhat smaller time-scale for phosphorus chemical shielding compared to the deuterium quadrupole interaction affects the conclusions because of the large extent, of motional averaging of the phosphorus line shape at 50°C’. The data presented here are too limited to provide a detailed description of the motions of solid B-form DNA. Even though the influence of specific models of rotational or jump motions on powder patterns can be calculated. the effectively isotropic averaging of the “P chemical shift pattern and the complete lack of averaging of the *H quadrupole pattern make such modelling difficult. However. the qualitative conclusions of independent re-orientations of the phosphodiester groups on a time scale faster than IO4 Hz and the rigidity of the bases are valid. Backbone motions in DSA clearly could have a significant influence on the interpretation of diffraction data. and it may be of interest to examine such data for a sample of B-form DNA at an elevated tempera~ture where these n.m.r. results would predict rigid bases and significant motions of the phosphodiester groups. It is important to keep the physical state of DNA in mind when discussing USA dynamics. At the present time, there is no evidence of motion in dehydrated DX.4 at 25°C or hydrated DNA at - 20°C. The solid B-form DNA shows large amplitude backbone but not base motions, especially at elevated temperatures. In contrast,. B-form DEA in solution behaves as a flexible polymer with phosphate motions having a rotational correlation time of about 1W6 seconds at 30°C (Opella ft al.. 1981). The phosphate motions in the solid state probably are slower or of limited amplitude compared to those observed for the polymer in solution. DNA backbone motions are significantly restricted by interaction with proteins. as seen for both prokaryotic (Cross et al., 1979; Opella et al.. 1980: Akutsu et al.. 1980; DiVerdi & Opella, 1981) and eukaryotic (J. A. DiVerdi, S. ,J. Opella. R. T. Ma. N. R. Kallenbach & 9. C. Seeman, unpublished results) nucleoprotein complexes.
LETTERS
TO THE
311
EDITOR
This research is being supported by grants from the American Cancer Society (NP-225) and the National Institutes of Health (GM-24266). One of us (S. J. 0.) is a fellow of the A. P. Sloan Foundation (1980-1982). J. A. DIVERDI
Department of Chemistry, University of Pennsylvania Philadelphia, Penn. 19104, U.S.A. Received 16 February
AND S. J. OPELLA
1981
REFERENCES Akutsu, H., Satake, H. & Franklin, R. M. (1980). Biochemistry, 19, 5264-5270. Arnott, S. (1970). Progr. Biophys. Mol. Biol. 21, 267-319. Barnes, R. G. & Bloom, J. W. (1972). J. Chem. Phys. 57, 3082-3086. Cross, T. A., DiVerdi, J. A., Wise, W. B. & Opella, S. J. (1979). In NMR and Biochemistry. pp. 67-74, Marcel Dekker, New York. DiVerdi, J. A. & Opella, S. J. (1981). Biochemistry, 20, 28&284. Doppler-Bernardi, F. & Felsenfeld, G. (1969). Biopolymers, 8, 733-741. Edzes, H. T., Rupprecht, A. & Berendsen, H. S. (1972). Biochem. Biophys. Res. Cwmmun. 46, 790-794.
Langridge,
R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. & Hamilton, L. D. (1960). J. 2, 19-37. Migchelsen, C., Berendsen, H. J. C. & Rupprecht, A. (1968). J. Mol. Biol. 37, 235-237. Munowitz, M. C., Dobson, C. M., Griffin, R. G. & Harrison, S. C. (1980). J. Mol. Biol. 141, 327-333. Opella, S. J., Cross, T. A., DiVerdi, J. A. & Sturm, C. F. (1980). Biophys. J. 32, 531-548. Opella, S. J., Wise, W. B. & DiVerdi, J. A. (1981). Biochemistry, 20, 284-290. Shindo, H., Wooten, J. B., Pheiffer, B. H. & Zimmerman, S. B. (1980). Biochemistry, 19,518526. Terao, T.. Matsui, S. & Akasaka, K. (1977). J. Amer. Chem. Sot. 99, 613-138. Watson, *J. D. & Crick, F. H. C. (1953). ,Vature (London), 171, 737-738. Mol.
Biol.