Two dimensional NMR studies on the solution structure of d-CTCGAGCTCGAG

Vol. 144, No. 1, 1987 April 14, 1987

TWO

DIMENSIONAL

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 26-34

NMR STUDIES ON THE OF d-CTCGAGCTCGAG

A n u Sheth, M. Ravikumar,

R.V.

SOLUTION

STRUCTURE

Hosur, Girjesh Govil,

Chemical Physics Group, Tata Institute of Fundamental Research, Homi B h a b h a Road, B o m b a y 400005, I N D I A and Tan-Zu-Kun

and H.T. Miles

National Institutes of Health, Bethesda, Maryland 20205

Received February i0, 1987

T w o dimensional (2D) ~ T - N M R investigations have been carried out on the self-complementary dodecanucleotide d - C T C G A G C T C G A G , which has cleavage sites for the restriction e n z y m e X h o I (between C and T). The central T C G portion is also k n o w n to show a preference for D N A a s e activity. Complete resonance assignments have been obtained for the non-exchangeable sugar and base protons of the oligonucleotide. Information regarding sugar geometries, glycosidic torsion angles and other structural parameters has been obtained from the relative intensities of the cross peaks in the C O S Y and N O E S Y spectra. T h e results indicate that deoxyribose rings of C1 and C7 adopt a conformation different from the remaining sugars in the double helical oligonucleotide. The central T C G portion also exhibits variations in the backbone structure. T h e base stacking in the double helix shows interesting sequence dependent effects suggesting that the sequence effects are not localised to nearest neighbours but extended over longer stretches. © 1987 Academic Press, Inc.

We have recently investigated by N M R , tides, d - G G A T C C G G A T C C

the structures of two oligonuclo-

[1,2] and d - G A A T T C G A A T T C

[3] which have reco-

ginition sites for restriction enzymes Barn HI and Eco RI, respectively.

In

both cases, it has been observed that the cleavage sites of the restriction enzymes exhibit conformational deviations from the rest of the molecule.

It

is of interest therefore, to check if such conformational heterogeniety is a characteristic feature of D N A

segments recognised by restriction enzymes.

have therefore undertaken investigations on a n u m b e r of D N A

segments eonta-

Abbreviations: C O S Y , Correlated Spectroscopy; NOESY , Nuclear Overhauser Effect Correlated Spectroscopy; FT-NMR , Fourier Transform Nuclear Magnetic Resonance

0006-291X/87 $1.50 Copyright © 1987 by Academic Press, lnc. All rights of reproduction in any form reserved.

26

We

VoI. 144, No. 1, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Xho

5' d - C

5

I

Xhol DNAase I

TC

G AGCT

C G A G

GAG C;TTCGAG C TC

12

10

8

6

4

ONAasel XhoI

Fig. i.

5'

Xho

I

The schematic representation of the self-complementary dcdecanucleotide d - C T C G A G C T C G A G studied in this paper. The cleavage sites of Xho I and DNase I in the D N A segments are marked by arrows.

ining specific cleavage sites for restriction e n z y m e s .

In this p a p e r w e report,

IH resonance assignments a n d some structural details on d - C T C G A G C T C G A G w h i c h has two cleavage sites (between C and T) for X h o I e n z y m e

(Fig. i).

EXPERIMENTAL Deoxy - (CTCGAGCTCGAG) w a s synthesised following procedures similar to those described previously [2]. 5 m g of the sample was dissolved in 0.5 ml of p h o s p h a t e buffer (0.02M) h a v i n g p H 7.2 a n d the total counterion concentration of 0.08 M. T h e solution w a s lyophilized arid redissolved in D ~ O twice a n d finally m a d e u p to 0.5 ml with 100% D 2 0 . ~ H N M R experimentLs were carried out using a B r u k e r A M 500 F T - N M R - s p e c t r o m e t e r operating at 500 M H z f r e q u e n c y for proton. T w o dimensional C O S Y [4] a n d N O E S Y [5] experiments w ere carried out with 2048 data points along the t2-axis and 512 data points along the tl-axis. T h e C O S Y pulse s e q u e n c e (90°-'tl-90o-t~) w a s modified b y introducing~a small fixed delay of 5 msecs, after each 90 ~ pulse, so as to e n h a n c e cross p e a k intensities in the two dimensional s p e c t r u m [6,7]. The NOESY s e q u e n c e u s e d w a s 90°-ti-90°- T -90ot~ w h e r e T is the mixing time. .m .m . . A relaxation delay of 1 sec w a s given in all ~ D experiments. T h e tlme domain data w e r e zero filled to 2048 points along the t2-axis, a n d 1024 points along the t.-axis a n d multiolied b v sine-sauare bell ann sine bell w i n d o w functions prior to Fourier transformation along the t2a n d t.-axis, respectively. Chemical shifts> (Table i) are e x p r e s s e d wi~h respec{ to sodium 3-trimethyl silyl (2,2,3,3-~H) propionate (TSP).

Table I.

Chemical Shifts of Non Exchangeable Protons in d - C T C G A G C T C G A G

Base

H6/H8

HI'

C1 T2 C3 G4 A5 G6 C7 T8 C9 GI0 All GI2

7.87 6.62 7.44 7.88 8.06 7.58 7.30 7.43 7.41 7.89 8.10 7.60

5.85 6.15 5.55 5.38 6.05 5.65 5.81 6.05 5.49 5.39 6.10 5.96

H2' 2.24 2.23 2.02 2.65 2.64 2.47 2.00 2.15 1.97 2.65 2.64 2.38

H2"

H3'

2.59 2.55 2.34 2.72 2.91 2.59 2.46 2.50 2.31 2.72 2.91 2.25

4.66 4.89 4.85 4.97 5.09 4.93 4.63 4.85 4.83 4.97 5.02 4.60

27

H4' 4.07 4.24 4.07 4.28 4.40 4.32 4.17 4.18 4.05 4.28 4.40 4.14

H5/H2/CH 3 5.92 1.66 5.62 7.66 5.14 1.52 5.64 7.80

Vol. 144, No. 1, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NOESY A5

II

5.4

I

I

,}ce ~

5"6

A5

f

c3H5

_

I

I

A5 T2 5"8-

H 1',H5

6.0

6.2a

i

I

I

80 117 8

8.2

IA]]

A5

G]OG4C] I[AI]

li4i

A51[TZGI2G6 C5T8C9

Base H6/H8 1.4

I

H2

C7t

: Base H6/H8

NOESY

CH3 CH3

0

~ - 0

I

T8

1.8

I

T2 C9

2.2 H2',H2"

C5

2.6

A11

<

GI2

A5 5.0 I

b

8.2

G4

G6 ,I,, 7'6

J[8.O )t .8 I

A]! A5

GIOG4 e l

1

I

T2 GI2 G6 C3 T8 C9

Base (H8/H6) 28

c7

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 144, No. 1, 1987

RESONANCE

ASSIGNMENTS

Individual assignments of IH resonance h a v e been obtained using 2D N M R techniques following the strategies described earlier [1-2,8-11]. First, the COSY

and NOESY

spectra are u s e d to identify the subsets of J-coupled protons

corresponding to the individual sugar residues. base protons are identified using J ( H 5 , H 6 )

T h e cytosine a n d the thymine

and J(CH3,H6)

correlations. In the

next stage, using the N O E ' s b e t w e e n base protons ( H 6 , H 8 , H 5 , C H 3) a n d

the

sugar resonances HI',H2' a n d H2", these sets are assigned to individual units. Four different types of sequential correlations h a v e been o b s e r v e d namely, (base) i --> (base)i+l(dl), (d 3) a n d

(base) i --> (Hl')i_l(d 2) (base) i - >

(base) i --> (H3')i_l(d4).

a n d some d I

connectivities.

As an illustration Fig. 2 s h o w s the d2,d 3

Stereospecific assignment of the H2',H2"

protons has b e e n obtained from the N O E S Y sities of HI'-(H2',H2")

(H2",H2')i_ 1

cross p e a k s

s p e c t r u m b y monitoring the inten-

(Fig. 3).

HI'-H2" cross peaks are always

stronger than HI'-H2' peaks. STRUCTURE

OF

The NOESY

CTCGAGCTCGAG s p e c t r u m provides vital clues on the overall structural

profile of the dodecanucleotide. strand N O E ' s

For example, w e observe a n u m b e r

of inter-

(Fig. 2a); b e t w e e n the H 2 proton of A5 a n d the HI' protons of

C9 a n d T8, b e t w e e n H 2 of A5 a n d H5 of C9, a n d b e t w e e n H2 of All and HI' of C3.

This proves that the A - T

base pairs are locked in h y d r o g e n

bonded

Fig. 2a.

The region (c0] = 7 - 8.2 ppm; c02 = 5 - 6.4 ppm) in the N O E S Y spectrum. The ~ spectrum shows cognplete intrastrand d~ sequential connectivities involving base (H6/H8) protons and the zHI t protons. The base protons are identified along the bottom of the figure, and the HI' protons along the horizontal lines. It may be noted that starting from GI2, one is able to jump to the preceding nucleotides sequentially without any breaks till CI, as expected from d2 distances in a right handed D N A . In the same section of the spectrum one also observes, an intrastrand base-base (d]) connectivity (dashed lines) involving H5 of C3 and H6 of the ~djacent T2. Finally, four interstrand cross peaks are observed. These involve (i) H2 of A5 and HI' of C9, (ii) H2 of A5 and HI' of T8, (iii) H2 of All and HI' of C3 and (iv) H2 of A5 and H5 of C9 (depicted by dashed lines). These cross-peaks confirm the existence of A .... T base pairing at all the four sites in the double helical D N A schematically shown in Fig. i.

Fig. 2b.

Sequential connectivities (dr) involving base (H6/H8) protons and sugar H2'/H2" protons. InJthis region one also observes cross peaks from the thymine (T2 & T8) methyl protons to the respective H6 protons and to the H6 proton of the preceding cytosine (d 1 connectivity, shown by dashed lines).

29

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 144, No. 1, 1987

~ C7HI'

1.4

NOESY

~- T8 CH3

I ~ - - - "~ T2 CH3 l

1.8

I

C1,H5 C7H2'f

~

,

C9H5 2.2

[~-12" GI2

~ C l H2' C3H2" ~

LH2 '~

T2 H2"

~CIH2"

2-6

AllH2"~

H2',H2"

C7 H2"

~ T 8 H2"

~

@ C 9 H2"

~G6

H2" [--"- H2' ( ~ H2'--l G1O ~ G4 ~--H 2" I~1~H2"~1

e, A5 H2"

3.0

I

I

I

I

I

6.2

6-0

5.8

5.6

5.4

H 1 ',H5 Fig. 3.

Expansion of the HIr-(H2 ', H2") of the N O E S Y spectrum of d-CTCGAGCTCGAG. The mixing time is 300 sec. and the digital resolution along the two axes is 7.9 Hz. It is expected that HI' H2" cross-peak for a nucleotide is more intense than the correspoding HI' - H2' peak. In fact, in seven cases (T2, C3, A5, G6, T8, C9 and All), the HI.' - H2' peaks are not visible at all. In this domain of the N O E S Y spectrum, one also observes the connectivities involving thymine methyl protons and the HI' (d~) or H5 (d L) proton on the adjacent cytosine nucleotide in the-same strand (depicted by dashed lines).

n e t - w o r k a n d the molecule is in the double helical form u n d e r the present experimental conditions.

Further, the o b s e r v e d sequential connectivities indi-

cate that the molecule adopts on overall right h a n d e d since all the cross peaks in t h e N O E S Y

helical structure. Finally,

s p e c t r u m have been assigned, only one

type of structure is present in a q u e o u s solution a n d coexistence of single stranded structures is excluded. Using the methodologies described previously, COSY

and NOESY

geometry,

[1-2,11-12] w e have u s e d

spectra to obtain detailed information regarding the sugar

glycosidic torsion angle a n d base-base stacking along the s e q u e n c e

of the molecule. S u G a r Geometries:

Information about the sugar ring g e o m e t r y is obtained from

2D C O S Y

[12],

spectrum

3O

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 144, No. 1, 1987

Fig. 4 shows expansions of all the domains of the C O S Y

spectrum of the

dodecanucleotide which are important for fixing the conformations of the deoxyribose rings. Several sugar residues show c o m m o n features hut a few show variations from the general pattern. O n e of the most striking observation is the complete absence of the H2"-H3' cross peaks in the C O S Y

spectrum for

ten of the twelve sugar residues (Fig. 4b). This implies that the value of J(H2"-H3') is small and it can be concluded that the conformation of deoxyribose rings of these ten nucleotides is restricted to the S domain of the pseudorotational wheel (P = 90 to 240°). The intensity patterns of the various cross peaks in Fig. 4 are consistent with the following conclusions (see ref.12). (a)

C7 is close to Ol'-endo.

(b)

T2, C3, A5, G6, T8, C9 and All have sugar geometries close to Cl'-exo.

(c)

G4 and GI0 have geometries in the range C2'-endo to C4'-endo.

(d)

C1 and GI2 exhibit sugar geometries in the range Ol'-endo to C4'-exo.

Glycosidic Dihedral Angle (X): NOESY

Information about X can be obtained from the

spectrum by monitoring the intensities of (H8/H6)---(HI')

and (HS/H6)

---(H2',H2") cross peaks. T h e behaviour of these cross peaks can be generally summarised

as

follows: (H8/H6) - HI' cross peaks are generally intense and those belonging to purines look relatively more intense than the ones belonging to the pyrimidines (Fig. 2a).

(HS/H6)

-- (H2', H2") (Fig. 2b) cross peaks, unfortunately

show too m u c h overlap in several cases and it is difficult to estimate their relative intensities.

Although from these spectra it is not possible to fix the

X angle with accuracy, a qualitative conclusion that the high anti domain seems reasonable.

X

angles are in

A detailed analysis with N O E

build-up is

necessary for more precise determination. Base-Base Stacking:

The sequential d I connectivities carry direct information

about base stacking patterns.

These N O E s

are not m u c h complicated by spin

diffusion processes, since for these, spin-diffusion paths are long and not very efficient. T h u s even a long mixing time N O E S Y structural information to a fair degree of accuracy.

can be used to derive In d - C T C G A G C T C G A G ,

d I connectivities are observed in the stretches CI-T2-C3, AII-GI2.

A5-G6-C7-T8

and

Of these, all except A 5 - G 6 and AII-GI2 are depicted by dashed 31

Vol. 144, No. 1, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

COSY

1.8

~ 2.2

2.6

T28

C7

~i~C]

"1"2

C3

~C9

c3~C

9

H2',H2"

"

G'IO~ G 4

GIOVG4 AI] ~ A 5 3.0 I

I

6"2

6.0

I

5'8

I

I

5.6

5-4

HI'

I

G12

4.61-

00 C] O0

o

00 H3' G6

T2

~G4,GIO All,AS

5'0 b I

i

:3.0

I

n

I

2.2

2.6

1"8

H2'.H2" Fig. 4.

a.

b.

Co

E x p a n d e d sections of.the 500 M H z C O S Y s p e c t r u m of d-CTCGAGCTCGAG at 2 5 ° C in D ~ O at p H 7.2. T h e digital resolution along both a x e s is 7.9 Hz/p~. HI' - (H2', H2")

cross peaks.

H2' - H3' cross peaks; in this region o n e also sees t w o H2" - H3' cross p e a k s c o r r e s p o n d i n g to C 1 a n d GI2. In the f o r m e r case the H2' - H3' p e a k is upfield with respect to the H 2 " - H3' cross p e a k , w h e r e a s in the latter case the situation is reversed. All the other ten H2" - H3' cross p e a k s are absent. H3' - H4' cross p e a k s w h i c h are o b s e r v e d for ten out of twelve nucleotides. T h e p e a k s c o r r e s p o n d i n g to G 4 a n d G I 0 are not observed.

32

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 144, No. 1, 1987

4.6

-

W T2 @

C3

H3'

G6

5,0

A5,A1 1

I

,

4,4

I 4.0

H4' Figure 4. continued.

lines in Figs. 2 and 3.

The NOEs

between A5 H8 and G6 H8 and between

All H8 and GI2 H8 protons occur in a different region of the N O E S Y which is not s h o w n here.

spectrum

T h e fact that d I connectivities are absent in the

stretches C 3 - G 4 - A 5 and T8-C9-GI0-AII, indicates that the base-base stacking in the major portion of the molecule deviates from that normally observed in the B D N A

models.

connectivities.

This conclusion is also supported b y the observed d 3

Examination of the d I patterns v i s a vis the sequence of the

molecule reveals an interesting sequence effect in the structure of the molecule. It is seen that the stretch -GAGCTCGAGof the molecule has a s y m m e t r y in the base sequence around the thymine in the centre.

Interestingly the d I connectivities are exactly complementary in

the two halves; connectivities are observed in the stretch A G C T the T C G A

stretch.

but not in

Similarly - G A - has no d I connectivity whereas - A G - at

the 3' end shows a d I connectivity. different as c o m p a r e d to the - C T C shows both the d I connectivities.

The -CTC-

stretch in the centre looks

fragment at the 5' - end; the latter These observations suggest that the sequ33

Vol. 144, No. 1, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ence effects on the structure are not limited to just the nearest neighbours but extend

over a longer stretch of the sequence.

the relevant sequence is five units long.

In the present case,

This provides a possible explanation

as to w h y the restriction enzymes require longer stretches of D N A

for specific

recognition, rather than just the structure around the cleavage site. ACKNOWLEDGEMENTS

The help provided by the 500 M H z F T - N M R

National Facility supported

by the Department of Science and Technology, Government of India, is gratefully acknowledged. REFERENCES i.

2. 3. 4. 5. 6. 7. 8. 9. i0. II. 12.

Hosur, R.V., Ravikumar, M., Roy, K.B., Tan-Zu-Kun, Miles, H.T. and Govil, G. (1985) 'Magnetic Resonance in Biology and Medicine', Tata McGraw-Hill Publishing C o m p a n y Limited, N e w Delhi, 243-260. Ravikumar, M., Hosur, R.V., Roy, K.B., Tan-Zu-Kun, Miles, H.T. and Govil, G. (1985), Biochemistry, 24, 7703-7711. Chary, K.V.R., Hosur, R.V., Govil, G. and T a n - Z u - K u n and Miles,H.T. (1987) Biochemistry (in press). Jeener, J. (1971) Ampere International S u m m e r School, Basko, Polje, Yugoslavia. Jeener, J., Meier, B.H., Bachmann, P. and Ernst, R.R., J. Chem. Phys. (1979) 71, 4546-4553. Anil Kumar, Hosur, R.V. and Chandrasekhar, K. (1984) J. Magn. Reson. 60, 143-148. Hosur, R.V., Chary, K.V.R., Anti K u m a r and Govil, G. (1985), J. Magn. Reson. 62, 123-127. Hare, D.R., Wemmer, D.L., Chou, S.H., Drobny, G. and Ried, B.R. (1983), J. Mol. Biol. 171, 319-336. Scheek, R.M., Boelens, R., Russo, N., van Boom, J.H. and Kaptein, R. (1984) Biochemistry 23, 1371-1376. Gronenborn, A.M. and Glore, G.M., Prog. N M R spectrc. (1985), 17,1-32. Hosur, R.V. (1986), Curr. Sci. 55, 597-605. Hosur, R.V., Ravikumar, M., Chary, K.V.R., Sheth, A., Govil, G., T a n - Z u - K u n and Miles, H.T. (1986) F E B S Lefts. 205, 71-76.

34