B-Z conformational transition and hydration of poly (dC-dG).poly (dC-dG) in fibres G. Aibiser and S. Pr6milat Laboratoire de Biophysique Mol~culaire, UA C N R S 494, Facultb des Sciences, Universitk de Nancy I, BP No. 239, 54506 Vandoeuvre les Nancy, France
(Received 19 July 1991; revised 16 January 1992) The B-Z transition o f poly( dC-dG).poly( dC-dG) has been studied by fibre X-ray diffraction and measurement o f fibre dimensions. The polymorphism o f the Z form is well observed as a function of variations o f the r.h. (relative humidity). The Z to B transition is obtained at very high r.h. values. The cooperative transition from B to Z is associated with a disorganization o f the fibre. Details about the hydration o f the polynucleotide during conformational transitions are presented and it is shown that a nucleotide in Z form can be associated with up to 16 water molecules and up to 22 when in the B form. Keywords: poly(dC-dG).poly(dC-dG);B-Z helical transition; hydration; X-ray diffraction;fibre dimensions
Introduction The left-handed double helical conformation of oligonucleotides composed of sequences o f alternating cytosine and guanine have been precisely determined by X-ray crystallography 1. This Z form of polynucleotides has also been observed in solutions 2 and in fibres by X-ray diffraction of poly(dC-dG).poly(dC-dG)3-7. Note that in these last cases, the left-handed helical conformation is frequently named the S form 8. Beside their interesting physicochemical properties, the biological role of left-handed conformations of DNA have been determined by in vivo experiments on different plasmids 9. The left-handed helical form of DNA intervenes in genetic recombination 1° and in the transcription process 11. It has also been shown that some proteins and ligands 12 can be more tightly bound to polynucleotide sequences when they are in the Z form rather than when in B conformation. Supercoiling, generated by biological processes, is a factor which can also stabilize stretches of Z-DNA in vivo as demonstrated in recent work 13. In order to gain information about the effects on the form transitions of varying external parameters, the study of poly(dC-dG).poly(dC-dG) molecules in fibres is particularly interesting as it can adopt left-handed as well as right-handed helical conformations depending on the physicochemical conditions 6. Moreover, the transition between these two families of double helixes can be followed by fibre X-ray diffraction associated with measurements of fibre dimensions, according to an experimental method previously established 14 and applied to the analysis of the hydration ~s of DNA during conformational transitions. This method is used in the present study and it allowed us to establish experimental curves for the B-Z transitions of poly(dC-dG).poly(dC-dG) and to determine the changes 0141-8130/92/030161-05 © 1992 Butterworth-HeinemannLimited
of hydration during the conformational modifications of the polynucleotide.
Experimental Lyophilized samples of poly(dC-dG).poly(dC-dG), purchased from Pharmacia, were used without any further purification to obtain, by addition of distilled water at pH 7, a gel from which fibres were drawn. As the salt concentration is important in determining the type of helical conformation adopted by the polynucleotide molecules, the following empirical procedure was adopted in order to achieve the appropriate concentration of NaC1 in the fibre. X-ray patterns were first obtained (conventional generator; K~ of Cu) to test the fibre and, according to the type of helix pattern observed, a small amount of a solution of 0.01 M NaC1 was added to the fibre which was then tested again by obtaining another X-ray pattern. This was made again until the desired helical conformation was achieved. The organization of the fibres is improved when they are submitted to cycles of variations in the r.h. (relative humidity) from very high (95%) to low values. During these cycles, the fibre is maintained vertical with a light tension applied to its free extremity 16. This tension is withdrawn when actual measurements are performed. The side of the X-ray camera facing the collimator has been made of a transparent plastic sheet which allows us to measure the fibre dimensions, before and after every X-ray exposure, with a binocular microscope. Fibre X-ray patterns associated with the different fibre lengths are therefore obtained under the same well controlled conditions of r.h. as for fibre dimension measurements 14. The determination of the number G of water molecules located in the vicinity of every nucleotide as well as its variations during conformational transitions is made
Int. J. Biol. Macromol., 1992, Vol. 14, June
161
B-Z conformational transition." G. Albiser and S. Prbmilat a c c o r d i n g to the p r o c e d u r e described previously is. W e therefore a p p l i e d the following r e l a t i o n : V
G=K-Nn where K = 1.67 1019 m m - 3 a n d where Vis the difference between the fibre v o l u m e m e a s u r e d at a given r.h. a n d at 0 % r.h.; N a n d n are the n u m b e r s of nucleotides respectively l o c a t e d a l o n g the fibre axis (between two p o i n t s chosen for the length m e a s u r e m e n t ) a n d in a section of the fibre. Values of N and n are o b t a i n e d from the d e t e r m i n a t i o n of the c r y s t a l l o g r a p h i c p a r a m e t e r s following the m e t h o d a l r e a d y used 15.
Results X-ray diffraction F i b r e s of p o l y ( d C - d G ) . p o l y ( d C - d G ) used in the present s t u d y are well organized. T h e y are n o t in the A helical form at 75% r.h. b u t in the Z c o n f o r m a t i o n . In Table1 are given the different characteristic p a r a m e t e r s of the d o u b l e helical c o n f o r m a t i o n s determ i n e d from X - r a y diffraction p a t t e r n s o b t a i n e d at
different r.h. values. O n e can see that m e a s u r e m e n t is possible even on p a t t e r n s o b t a i n e d at very low r.h. values. Actually, the X - r a y p a t t e r n s d o present a g o o d definition for r.h. between 35 a n d 85% (Figure la, b); we then get the Z form of the p o l y n u c l e o t i d e . W h e n the r.h. has risen to values between 85 a n d 9 5 % , the diffraction spots b e c o m e m o r e diffused (Figure lc) a n d the p a t t e r n o b t a i n e d at values of the r.h. higher t h a n 9 5 % is characteristic of the B form (Figure ld) with the classic helical p a r a m e t e r s P = 33.8 A a n d p = 3 . 3 9 A . O n e should note t h a t the present e x p e r i m e n t a l o b s e r v a t i o n s are in a g r e e m e n t with results a l r e a d y o b t a i n e d on fibres of p o l y ( d C - d G ) . p o l y ( d C - d G ) with s y n c h r o t r o n r a d i a t i o n 7. W e n o t e d an e v o l u t i o n of the p a r a m e t e r s P a n d p (Table 1 ) in the Z form while the r.h. was increased. But the r a t i o Pip r e m a i n e d a l m o s t c o n s t a n t with a value very n e a r to 6. D u r i n g these modifications of the Z form, the helical p a r a m e t e r s c o r r e s p o n d i n g to the two forms are defined as Z 1 (or S l ) a n d Z 2 (or $2) 7. These c o n f o r m a t i o n s , o b s e r v e d at 4 0 % a n d 88% r.h. respectively, c o r r e s p o n d to P = 43 A a n d P = 45 A. W h e n the p o l y n u c l e o t i d e is in its Z form, the m e r i d i a n spots o b s e r v e d on the X - r a y p a t t e r n are characteristic of
Table 1 Z-B-Z transitions as functions of the relative humidity R.h. (%) 6 25 47 75 85 90 95 97.5 90.5 88 65 18
Form
a = b (A)
C = P (A)
p (A)
Pm (A)
Z1 Z1 Z1 Z1 Z2 Z2 Z2 B B Z2 B Zz Z1 Z1
17.0 17.3 18.0 18.7 19.5 21.6 23.8 47.0 42.0
42.0 42.5 43.5 43.8 44.2 44.2 46.5 33.8 33.8 45.2 33.8 44.4 43.4 42.0
6.85 6.92 7.13 7.25 7.32 7.36 6.46
3.42 3.46 3.51 3.62 3.66 3.68 3.78 3.39 3.37 3.75 3.37 3.63 3.60 3.45
40.0 18.5 17.5
6.46 6.40 7.27 7.20 6.90
u
G 6.1 6.1 6 1 6.0 6.0 6.0
10.0 10.0 10.0 6.1 6.0 6.0
0.3 1.5 3.5 6.1 9.8 16.6 22.7 15.3 10.8 3.0 0.1
Crystallographic parameters: a = b = parameter of the hexagonal cell; c = P = helix pitch; p = axial separation of the repeat!ng unit (dinucleotide); Pm = axial separation of the repeating unit (one nucleotide); u = Pip = number of repeating units per helical turn; G = number of water molecules per nucleotide
(a)
(b)
Figure 1 Z-B transition. X-ray patterns obtained at: (a) 23% r.h. form; (d) 97.5% r.h. - B form
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Int. J. Biol. M a c r o m o l . , 1992, Vol. 14, June
(c)
(d)
Z 1 form; (b) 86% r.h. - Z 2 form; (c) 95% r.h. - limit of
Z2
B-Z conformational transition." G. Albiser and S. Prbmilat a dinucleotide as the repeating unit. These spots are modified with increasing r.h. and finally disappear for r.h. values higher than 95%. We then have a change of conformation; the meridian spot which corresponds to 7.5 A disappears and the one associated with a distance of 6.4 A is observed on X-ray patterns. When the B form is obtained at 97.5% r.h., only one meridian spot at 3.4 A is present; the repeating unit is then one nucleotide in one helical conformation. The curve in Figure2 represents the variations with the r.h. of the value Pm of the mean axial projection of a nucleotide. We can see
L(mm) 1.60
1.55
1,50
1~45
1.40
T
3.z' Pg~"
/ I/ 1/
3.6 / /
f
"//
Z2
Fibre dimensions
I I
/ J
i I ~B
J
3.5 Z 1
1
34 I O
that this parameter gives increasing values when the r.h. rises from 0 to 95% and it decreases very steeply for r.h. higher than 95%. Moreover, we note that when the Z form is observed, the parameter a of the hexagonal unit cell varies from 17 to 23.8 A. The unit cell then comprises only one double helix 7 and the value of a therefore represents the distance between the axes of the nearest neighbour helixes. With the transition from Z to B, the distance between axes of helixes in the fibre varies from 24 to 27 A and the cell contains then three double helixes 17, with a = 47 A. To study the reverse transition from B to Z, the r.h. was lowered very slowly from the value of 97%. When the r.h. is near to 90% a mixture of B and Z forms is still observed on the X-ray patterns (Figure 3a). One can see a meridian spot at 3.4 A (B form), a meridian at 6.46 A associated with the alternating sequence of dinucleotides and only one meridian spot at 3.74 A characterizing the Z form. At 88% r.h. the meridian spot at 7.27 A (Z form) reappears on the patterns (Figure 3b) and at 80% the Z form alone is observed (Figure 3c). We noted that when the r.h. is lowered suddenly from 97.5% (the polynucleotide is then in the B form) to 30%, one then obtains a poor X-ray pattern with a meridian spot at 3.32 A and an important second layer line which allows us to determine a helical pitch of 30/~ (see Figure 3d). This distorted B conformation (or C form) 14, which corresponds to nine nucleotide pairs per helical turn, is modified when the r.h. is increased and one again gets the Z form at 80% r.h. but the fibre is then very much disorganized.
2tO
410
'
6tO
80
'
100
r.h.%
Figure 2 Variations of the fibre length L ( H ) for the Z-B-Z transitions and of the rise per nucleotide Pm ( n ) for the Z-B transition as functions of the relative humidity
(a)
(b)
Measurements of fibre dimensions during the Z to B and B to Z transitions have given values presented in Figures 2 and 4. These measurements are performed under the same conditions of r.h. as those realized for X-ray diffraction. In Figure 2, we can see how the fibre length increases up to 95% r.h. and it then suddenly decreases when the r.h. is higher than 95%. At 97.5% r.h. the fibre length is even lower than at 0%0. Conversely the fibre diameter (Figure 4) increases slightly for r.h. up to 80% and steeply thereafter. When the r.h. is decreased from 97.5%, the fibre length remains almost constant (Figure 2) for r.h. up to 88% (note that at such a value of r.h., X-ray patterns show
(c)
(d)
Figure 3 B-Z transition. X-ray patterns obtained at: (a) 90% r.h. - B-Z forms; (b) 85% r.h. - Z2 form; (c) 40% r.h. - Z1 form; (d) result of a sudden decrease of r.h. from 97% to 50%
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163
B-Z conformational transition: G. Albiser and S. Prbmilat This fact is observed when poly(dC-dG).poly(dC-dG) is in its Z form as well as when the transition to the B form occurs. We also noted that the change of conformation from Z to B appears when the distance between helix axes has a value of at least 23.8/~ and that distance increases to 27/~ for the B form. Besides, we know ~ that the diameter of the double helixes of poly(dC-dG).poly(dC-dG) in the Z form is near to 18 ~ and that it is 20 A in the B conformation 24. We therefore see that relatively large interhelical distances are necessary in order to allow the important molecular movements inducing the transition between left- and right-handed helical conformations of poly(dC-dG).poly(dC-dG). Such enlargement of the packing distances of the double helixes is due to water molecules and is obtained by raising the r.h. to values very near to 100%.
0.26 0.23
0.20
0.17 B
26
/ /
z~
23 /.a
20 17
/.ti
j._
_
-
-!
.
I
20
.
.
.
--
d
--
I
40
_
B
--
.
--
--
--
I
I
I
60
80
100
r.h.%
Figure 4 Variations of the fibre diameter D ( 0 - - 0 ) and the distance between helix axes d.a (ll---m) for the Z-B transition as functions of the relative humidity mixtures of B and Z forms). For r.h. lower than 80% the fibre length decreases but one does not obtain the values for the length that we had at the beginning of the process (the fibre length is 5% smaller). Nevertheless, the fibre diameter again takes its initial value (as the crystallographic cell parameter). An additional cycle of r.h. variations gives the same effect and the fibre length is .again lowered when the r.h. is at 0%. Moreover, when the r.h. is suddenly decreased from 97.5 to 30% such a loss of the fibre length is even larger (near to 10% ).
Hydration of poly( dC-dG).poly( dC-dG) Results obtained simultaneously from X-ray diffraction and measurements of variations of the fibre dimensions with the r.h., allowed us to follow the changes in the number of water molecules associated with a nucleotide during the conformational transitions ~5. We therefore determined (Table 1 ) that there are two water molecules per nucleotide when the poly(dC-dG).poly(dC-dG) is in the Z~ form. Note that these figures do not take into account water which remains in the fibre at 0% r.h. ~8. This number increases regularly during the evolution from Z1 to Z 2 and there are 16 water molecules per nucleotide in this last conformation (just before the transition to the B form). The complete transition from Z 2 to B necessitates the addition of six water molecules per nucleotide which then have 22 water molecules in their vicinity; this last number is even increased when the r.h. is more than 97.5%. These results are in agreement with data deduced from studies on poly (dC-dG).poly (dC-dG) films by infrared spectroscopy 19 and with values obtained from studies on solutions by dielectric relaxation 2°. Moreover, the present observations concerning the variations of the number of water molecules during the B-Z conformational transition are complementary to those obtained from X-ray crystallography where the precise position of water in the Z double helix can be estimated 2a-23. The comparison of results obtained from X-ray diffraction with the measurement of variations of the fibre dimensions indicates a proportionality between the interhelical distance and the fibre diameter (see Figure 4).
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Int. J. Biol. Macromol., 1992, Vol. 14, June
Discussion These results, obtained simultaneously from X-ray diffraction and measurement of dimensions of poly(dCdG).poly(dC-dG) fibres, clearly show the reversibility of the B-Z transition. However, we noted that the form transition is realized at very high and in a narrow interval of r.h. values (mainly for the Z z to B transition). At very low r.h., the polynucleotide is in the Z 1 form and it evolves progressively, as the r.h. rises, towards the Z z form until it changes steeply to the B form. The application of the present experimental method ~4 allowed us to determine that the Z form of poly(dCdG).poly(dC-dC) can be associated with up to 16 water molecules per nucleotide and that six more are necessary to achieve the Z to B transition. An almost perfect proportionality between the fibre length and the helical parameter p (an average value for Z) is observed (see Figure 2) for the transitions Z1-Z2-B. This fact clearly indicates the polymorphism of the Z form 25 of poly(dC-dG).poly(dC-dG). But it should be noted that, even if p varies with the r.h., the number of dinucleotides per helical turn remains equal to six. Moreover, the Z 1 helical conformation corresponds to very low r.h. while Z z is obtained at very high r.h., just before the transition to the B form. We can add that the continuous Z ~-Z2 change of form is perfectly reversible. Conversely, the reverse transition from B to Z, observed when the r.h. is lowered, is much slower as already noted in studies on solutions z6 or on fibres ~9. Moreover, under the present experimental conditions, this change of form is obtained in a larger interval of r.h. We first observed a mixture of B and Z 2 forms when the r.h. is decreased and, thereafter, a progressive change from Z 2 to Z1. The transition from B to Z is always associated with a disorganization of the fibre and that effect is more important when the r.h. is suddenly lowered. As a consequence, we observed that, even for very slow variations of the r.h., there is no proportionality between the fibre length and the helical parameter p. Larger lengths of the fibre should correspond to the transition from B to Z2, but the disorganization of the fibre is superimposed on that transition and any observation of the fibre length variation becomes impossible when the r.h. is lowered from the value of 97%. We can add that a tension applied on the fibre ~6 improves the B-Z transition and actually maintains the fibre organization, but it also induces sliding of the
B - Z c o n f o r m a t i o n a l transition." G. A l b i s e r a n d S. P r b m i l a t
polynucleotide helixes past one another and that makes the fibre length measurements irrelevant. Nevertheless, it appears that the change from B to Z conformation remains cooperative because no well organized form, different from B or Z, can actually be observed on X-ray patterns during the transition. This is in agreement with results obtained from studies on solutions 27 29 and also on films 19 of poly(dC-dG).poly(dC-dG).
10 11 12 13 14 15 16
References 1 2 3 4 5 6 7 8 9
Wang, A. H. J., Quigley, G. J., Kolpak, F. J., Crawford, J. L., Van Boom, J. H., Van der Marel, G. and Rich, A. Nature 1979, 282, 680 Pohl, F. M. and Jovin, T. M. J. Mol. Biol. 1972, 67, 375 Arnott, S., Chandrasekaran, R., Birdsall, D. L., Leslie, A. G. W. and Ratliff, R. L. Nature 1980, 283, 743 Behe, M., Zimmerman, S. and Felsenfeld, G. Nature 1981, 293, 233 Sasisekharan, V. and Brahmachari, S. K. Curr. Sci. 1981,50, 10 Mahendrasingam, A., Pigram, W. J., Fuller, W., Brahms, J. and Vergne, J. J. Mol. Biol. 1983, 168, 897 Manhendrasingam, A., Denny, R. C., Forsyth, V. T., Greenall, R. J., Pigram, W. J., Papiz, M. Z. and Fuller, W. Inst. Phys. Conf. 1990, 101,225 Leslie, A. G. W., Arnott, S., Chandrasekaran, R. and Ratliff, R. L. J. Mol. Biol. 1980, 143, 49 Jaworski, A., Hsieh, W. T., Blaho, J. A., Larson, J. E. and Wells, R. D. Science 1987, 238, 773
17 18 19 20 21 22 23 24 25 26 27 28 29
Kmiec, E. B. and Holloman, W. K. Cell 1986, 44, 445 Vasicek, T. J., McDevitt, B. E., Freeman, M. W., Fennick, B. J., Hendy, G. N., Potts, J. T., Rich, A. and Kronenberg, H. M. Proc. Natl. Acad. Sci. USA 1983, 80, 2127 Fishel, R. A., Detmer, K. and Rich, A. Proc. Natl. Acad. Sci. USA 1988, 85, 36 Rahmouni, A. R. and Wells, R. D. Science 1989, 246, 368 Premilat, S., Harmouchi, M. and Albiser, G. Biophys. Chem. 1990, 35, 37 Harmouchi, M., Albiser, G. and Pr6milat, S. Eur. Biophys. J. 1990, 19, 87 Albiser, G., Harmouchi, M. and Pr~milat, S. J. Biomol. Struct. Dynam. 1988, 6, 359 Langridge, R., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. and Hamilton, L. D. J. Mol. Biol. 1960, 2, 19 Tao, N. J., Lindsay, S. M. and Rupprecht, A. Biopolymers 1989, 28, 1019 Pilet, J. and Leng, M. Proc. Natl. Acad. Sci. USA 1982, 79, 26 Umehara, T., Kuwabara, S., Mashimo, S. and Yagihara, S. Biopolymers 1990, 30, 649 Westhof, E. Int. J. Biol. Macromol. 1987, 9, 185 Saenger, W., Hunter, W. N. and Kennard, O. Nature 1986, 324, 385 Gessner, R. V., Frederick, C. A., Quigley, G. J., Rich, A. and Wang, A. H. J. J. Biol. Chem. 1989, 264, 7921 Pr6milat, S. and Albiser, G. Nucl. Acids Res. 1983, 11, 1897 Drew, H.R. andDickerson, R.E.J. Mol. Biol. 1981,152,723 Goto, S. Biopolymers 1984, 23, 2211 Soumpasis, D. M., Robert-Nicoud, M. and Jovin, T. M. FEBS Lett. 1987, 213, 341 Preisler, R. S. Biochem. Biophys. R. Comm. 1987, 148, 609 Hamori, E. and Jovin, T. M. Biophys. Chem. 1987, 26, 375
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(Received 19 July 1991; revised 16 January 1992) The B-Z transition o f poly( dC-dG).poly( dC-dG) has been studied by fibre X-ray diffraction and measurement o f fibre dimensions. The polymorphism o f the Z form is well observed as a function of variations o f the r.h. (relative humidity). The Z to B transition is obtained at very high r.h. values. The cooperative transition from B to Z is associated with a disorganization o f the fibre. Details about the hydration o f the polynucleotide during conformational transitions are presented and it is shown that a nucleotide in Z form can be associated with up to 16 water molecules and up to 22 when in the B form. Keywords: poly(dC-dG).poly(dC-dG);B-Z helical transition; hydration; X-ray diffraction;fibre dimensions
Introduction The left-handed double helical conformation of oligonucleotides composed of sequences o f alternating cytosine and guanine have been precisely determined by X-ray crystallography 1. This Z form of polynucleotides has also been observed in solutions 2 and in fibres by X-ray diffraction of poly(dC-dG).poly(dC-dG)3-7. Note that in these last cases, the left-handed helical conformation is frequently named the S form 8. Beside their interesting physicochemical properties, the biological role of left-handed conformations of DNA have been determined by in vivo experiments on different plasmids 9. The left-handed helical form of DNA intervenes in genetic recombination 1° and in the transcription process 11. It has also been shown that some proteins and ligands 12 can be more tightly bound to polynucleotide sequences when they are in the Z form rather than when in B conformation. Supercoiling, generated by biological processes, is a factor which can also stabilize stretches of Z-DNA in vivo as demonstrated in recent work 13. In order to gain information about the effects on the form transitions of varying external parameters, the study of poly(dC-dG).poly(dC-dG) molecules in fibres is particularly interesting as it can adopt left-handed as well as right-handed helical conformations depending on the physicochemical conditions 6. Moreover, the transition between these two families of double helixes can be followed by fibre X-ray diffraction associated with measurements of fibre dimensions, according to an experimental method previously established 14 and applied to the analysis of the hydration ~s of DNA during conformational transitions. This method is used in the present study and it allowed us to establish experimental curves for the B-Z transitions of poly(dC-dG).poly(dC-dG) and to determine the changes 0141-8130/92/030161-05 © 1992 Butterworth-HeinemannLimited
of hydration during the conformational modifications of the polynucleotide.
Experimental Lyophilized samples of poly(dC-dG).poly(dC-dG), purchased from Pharmacia, were used without any further purification to obtain, by addition of distilled water at pH 7, a gel from which fibres were drawn. As the salt concentration is important in determining the type of helical conformation adopted by the polynucleotide molecules, the following empirical procedure was adopted in order to achieve the appropriate concentration of NaC1 in the fibre. X-ray patterns were first obtained (conventional generator; K~ of Cu) to test the fibre and, according to the type of helix pattern observed, a small amount of a solution of 0.01 M NaC1 was added to the fibre which was then tested again by obtaining another X-ray pattern. This was made again until the desired helical conformation was achieved. The organization of the fibres is improved when they are submitted to cycles of variations in the r.h. (relative humidity) from very high (95%) to low values. During these cycles, the fibre is maintained vertical with a light tension applied to its free extremity 16. This tension is withdrawn when actual measurements are performed. The side of the X-ray camera facing the collimator has been made of a transparent plastic sheet which allows us to measure the fibre dimensions, before and after every X-ray exposure, with a binocular microscope. Fibre X-ray patterns associated with the different fibre lengths are therefore obtained under the same well controlled conditions of r.h. as for fibre dimension measurements 14. The determination of the number G of water molecules located in the vicinity of every nucleotide as well as its variations during conformational transitions is made
Int. J. Biol. Macromol., 1992, Vol. 14, June
161
B-Z conformational transition." G. Albiser and S. Prbmilat a c c o r d i n g to the p r o c e d u r e described previously is. W e therefore a p p l i e d the following r e l a t i o n : V
G=K-Nn where K = 1.67 1019 m m - 3 a n d where Vis the difference between the fibre v o l u m e m e a s u r e d at a given r.h. a n d at 0 % r.h.; N a n d n are the n u m b e r s of nucleotides respectively l o c a t e d a l o n g the fibre axis (between two p o i n t s chosen for the length m e a s u r e m e n t ) a n d in a section of the fibre. Values of N and n are o b t a i n e d from the d e t e r m i n a t i o n of the c r y s t a l l o g r a p h i c p a r a m e t e r s following the m e t h o d a l r e a d y used 15.
Results X-ray diffraction F i b r e s of p o l y ( d C - d G ) . p o l y ( d C - d G ) used in the present s t u d y are well organized. T h e y are n o t in the A helical form at 75% r.h. b u t in the Z c o n f o r m a t i o n . In Table1 are given the different characteristic p a r a m e t e r s of the d o u b l e helical c o n f o r m a t i o n s determ i n e d from X - r a y diffraction p a t t e r n s o b t a i n e d at
different r.h. values. O n e can see that m e a s u r e m e n t is possible even on p a t t e r n s o b t a i n e d at very low r.h. values. Actually, the X - r a y p a t t e r n s d o present a g o o d definition for r.h. between 35 a n d 85% (Figure la, b); we then get the Z form of the p o l y n u c l e o t i d e . W h e n the r.h. has risen to values between 85 a n d 9 5 % , the diffraction spots b e c o m e m o r e diffused (Figure lc) a n d the p a t t e r n o b t a i n e d at values of the r.h. higher t h a n 9 5 % is characteristic of the B form (Figure ld) with the classic helical p a r a m e t e r s P = 33.8 A a n d p = 3 . 3 9 A . O n e should note t h a t the present e x p e r i m e n t a l o b s e r v a t i o n s are in a g r e e m e n t with results a l r e a d y o b t a i n e d on fibres of p o l y ( d C - d G ) . p o l y ( d C - d G ) with s y n c h r o t r o n r a d i a t i o n 7. W e n o t e d an e v o l u t i o n of the p a r a m e t e r s P a n d p (Table 1 ) in the Z form while the r.h. was increased. But the r a t i o Pip r e m a i n e d a l m o s t c o n s t a n t with a value very n e a r to 6. D u r i n g these modifications of the Z form, the helical p a r a m e t e r s c o r r e s p o n d i n g to the two forms are defined as Z 1 (or S l ) a n d Z 2 (or $2) 7. These c o n f o r m a t i o n s , o b s e r v e d at 4 0 % a n d 88% r.h. respectively, c o r r e s p o n d to P = 43 A a n d P = 45 A. W h e n the p o l y n u c l e o t i d e is in its Z form, the m e r i d i a n spots o b s e r v e d on the X - r a y p a t t e r n are characteristic of
Table 1 Z-B-Z transitions as functions of the relative humidity R.h. (%) 6 25 47 75 85 90 95 97.5 90.5 88 65 18
Form
a = b (A)
C = P (A)
p (A)
Pm (A)
Z1 Z1 Z1 Z1 Z2 Z2 Z2 B B Z2 B Zz Z1 Z1
17.0 17.3 18.0 18.7 19.5 21.6 23.8 47.0 42.0
42.0 42.5 43.5 43.8 44.2 44.2 46.5 33.8 33.8 45.2 33.8 44.4 43.4 42.0
6.85 6.92 7.13 7.25 7.32 7.36 6.46
3.42 3.46 3.51 3.62 3.66 3.68 3.78 3.39 3.37 3.75 3.37 3.63 3.60 3.45
40.0 18.5 17.5
6.46 6.40 7.27 7.20 6.90
u
G 6.1 6.1 6 1 6.0 6.0 6.0
10.0 10.0 10.0 6.1 6.0 6.0
0.3 1.5 3.5 6.1 9.8 16.6 22.7 15.3 10.8 3.0 0.1
Crystallographic parameters: a = b = parameter of the hexagonal cell; c = P = helix pitch; p = axial separation of the repeat!ng unit (dinucleotide); Pm = axial separation of the repeating unit (one nucleotide); u = Pip = number of repeating units per helical turn; G = number of water molecules per nucleotide
(a)
(b)
Figure 1 Z-B transition. X-ray patterns obtained at: (a) 23% r.h. form; (d) 97.5% r.h. - B form
162
Int. J. Biol. M a c r o m o l . , 1992, Vol. 14, June
(c)
(d)
Z 1 form; (b) 86% r.h. - Z 2 form; (c) 95% r.h. - limit of
Z2
B-Z conformational transition." G. Albiser and S. Prbmilat a dinucleotide as the repeating unit. These spots are modified with increasing r.h. and finally disappear for r.h. values higher than 95%. We then have a change of conformation; the meridian spot which corresponds to 7.5 A disappears and the one associated with a distance of 6.4 A is observed on X-ray patterns. When the B form is obtained at 97.5% r.h., only one meridian spot at 3.4 A is present; the repeating unit is then one nucleotide in one helical conformation. The curve in Figure2 represents the variations with the r.h. of the value Pm of the mean axial projection of a nucleotide. We can see
L(mm) 1.60
1.55
1,50
1~45
1.40
T
3.z' Pg~"
/ I/ 1/
3.6 / /
f
"//
Z2
Fibre dimensions
I I
/ J
i I ~B
J
3.5 Z 1
1
34 I O
that this parameter gives increasing values when the r.h. rises from 0 to 95% and it decreases very steeply for r.h. higher than 95%. Moreover, we note that when the Z form is observed, the parameter a of the hexagonal unit cell varies from 17 to 23.8 A. The unit cell then comprises only one double helix 7 and the value of a therefore represents the distance between the axes of the nearest neighbour helixes. With the transition from Z to B, the distance between axes of helixes in the fibre varies from 24 to 27 A and the cell contains then three double helixes 17, with a = 47 A. To study the reverse transition from B to Z, the r.h. was lowered very slowly from the value of 97%. When the r.h. is near to 90% a mixture of B and Z forms is still observed on the X-ray patterns (Figure 3a). One can see a meridian spot at 3.4 A (B form), a meridian at 6.46 A associated with the alternating sequence of dinucleotides and only one meridian spot at 3.74 A characterizing the Z form. At 88% r.h. the meridian spot at 7.27 A (Z form) reappears on the patterns (Figure 3b) and at 80% the Z form alone is observed (Figure 3c). We noted that when the r.h. is lowered suddenly from 97.5% (the polynucleotide is then in the B form) to 30%, one then obtains a poor X-ray pattern with a meridian spot at 3.32 A and an important second layer line which allows us to determine a helical pitch of 30/~ (see Figure 3d). This distorted B conformation (or C form) 14, which corresponds to nine nucleotide pairs per helical turn, is modified when the r.h. is increased and one again gets the Z form at 80% r.h. but the fibre is then very much disorganized.
2tO
410
'
6tO
80
'
100
r.h.%
Figure 2 Variations of the fibre length L ( H ) for the Z-B-Z transitions and of the rise per nucleotide Pm ( n ) for the Z-B transition as functions of the relative humidity
(a)
(b)
Measurements of fibre dimensions during the Z to B and B to Z transitions have given values presented in Figures 2 and 4. These measurements are performed under the same conditions of r.h. as those realized for X-ray diffraction. In Figure 2, we can see how the fibre length increases up to 95% r.h. and it then suddenly decreases when the r.h. is higher than 95%. At 97.5% r.h. the fibre length is even lower than at 0%0. Conversely the fibre diameter (Figure 4) increases slightly for r.h. up to 80% and steeply thereafter. When the r.h. is decreased from 97.5%, the fibre length remains almost constant (Figure 2) for r.h. up to 88% (note that at such a value of r.h., X-ray patterns show
(c)
(d)
Figure 3 B-Z transition. X-ray patterns obtained at: (a) 90% r.h. - B-Z forms; (b) 85% r.h. - Z2 form; (c) 40% r.h. - Z1 form; (d) result of a sudden decrease of r.h. from 97% to 50%
Int. J. Biol. Macromol., 1992, Vol. 14, June
163
B-Z conformational transition: G. Albiser and S. Prbmilat This fact is observed when poly(dC-dG).poly(dC-dG) is in its Z form as well as when the transition to the B form occurs. We also noted that the change of conformation from Z to B appears when the distance between helix axes has a value of at least 23.8/~ and that distance increases to 27/~ for the B form. Besides, we know ~ that the diameter of the double helixes of poly(dC-dG).poly(dC-dG) in the Z form is near to 18 ~ and that it is 20 A in the B conformation 24. We therefore see that relatively large interhelical distances are necessary in order to allow the important molecular movements inducing the transition between left- and right-handed helical conformations of poly(dC-dG).poly(dC-dG). Such enlargement of the packing distances of the double helixes is due to water molecules and is obtained by raising the r.h. to values very near to 100%.
0.26 0.23
0.20
0.17 B
26
/ /
z~
23 /.a
20 17
/.ti
j._
_
-
-!
.
I
20
.
.
.
--
d
--
I
40
_
B
--
.
--
--
--
I
I
I
60
80
100
r.h.%
Figure 4 Variations of the fibre diameter D ( 0 - - 0 ) and the distance between helix axes d.a (ll---m) for the Z-B transition as functions of the relative humidity mixtures of B and Z forms). For r.h. lower than 80% the fibre length decreases but one does not obtain the values for the length that we had at the beginning of the process (the fibre length is 5% smaller). Nevertheless, the fibre diameter again takes its initial value (as the crystallographic cell parameter). An additional cycle of r.h. variations gives the same effect and the fibre length is .again lowered when the r.h. is at 0%. Moreover, when the r.h. is suddenly decreased from 97.5 to 30% such a loss of the fibre length is even larger (near to 10% ).
Hydration of poly( dC-dG).poly( dC-dG) Results obtained simultaneously from X-ray diffraction and measurements of variations of the fibre dimensions with the r.h., allowed us to follow the changes in the number of water molecules associated with a nucleotide during the conformational transitions ~5. We therefore determined (Table 1 ) that there are two water molecules per nucleotide when the poly(dC-dG).poly(dC-dG) is in the Z~ form. Note that these figures do not take into account water which remains in the fibre at 0% r.h. ~8. This number increases regularly during the evolution from Z1 to Z 2 and there are 16 water molecules per nucleotide in this last conformation (just before the transition to the B form). The complete transition from Z 2 to B necessitates the addition of six water molecules per nucleotide which then have 22 water molecules in their vicinity; this last number is even increased when the r.h. is more than 97.5%. These results are in agreement with data deduced from studies on poly (dC-dG).poly (dC-dG) films by infrared spectroscopy 19 and with values obtained from studies on solutions by dielectric relaxation 2°. Moreover, the present observations concerning the variations of the number of water molecules during the B-Z conformational transition are complementary to those obtained from X-ray crystallography where the precise position of water in the Z double helix can be estimated 2a-23. The comparison of results obtained from X-ray diffraction with the measurement of variations of the fibre dimensions indicates a proportionality between the interhelical distance and the fibre diameter (see Figure 4).
164
Int. J. Biol. Macromol., 1992, Vol. 14, June
Discussion These results, obtained simultaneously from X-ray diffraction and measurement of dimensions of poly(dCdG).poly(dC-dG) fibres, clearly show the reversibility of the B-Z transition. However, we noted that the form transition is realized at very high and in a narrow interval of r.h. values (mainly for the Z z to B transition). At very low r.h., the polynucleotide is in the Z 1 form and it evolves progressively, as the r.h. rises, towards the Z z form until it changes steeply to the B form. The application of the present experimental method ~4 allowed us to determine that the Z form of poly(dCdG).poly(dC-dC) can be associated with up to 16 water molecules per nucleotide and that six more are necessary to achieve the Z to B transition. An almost perfect proportionality between the fibre length and the helical parameter p (an average value for Z) is observed (see Figure 2) for the transitions Z1-Z2-B. This fact clearly indicates the polymorphism of the Z form 25 of poly(dC-dG).poly(dC-dG). But it should be noted that, even if p varies with the r.h., the number of dinucleotides per helical turn remains equal to six. Moreover, the Z 1 helical conformation corresponds to very low r.h. while Z z is obtained at very high r.h., just before the transition to the B form. We can add that the continuous Z ~-Z2 change of form is perfectly reversible. Conversely, the reverse transition from B to Z, observed when the r.h. is lowered, is much slower as already noted in studies on solutions z6 or on fibres ~9. Moreover, under the present experimental conditions, this change of form is obtained in a larger interval of r.h. We first observed a mixture of B and Z 2 forms when the r.h. is decreased and, thereafter, a progressive change from Z 2 to Z1. The transition from B to Z is always associated with a disorganization of the fibre and that effect is more important when the r.h. is suddenly lowered. As a consequence, we observed that, even for very slow variations of the r.h., there is no proportionality between the fibre length and the helical parameter p. Larger lengths of the fibre should correspond to the transition from B to Z2, but the disorganization of the fibre is superimposed on that transition and any observation of the fibre length variation becomes impossible when the r.h. is lowered from the value of 97%. We can add that a tension applied on the fibre ~6 improves the B-Z transition and actually maintains the fibre organization, but it also induces sliding of the
B - Z c o n f o r m a t i o n a l transition." G. A l b i s e r a n d S. P r b m i l a t
polynucleotide helixes past one another and that makes the fibre length measurements irrelevant. Nevertheless, it appears that the change from B to Z conformation remains cooperative because no well organized form, different from B or Z, can actually be observed on X-ray patterns during the transition. This is in agreement with results obtained from studies on solutions 27 29 and also on films 19 of poly(dC-dG).poly(dC-dG).
10 11 12 13 14 15 16
References 1 2 3 4 5 6 7 8 9
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