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Hydration of DNA
J. Mol. Biol. (1963) 37,236-237
Hydration of DNA Comparison of Nuclear Magnetic Resonance Results for Oriented DNA intheR,BandCForm The nuclear magnetio resonance teahnique was used by Berendsen (1960,1962u,b) for a study of the hydration of oriented collagen fibres. The observed NMRt spectrum of water protons was superposed on a very broad signal from protons of the macromolecules. Owing to the large diEertmce in line width, it was possible to study this water signal separately. The water spectrum showed a splitting into three lines with the separation of the outer lines following the angular dependence (3 co&3 -1) for a dipole-dipole interaction between two magnetia nuclei, when the angle B between fibre axis and magnetic field was varied. This result indicated an average H-H interaction in the fibre direction. It was suggested that the oollagen macromolecule, because of the excellent fit of its axial repeat distance to a multiple of the repeat distance of ice I, would stabilize chains of reorienting water moleoules along the maoromolecule by hydrogen bonding at appropriate sites. At higher water oontents a single water line with an angular dependent line shape was observed, which could qualitatively be interpreted in agreement with the suggested model. This work has been extended to variable temperature measurements (Berendsen & Migchelsen, 1966) and to the study of oriented collagen samples hydrated with heavy water (Migohelsen & Berendsen, 1967) in order to obtain further information about the dynamic behaviour of water moleoules and protons in the water of hydration. The NMR spectrum of deuterons in collagen hydrated with heavy water showed a splitting into two lines with the same angular dependence but uaused by quadrupole interaction of the deuterium nuclei, with the average electric field gradient at the sites of the deuterium nuclei. This result indicated an average electric field gradient along the collagen fibre direction and gave further support to the suggested hydration model. Measurements have also been performed on oriented fibres of silk fibroin, keratin and salmon sperm DNA (Berendsen & Migchelsen, 1966) with results which, except for DNA, could be interpreted in agreement with the macromolecular structures. The expected H-H interaction in the fibre dire&ion of DNA was, however, reported later by Ruppreoht (1966~~)who had developed a wet-spinning method for preparation of films of highly oriented DNA (Rupprecht, 1968b). The calf thymus NaDNA sample studied, whioh was in the A form of DNA, showed a single line with a similar angular dependence to that of wet collagen. We have now established co-operation and have extended the NM% study of oriented NaDNA to variable temperature measurements and to samples hydrated with D,O. This work, which will be published in full separately (Migohelsen, Berendsen & Rupprecht, manuscript in preparation), has confirmed the correctness of Ruppreoht’s preliminary result also for samples of salmon sperm NaDNA. t Abbrewintiona
umd: NMR,
nuolear megnetio
resonance.
236
236
C. MIGCHELSEN,
H.
J.
C. BERENDSEN
AND
A.
RUPPRECHT
The structures of the three DNA conformations are well-known through the extensive X-ray diffraction studies performed by Wilkins and collaborators (Davies, 1967). The hydration of the B form has been the subject of special interest (Jacobson, 1953; Kavanau, 1964; Lewin, 1967) since this conformation of DNA exists i,n viva in the chromosomes and is also believed to exist in water solutions. Already in 1953 Jacobson pointed out the excellent fit of the Watson-Crick structure (Watson & Crick, 1953) to the structure of ice I. The B form with its pitch of 33.7 A fits within 0.5 A to seven repeats of4*74& which can be taken as the second neighbour distance in water and which should be the repeating distance in a chain of water molecules. The A form with a pitch of 28.15 A fits with a similar accuracy (0.3 b) to six water repeats. However, the C form with a pitch of 31.0 A does not fit well to either six or seven repeats of 4.74 A. Recently the hydration of LiDNA in the B and C form has been studied at room temperature for both H,O and D,O hydration. In this paper we compare the different results obtained for the three DNA conformations. For sample preparation the electrolyte contents of the DNA films were chosen to give the desired DNA configurations (Rupprecht & Forslind, MS. in preparation). Samples were prepared in a moist atmosphere by folding 8*5-mm wide films of oriented DNA back and forth to form concertina-like packs which were slightly compressed into square pieces of oriented DNA with the approximate dimensions 8.5 mm x 8.5 mm x 2 mm. The NMR measurements were carried out at room temperature with a Varian wide line NMR spectrometer operating at 60 MHz for proton spectra and at 10 MHz for deuteron resonance. The proton NMR spectra of oriented LiDNA in the B and C conformation showed a splitting into two lines at room temperature, in contrast to NaDNA in the A form, which showed only an angular dependent single line at room temperature. At lower temperatures the A form also gave a splitting. The angular dependence of the spectra of these three forms clearly demonstrated an average H-H interaction in the fibre direction but with different degrees of anisotropy. The deuteron NM.R spectra showed an angular dependent splitting into two lines for all three DNA conformations. The splittings were proportional to (3 co&?-l), the maximum values again indicating different degrees of anisotropy for the three forms. The values of maximum splitting, i.e. with the fibre direction parallel to the magnetic field, of the three forms of DNA are given in Table 1, together with their water contents. TABLE
1
Maximum splitting observed at room temperature in NMR spectra of oriented DNA in the A, B and C form
Salt
NaDNA LiDNA LiDNA
DNA conformation
A B c
Maximum Proton
splitting?
NMR
Single lines 0~09%0*14 0.21-0.28
7 Measured on some different samples. $ Splitting below + 10% N 0.09 gauss.
(gauss)
Deuteron 0.8-1.7 3-4 6.2-6.5
NMR
Relative humidity (%I 75 66 66
water content g/100 g &Y DNA - 43 N 41 N 34
LETTERS
TO
THE
237
EDITOR
These results indicate hydration structures in DNA similar to those found in collagen at various water contents. The average H-H interaction in the fibre direction is, however, smaller for DNA than for collagen with the same water content, as indicated by the lower values of maximum splittings. The results further indicate that proton exchange processes occur in NaDNA (Migchelsen et al., manuscript in preparation) which remove the cause in the dipole splitting. Such exchange protons between water molecules has also been found to occur in collagen (Migchelsen & Berendsen, manuscript in preparation). The model developed for collagen, i.e. the existence of chain-like structures of water molecules in which rotations are anisotropic as a result of the anisotropic distribution of llydrogen bonds, is applicable to DNA as well. It is doubtful, however, whether the fit of macromolecular repeating distances to multiples of the repeating distance in a water chain acts as a stabilizing factor. While the A and B forms show an excellent fit, the C form does not; nevertheless the C form shows the same type of hydration with a degree of anisotropy even higher than that of the A and B form. In this connection, reference should be given to a recent NMR study of the hydration of rayon fibres by Dehl (1968). The present work will be extended to variable temperature NMR measurements on oriented LiDNA and a full report will be given later. One of us (A. R.) wishes to acknowledge the support of the Carl-Bertel Nathhorst Scientific Foundation, the State Council of Technical Research and the Swedish Natural Science Research Council. A four-week EMBO fellowship made possible part of t~he co-operative work.
C.MIWHELSEN BERENDSEN
Laboratory of Physical Chemistry The University of Groningen Bloemsingel 10, Groningen The Netherlands
H.J.C.
A. RUPPRECHT
Bacteriological Bioengineering Department of Bacteriology Karolinska Institutet Stockholm, Sweden Received
17 June
1968 REFERENCES
Berendsen, H. J. C. (1960). Biol. Bull. 119, 287. Berendsen, H. J. C. (1962a). Thesis, University of Groningen. Berendsen, H. J. C. (19626). J. Chem. P&p. 36, 3297. Berendsen, H. J. C. & Migchelsen, C. (1965). Ann. N.Y. Acud. Sci. 125, 365. Berendsen, H. J. C. t Migchelsen, C. (1966). Fed. Proc. 25, 998. Davies, D. R. (1967). Ann. Rev. Biochem. 36, part I, 321. Dehl, R. E. (1968). J. Chem. Phys. 48, 831. Jacobson, B. (1953). Nature, 172, 666. Kavanau, J. L. (1964). Water and Solute-Water Itinteractione. San Francisco, Calif: HoldenDay, Inc. Lewin, S. (1967). J. Theoret. Biol. 17, 181. Migchelsen, C. & Berendsen, H. J. C. (1967). In Magnetic Resonance and Relaxation, ed. by North-Holland Publ. Co. R. Blinc, p. 761. Amsterdam: Rupprecht, A. (1966a). Acta Chem. Stand. 20, 582. Rupprecht, A. (19666). Acta Chem. Stand. 20, 494. Watson, J. D. & Crick, F. H. C. (1963). Nature, 171, 737.
Hydration of DNA Comparison of Nuclear Magnetic Resonance Results for Oriented DNA intheR,BandCForm The nuclear magnetio resonance teahnique was used by Berendsen (1960,1962u,b) for a study of the hydration of oriented collagen fibres. The observed NMRt spectrum of water protons was superposed on a very broad signal from protons of the macromolecules. Owing to the large diEertmce in line width, it was possible to study this water signal separately. The water spectrum showed a splitting into three lines with the separation of the outer lines following the angular dependence (3 co&3 -1) for a dipole-dipole interaction between two magnetia nuclei, when the angle B between fibre axis and magnetic field was varied. This result indicated an average H-H interaction in the fibre direction. It was suggested that the oollagen macromolecule, because of the excellent fit of its axial repeat distance to a multiple of the repeat distance of ice I, would stabilize chains of reorienting water moleoules along the maoromolecule by hydrogen bonding at appropriate sites. At higher water oontents a single water line with an angular dependent line shape was observed, which could qualitatively be interpreted in agreement with the suggested model. This work has been extended to variable temperature measurements (Berendsen & Migchelsen, 1966) and to the study of oriented collagen samples hydrated with heavy water (Migohelsen & Berendsen, 1967) in order to obtain further information about the dynamic behaviour of water moleoules and protons in the water of hydration. The NMR spectrum of deuterons in collagen hydrated with heavy water showed a splitting into two lines with the same angular dependence but uaused by quadrupole interaction of the deuterium nuclei, with the average electric field gradient at the sites of the deuterium nuclei. This result indicated an average electric field gradient along the collagen fibre direction and gave further support to the suggested hydration model. Measurements have also been performed on oriented fibres of silk fibroin, keratin and salmon sperm DNA (Berendsen & Migchelsen, 1966) with results which, except for DNA, could be interpreted in agreement with the macromolecular structures. The expected H-H interaction in the fibre dire&ion of DNA was, however, reported later by Ruppreoht (1966~~)who had developed a wet-spinning method for preparation of films of highly oriented DNA (Rupprecht, 1968b). The calf thymus NaDNA sample studied, whioh was in the A form of DNA, showed a single line with a similar angular dependence to that of wet collagen. We have now established co-operation and have extended the NM% study of oriented NaDNA to variable temperature measurements and to samples hydrated with D,O. This work, which will be published in full separately (Migohelsen, Berendsen & Rupprecht, manuscript in preparation), has confirmed the correctness of Ruppreoht’s preliminary result also for samples of salmon sperm NaDNA. t Abbrewintiona
umd: NMR,
nuolear megnetio
resonance.
236
236
C. MIGCHELSEN,
H.
J.
C. BERENDSEN
AND
A.
RUPPRECHT
The structures of the three DNA conformations are well-known through the extensive X-ray diffraction studies performed by Wilkins and collaborators (Davies, 1967). The hydration of the B form has been the subject of special interest (Jacobson, 1953; Kavanau, 1964; Lewin, 1967) since this conformation of DNA exists i,n viva in the chromosomes and is also believed to exist in water solutions. Already in 1953 Jacobson pointed out the excellent fit of the Watson-Crick structure (Watson & Crick, 1953) to the structure of ice I. The B form with its pitch of 33.7 A fits within 0.5 A to seven repeats of4*74& which can be taken as the second neighbour distance in water and which should be the repeating distance in a chain of water molecules. The A form with a pitch of 28.15 A fits with a similar accuracy (0.3 b) to six water repeats. However, the C form with a pitch of 31.0 A does not fit well to either six or seven repeats of 4.74 A. Recently the hydration of LiDNA in the B and C form has been studied at room temperature for both H,O and D,O hydration. In this paper we compare the different results obtained for the three DNA conformations. For sample preparation the electrolyte contents of the DNA films were chosen to give the desired DNA configurations (Rupprecht & Forslind, MS. in preparation). Samples were prepared in a moist atmosphere by folding 8*5-mm wide films of oriented DNA back and forth to form concertina-like packs which were slightly compressed into square pieces of oriented DNA with the approximate dimensions 8.5 mm x 8.5 mm x 2 mm. The NMR measurements were carried out at room temperature with a Varian wide line NMR spectrometer operating at 60 MHz for proton spectra and at 10 MHz for deuteron resonance. The proton NMR spectra of oriented LiDNA in the B and C conformation showed a splitting into two lines at room temperature, in contrast to NaDNA in the A form, which showed only an angular dependent single line at room temperature. At lower temperatures the A form also gave a splitting. The angular dependence of the spectra of these three forms clearly demonstrated an average H-H interaction in the fibre direction but with different degrees of anisotropy. The deuteron NM.R spectra showed an angular dependent splitting into two lines for all three DNA conformations. The splittings were proportional to (3 co&?-l), the maximum values again indicating different degrees of anisotropy for the three forms. The values of maximum splitting, i.e. with the fibre direction parallel to the magnetic field, of the three forms of DNA are given in Table 1, together with their water contents. TABLE
1
Maximum splitting observed at room temperature in NMR spectra of oriented DNA in the A, B and C form
Salt
NaDNA LiDNA LiDNA
DNA conformation
A B c
Maximum Proton
splitting?
NMR
Single lines 0~09%0*14 0.21-0.28
7 Measured on some different samples. $ Splitting below + 10% N 0.09 gauss.
(gauss)
Deuteron 0.8-1.7 3-4 6.2-6.5
NMR
Relative humidity (%I 75 66 66
water content g/100 g &Y DNA - 43 N 41 N 34
LETTERS
TO
THE
237
EDITOR
These results indicate hydration structures in DNA similar to those found in collagen at various water contents. The average H-H interaction in the fibre direction is, however, smaller for DNA than for collagen with the same water content, as indicated by the lower values of maximum splittings. The results further indicate that proton exchange processes occur in NaDNA (Migchelsen et al., manuscript in preparation) which remove the cause in the dipole splitting. Such exchange protons between water molecules has also been found to occur in collagen (Migchelsen & Berendsen, manuscript in preparation). The model developed for collagen, i.e. the existence of chain-like structures of water molecules in which rotations are anisotropic as a result of the anisotropic distribution of llydrogen bonds, is applicable to DNA as well. It is doubtful, however, whether the fit of macromolecular repeating distances to multiples of the repeating distance in a water chain acts as a stabilizing factor. While the A and B forms show an excellent fit, the C form does not; nevertheless the C form shows the same type of hydration with a degree of anisotropy even higher than that of the A and B form. In this connection, reference should be given to a recent NMR study of the hydration of rayon fibres by Dehl (1968). The present work will be extended to variable temperature NMR measurements on oriented LiDNA and a full report will be given later. One of us (A. R.) wishes to acknowledge the support of the Carl-Bertel Nathhorst Scientific Foundation, the State Council of Technical Research and the Swedish Natural Science Research Council. A four-week EMBO fellowship made possible part of t~he co-operative work.
C.MIWHELSEN BERENDSEN
Laboratory of Physical Chemistry The University of Groningen Bloemsingel 10, Groningen The Netherlands
H.J.C.
A. RUPPRECHT
Bacteriological Bioengineering Department of Bacteriology Karolinska Institutet Stockholm, Sweden Received
17 June
1968 REFERENCES
Berendsen, H. J. C. (1960). Biol. Bull. 119, 287. Berendsen, H. J. C. (1962a). Thesis, University of Groningen. Berendsen, H. J. C. (19626). J. Chem. P&p. 36, 3297. Berendsen, H. J. C. & Migchelsen, C. (1965). Ann. N.Y. Acud. Sci. 125, 365. Berendsen, H. J. C. t Migchelsen, C. (1966). Fed. Proc. 25, 998. Davies, D. R. (1967). Ann. Rev. Biochem. 36, part I, 321. Dehl, R. E. (1968). J. Chem. Phys. 48, 831. Jacobson, B. (1953). Nature, 172, 666. Kavanau, J. L. (1964). Water and Solute-Water Itinteractione. San Francisco, Calif: HoldenDay, Inc. Lewin, S. (1967). J. Theoret. Biol. 17, 181. Migchelsen, C. & Berendsen, H. J. C. (1967). In Magnetic Resonance and Relaxation, ed. by North-Holland Publ. Co. R. Blinc, p. 761. Amsterdam: Rupprecht, A. (1966a). Acta Chem. Stand. 20, 582. Rupprecht, A. (19666). Acta Chem. Stand. 20, 494. Watson, J. D. & Crick, F. H. C. (1963). Nature, 171, 737.