Volume 4 Number 9 September

1977

Nucleic Acids Research

A theoretical study on the effect of "bound" water on the proton chemical shifts of the nucleic

acid bases Claude Giessner-Prettre, Femando Ribas Prado and Bernard Pullman

Institut de Biologie Physico-Chimique, Laboratoire de Biochimie Theorique associe au C.N.R.S., 13, rue P. et M. Curie, 75005 Paris, France.

Received 8 July 1977

ABSTRACT

Computations are performed on the proton chemical shifts due to hydrogen bonding between the purine and pyrimidine bases of the nucleic acids and water molecules of their first hydration shell. The water molecules should produce measurable shifts essentially for protons of the bases located close to the site of interaction. For the imino protons of the bases G-N1H and U-N3H participating in hydrogen bonding, the calculated A6 is larger for the interaction of a base with a complementary base than for its interaction with water. Base pairing will thus produce a downfield shift in water but the measured Ad due to pairing in this solvent will be smaller than in an inert solvent. Also, the chemical shift difference between G-N1H and U-N3H in water will be larger if the molecules are engaged in pairs than if they are not. I

The chemical shifts of protons in a molecule may have different values

in the free and the solvated species. This difference is known to be large

for protons engaged in intermolecular hydrogen bonding but is generally unknown for the

other protons in the molecule.

In our previous work on the dependence of the chemical shifts of the

various protons of the nucleic acid bases upon their hydrogen bonding or

upon the secondary structure of the polymers we have considered the effect of the interaction between the bases but neglected the possible role of the surrounding solvent molecules (1). Now, the formation of an ordered secondary structure from a random coil or of molecular aggregates from monomeric units will perturbe the solute-solvent interactions of polar molecules. In the case of the nucleic acid bases in which we are interested here, most of the proton magnetic measurements are made in dimethylsulfoxide (2-3) and water (4-7), two polar solvents which can form hydrogen bonds with them. In the absence of experimental data, theoretical calculations can be useful for investigating the influence of solvent molecules on the variation of the proton shifts which are observed upon helix-coil transition or

C' Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

3229

Nucleic Acids Research upon the formation

tion on the role of

of base pairs or triplets since they can give informasolvent molecules of the solvation shell on the chemi-

cal shift of any proton of the solute. In the present case previous calculations (1) have shown that the formation of the Watson-Crick A-U and G-C pairs produces variations of the chemical shift of guanine N H and

uracil

N3H

which have the same value. This result implies that the dif-

ferences between the chemical shifts of the two protons are the same when the bases are free and when they are in their respective complementary pairs. Some

of the experimental data seem to indicate, however, that this

difference may be

larger for the imino protons of the molecules engaged

in-pairs(8-9) than for the unpaired bases (2) (3) (10).This situation could be due to a differential effect of the solvent water upon the two shifts and the exploration of this possibility was one of the main incentives for this work. For the sake of carrying out such an investigation one needs to have

information about the structure of the hydration shell around the compounds studied. Such an information is available for the nucleic acid bases from the work of A.

Pullman (11-12). This makes it possible to under-

take the calculation of the

influence of solvent molecules on the chemi-

cal shifts of the protons of these compounds for the different intermolecular associations which have been found to be the most favorable ones. These computations should thus provide information on the chemical shift variations which may be produced

by the rupture or the creation of a base-

water intermolecular bond.

Computational procedure and input data The method of calculation is the one used in our study on the hydrogenbonded nucleic acid bases (1). Since water is not an aromatic molecule there is no ring current effect of this molecule on the protons of the

bases; the atomic magnetic anisotropy term and the polarization effect are therefore here the two contributions to the calculated chemical shift variations. The chemical shift variations ( A6 in p.p.m.) that we compute are

those which would be observed between the chemical shifts of the protons of the isolated nucleic acid bases and the chemical shifts of the same protons in the complex formed by the base and a water molecule in the spe-

cified site. The geometrical input data are those of Pullman's study (11) on the

3230

Nucleic Acids Research hydration of the bases except in the case of uracil. For this last molecu-

le we have taken her data on thymine and replaced the methyl group by a hy-

drogen atom. Results and discussion

Fig. 1 (a-d) reports the positions of a water molecule for which we

V6 11

7

a

k.

4

10

3/'-

H

H 9

2\11 I

10

12

H

N

rs

i~~ I

-

4 2

(a)

(b)

A7

[6 7__

5\

6>

3

H/b

H O

4Jr

Zs

9

N

N

H

8

3

2 N

H

9

H

2

H,

H

s12

10

\1

13

11

(c)

Figure 1.

-

Positions

(11) for which Ad's

1>

1 10 (d)

of the water molecule around the nucleic acid bases

are

reported in the Tables I-IV. a) adenine, b) uracil,

c) guanine, d) cytosine. 3231

Nucleic Acids Research have calculated the A6 of the protons of the bases. Only "in plane" plexes (with the

atom of the water molecule in the plane of the

oxygen

base) have been considered since they (11). In Tables I-IV

com-

are

are

by far the most favorable

reported, for each position of water, its

ones.

energy

of interaction with the base, the mutual orientation of the planes of the two molecules and the chemical shift variations for the

protons

of

the base. Adenine. molecules which

The water

amino

(positions 3 to 6

group

will be engaged in

a

are

on

located in the region of N1 and of the

Fig. la) will be removed when adenine

pairing of the Watson-Crick type.. We

I that the calculated values of A6 for these positions of

small for

are very

tions find

the amino

a

C8H

a

water molecule

and for C2H. For positions 4, 5 and 6

large chemical shift variation for

group.

Since the formation of

a

one

from Table

see

our

computa-

of the protons of

Watson-Crick A.U pair from

a

hy-

drated adenine will be accompanied for the protons of adenine by chemical shift variations which are the differences between the shift variations

due to pairing and the

ones

due to hydration, the set of values reported

here show that the pairing will produce than in

ler in water

an

a

A6 for the amino protons smal-

inert solvent, while for the CH protons the varia-

TABLE I

Interaction energy, dihedral angle between the two molecular planes and proton chemical shift variations for the hydration of adenine at the sites indicated.

Position of the H20 molecule

E(kcal/mole)

1

-3.9

2

-2.8 -7.6 -9.1

3 4 5

6 7 8 9 10 11

12 13

3232

A6 (p.p.m.)

f (dO)

C2H

-8.3 -5.2 -9.9 -7.8 -5.0 -8.3 -10.8 -12.0 -9.3

NH2a

NH 2

C8H

NH

-0.02 0. -0.02

-0.01 -0.02

0.

-0.41 0. -0.47 -0.01 -0.06 -0.04

0.

-0.03

0.03

-1.53

0.

0.

90.

-0.03

0.11

-0.62

0.

0.

90.

0.03

-1.16

0.02

0.02

90. 0. 90. 90. 90.

0.

0.05 -0.61

0.10 -0.08

-0.04

-0.02 -0.16

-0.05 -0.08

0.01 0.01 0.02 0.01 -0.01 0. -0.02 -0.02 -0.07 -0.07

0.02 -1.18

90. 90.

0. 0.

0.03

0.01

-0.20 -0.02

0.02

0.23

-0.06 -0.04 -1.34

0.01

0.03

-0.77

0.

0.01

-1.46

-0.02 -0.03

-0.28

Nucleic Acids Research tions will be equal in the two types of solvent. The water molecule loca-

ted near N7 (positions 7 and 8 on Fig. la) produces a measurable A6

C8H

and on the amino protons but a negligeable one on

of the

C2H.

on

A base pairing

Hoogsteen type will produce, if it occurs in water, a Ad for the

protons of

adenine which will be slightly decreased (C 8H and amino protons)

or unchanged (C2H) with respect to those which would be observed in an

inert solvent. For the third and strongest hydration site of adenine around

N9H, our calculations show that the

N9H

N3

and

imino proton should undergo a

large shift by water molecules at positions 9, 10 or 11. Our previous results on hydrogen-bonded bases (1) have shown that a is accompanied by a downfield shift of C 2H, while a

Watson-Crick pairing

Hoogsteen pairing will produce a downfield shift of C 8H. The present re-

sults show that this conclusion is valid in the presence of water since

the locations of the molecules of water which have the strongest interaction energies with the base (4, 7 and 1 2) produce A6's on the CH protons

appreciably smaller

than those due to the pairing (of the order of -.05

p.p.m. and -.5p.p.m., respectively). For the CH protons only positions such as 2 and 9 would produce a lar-

ge downfield shift. They correspond, however, to low interaction energies

and statistically have

thus little chance to occur since the water-water

interaction energy (6.7 kcal/mole (14)) is larger than the adenine-water

interaction energy for these positions. For the amino protons the presence of water decreases significantly the A6 due to pairing.

Uracil For this molecule the results of A. Pullman (11)

(Fig. lb and Table

stable hydration sites are located in the vicinity of the imino protons. These strongly interacting water molecules produce large variations of the chemical shift (> 1.p.p.m.) of N 1H and N3H, the pro-

II) show that the

ton

engaged in one of the

hydrogen bonds formed when the base pairs with

adenine and which is used by the experimentalists to determine the secon-

dary structure of polynucleotides and RNA's (4-8) (14). For this imino proton the A6 due to pairing is thus smaller by about 1 p.p.m. in water than in an inert solvent. the water molecules which give complexes with a large interaction enerqy have an almost negligeable influence on the CH pro-

All positions

of

ton chemical shifts. The A6 's calculated for positions 5 and 11 show that

3233

Nucleic Acids Research TABLE II Interaction energy; dihedral angle between the two molecular planes and proton chemical shift variations for the hydration of uracil at the sites indicated. Position of the H20 nilecule

E(kcal/mole)

Ad(p.p.m.)

f (d°)

N1H

N3H

C5H

C6H

1

-5.0

90.

0.05

0.03

0.06

2

-8.1

90.

-1.59

0.04

0.03

-1.77 0.02

3 4 5 6

-9.9 -11.9 -6.0 -9.6 -9.8 -7.4 -8.8 -9.1 -6.6

90.

-1.14

0.07

0.03

0.03

0. 0. 0.

-1.54

0. 0.04

0.04 -0.08 -1.56

0.02 -0.02 0.02

-0.02 0.02

120. 0. 0. 90. 0.

0.06 0.03

-0.91

0.03

0.02

-1.77

0.03

0.03

7 8 9

10 11

0.03

0.02

-1.39

0.03

-0.86

0.04 0.05

0.02 0.03

-0.02

-0.12

-0.01

-0.02

a water molecule bound to one of the carbonyl oxygens by a "conventional"

Ad's of the imino protons which are of the most favorable case. The calculated Ad for position 1 shows that the CH protons will undergo a large chemical shift variation only if the solvent molecule is lo-

hydrogen bond (11) produces order of -0.1 p.p.m. in

the

cated in their vicinity; the interaction energy for such cases is, as for adenine, too small to be of significance. Guanine.

The values reported in Table III show that for this base there are favorable positions of the water molecule which produce large downfield variations of the imino proton N 1H. One of these positions, 3, (Fig. lc) produces also a large shift on one of the amino protons. As in the case

of adenine the water molecules near N

cause a very small Ad on C 8H.

These results show that the chemical shift variation due to the pairing of guanine will be smaller in water than in an inert solvent for the amino and imino protons. For C 8H the Ad will be practically the same in both ca-

ses.

The very stable position 7 where the water molecule forms a bridge

between

06

and

N7

induces a

AM for C 8H which is large enough to influence

the shift which is measured for this proton, e.g. when the C.G.C

trimer

is formed. Since a water molecule in this position 7 does not produce any

large shift on N 1H or on the amino protons, the chemical shifts of these 3234

Nucleic Acids Research TABLE III Interaction energy, dihedral angle between the two molecular planes and proton chemical shift variations for the hydration of guanine at the sites indicated. Position of the

E(kcal/mole)

v(d°)

H20 molecule

2a

1 2

-7.1 -8.0

90. 90.

3

-11.4 -10.4 -9.0

90. 90. 0. 0.

4

5 6

-7.7

-11.0 -8.8 -4.3 -8.2 -9.8 -10.6 -7.5

7

8 9

10 11

12 13

0. 0. 90. 90.

90. 0. 0.

-1.29 0.12 0.15 0.06

0.02 -0.01 -0.02 -0.02 0. 0.01 0. 0.02 0.07

NH2b

AW(p.p.m.) N1H C8H

0.08 0.01 -1.66 -0.08 -0.75 -1.09 -0.14 -0.92 -0.09 -0.56 -0.02 -0.06

-0.02 -0.01 0.01 0.02 -0.07 -0.07 -0.11

N9H

0.02 0.05 0.01 0.01 0.02 0.01 0.01 0.01 -0.01 0.01 -0.03 -0.03

-0.06 -0.10 -0.06 -0.03 -0.33 -0.06 0.02 -0.50 -0.14 0.02 0.07 -1.78 -0.03 0.02 -0.56 -0.03 -0.03 -1.47 -0.05 -0.03 0.29

protons will not be sensitive to a possible hydration of N7 or of 06 when

guanine is engaged in a G.C

pair.

The water positions 10 to 13 in the

N3-N9H

region, which are relatively

stable have a large influence on the chemical shift of the imino

N9H

proton

and a small effect on one of the amino protons. Table III shows that

position 9 is the only one which produces a lar-

ge shift of the C 8H proton of the base, but the low value of the interac-

tion energy for this position precludes its involvement in a stable association.

Cytosine The formation of a G.C pair requires the removal of the water molecu-

les located near

C202'

N3 and a part of the amino group, which correspond

to positions 2-6 (Fig. ld). Table IV shows that a water molecule in any of these positions induces a downfield shift only for one of the amino protons and that this shift is smaller than -0.5 p.p.m. for the most favorable positions 4 and 5. The hydration of the second amino proton (position 8) produces a downfield shift on C 5H which is of the order of magnitu-

de of the one due to the G.C pairing (1). Stable positions such as 1 and 10

close to the imino proton have a large influence on N 1H and a smaller one onC

6H. 3235

Nucleic Acids Research TABLE IV

Interaction energy, dihedral angle between the two molecular planes and proton chemical shift variations for the hydration of cytosine at the sites indicated. Position of the molecule H2O 2

E(kcal/mole)

A6 (p.p.m.)

p (dO) NH

N1I

1

-9.9

2

-7.8

0. 0.

-1.46 -0.06

NH 2a

N

2b

0.01 -0.01 0.

CH 5

CH 6

0.01 -0.03

-0.03 -0.02 -0.03

3

-8.9

0.

-0.06

-0.04 -0.05 -0.04 -0.04

4 5

-11.1

0.

-0.05

-0.18 -0.05 -0.05 -0.04

-11.1

0.

-0.05

6

-7.6

0.

-0.01

7

-5.2 -6.8

90.

0.02

8

90.

0.04

-0.44 -0.07 -0.05 -0.04 -1.42 0.01 -0.03 -0.01 -0.36 -0.18 0.01 0.02 0.14 -1.64 -0.16 0.08

9

-2.8

0.05

10

-7.7

90. 90.

-0.95

0.06 -0.06 -1.33 0.02

0.01

0.09

0.03 -0.11

General comments The results obtained for the four bases have a number of features in common. It is evident that a water molecule hydrogen-bonded to a base will produce a measurable shift for protons located on the part of the base which is close to

the

site of interaction. This type of effect was alrea-

dy present in our results on the hydrogen-bonded bases (1) but is more pronounced in the case of water, probably because of the small size of this molecule. This finding implies that the chemical shift of the imino protons

engaged in the pairing of the bases (particularly well studied in NMR of nucleic acids (2-10))will not be sensitive to the formation of hydrogen bonds between the solvent and atoms of the base which are not participa-

ting in the hydrogen bonds responsible for this pairing. On the other hand for the imino protons participating in the hydrogen bonding, the calculated values of A6 are larger if the base is interacting with another base than if it is interacting with a water molecule. This result implies that base pairing will produce a downfield shift if it occurs in water, but that the measured A6 due to pairing will, in this solvent, be smaller than if this quantity is measured in an inert solvent. The calculated values of the differences between the Ad due to basepairing and the one due to hydration of the base should be considered as a valid first approximation in spite of the fact that we have considered only the interaction of individual water molecules with the bases. The validity of the conclusions drawn from this study is supported by the results on hydration of formamide : the study of the complete hydration of that molecule (15) has shown that the structure of the first solvation

3236

Nucleic Acids Research shell is not gondamentally different from the superposition of the stable positions computed for a molecule of water interacting with it (12) (16). For uracil and guanine there are stable positions of water molecules which produce a large downfield A6 of the imino protons of the two mole-

cules, N3H and N1H, respectively. For uracil the most favorable positions are 7 and 10 which produce a shift of -0.91 and -0.86 p.p.m. of N 3H. For

guanine the corresponding data are for positions 3 and 4 which give a shift

of -1.09 and -0.92 p.p.m. of

larger than for the

N1H

N 1H. These Ad's for guanine are in both cases

uracil and this result shows that the chemical shift of

imino proton of guanine is greater than that of the N3H imino pro-

ton of uracil. This difference

means that base-pairing in water will pro-

duce a chemical shift variation of these imino protons which tends to be smaller for guanine than for uracil, if the intrinsic chemical shift variation due to pairing

are equal for

the two protons. Consequently the chemical shift difference between guanine N1H and uracil N 3H in water will be

larger if the

molecules are engaged in pairs than if they are not. Experi-

mental data tend to the same conclusion (10) The calculated chemical shift variations of the amino proton of ade-

nine which is hydrogen bonded to the water molecule for positions 4 and 5 of the solvent molecule show that position 4 which corresponds to an arrangement in which the hydrogen bonded proton of the base is in the plane of the water molecule produces a A6 larger than the one calculated for position 5. Positions 11 and 12 for adenine, 9 and 10 for uracil and 11 and 12 for guanine show the same phenomenon. Ditchfield (17) has obtained similar

results for the water dimer

from his elaborate ab-initio GIAO-CHF calcula-

tions with a split basis set. In our method of computation the difference between the A6 for protons in the plane and out of plane of water is due to the contribution of the local diamagnetic anisotropy of the oxygen atom of the molecule which has

strongly directional properties. Acknowledgements One of us (F.R.P.) wishes to acknowledge the support of a doctoral fellowship from Conselho Nacional de Desenvolvimento Cientifico e

Tecnologi co-Brazil.

References 1. Giessner-Prettre, C., Pullman, B. and Caillet, C. (1977) Nucl. Acid. Res. 4, 99-116. 3237

Nucleic Acids Research 2. Shoup, R.R., Miles, H.T. and Becker, E.D. (1966) Biochem. Biophys. Res. Comm. 23, 194-201.

3. Wang, S.M. and Li, N.C. (1968) J. Am. Chem. Soc. 90, 5069-5074. 4. Jones, C.R., Kearns, D.R. and Muench, K.H. (1973) J. Mol. Biol. 103, 747-764.

5. Krugh, T.R., Laing, T.R. and Young, M.A. (1976) Biochemistry, 15, 12241228. 6. Patel, D.J. and Canuel, L. (1976) Proc. Nat. Acad. Sci. 73, 674-678. 7. Kan, L.S. and Ts'o, P.O.P. (1977) Nucl. Acid. Res. 4, 1633-1647 and references therein. 8. Lightfoot, D.R., Wong, K.L., Kearns, D.R., Reid, B. and Schulman, R.G. (1973) J. Mol. Biol. 78, 71-89.

9. Wong, K.L., Wong, Y.P. and Kearns, D.R. (1975) Biopolymers, 14, 749762. 10. Robillard, G.T., Hilbers, C.W., Reid, B.R., Gangloff, J., Dirheimer, G. and Schulman, R.G. (1976) Biochemistry, 15, 1883-1888. 11. Pullman, A. (1975) in "Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions" (Sundaralingam, M. and Rao, S.T.

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12. 13. 14. 15. 16.

Pullman, A. and Pullman,

Berthod, H. and Pullman, A., unpublished results. Harpool, R.D. (1977) J. Am. Chem. Soc. 99, 349-353.

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17. Ditchfield, R. (1976)

3238

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