Eur. J. Biochem. 74, 337-341 (1977)

Determination of the Solution Conformation of Adenosine 2' : 3'-Monophosphate by Nuclear Magnetic Resonance with Lanthanide Probes G . Victor FAZAKERLEY and Melanie A. WOLFE Department of Inorganic Chemistry, University of Cape Town, Rondebosch (Received September 22, 1976)

The aqueous solution conformation of adenosine 2' : 3'-monophosphate at pH 2.5 has been determined by a nuclear magnetic resonance method utilizing lanthanide ions as shift and relaxation probes. The ribose conformation is best described as a rapid equilibrium of 2'-endo(3'-exo) and 3'endo(2'-exo) conformations in a ratio of approximately 2 to 1. The orientation of the base relative to to ribose is restricted to a narrow range about XCN = - 70". In the breakdown of ribonucleic acids catalyzed by pancreatic ribonuclease the first step involves the formation of 2':3' cyclic nucleotides with subsequent hydrolysis to 3'-ribonucleotides. Studies on the solution conformation of the intermediate 2': 3' cyclic nucleotides have been limited to NMR coupling constant interpretation of the ribose conformation [l, 21 and potential energy calculations in the overall molecular conformation 133. No X-ray crystallographic data are available on the purine nucleotides although cytidine 2': 3'-monophosphate [4,5] and uridine 2': 3'-O,O-cyclophosphorothiolate [6,7] have been reported. We describe in this paper the conformation of adenosine 2' : 3'-monophosphate determined with the aid of lanthanide shift and relaxation probes. The 3':5' cyclic nucleotides have been studied with the aid of lanthanide probes with conflicting conclusions on the conformation about the glycosidic bond [8 - 101. In these molecules the ribose is held relatively rigid in the region 3T4 to 4T3. It is difficult to distinguish between these from the data available from shift and relaxation experiments. However, the cyclic phosphate ring imposes much less restraint upon the ribose conformation in the 2': 3' nucleotides and a number of conformations of which 2'-endo, 3'-exo, 3'-endo, and 2'-ex0 seem the most likely, have been predicted [ll]. This is in disagreement with the cytidine 2': 3'-monophosphate study in the solid state where the two molecules in the asymmetric unit were found with O(1') endo and nearly planar ribose conformations. However, there is no reason Abbreviation. NMR, nuclear magnetic resonance.

to assume that those conformations would be retained in solution or that they would be observed in adenosine 2' :3'-monophosphate. Coupling constant data [l] suggest a tendency towards 3'-endo(2'-exo) ribose conformation in the pyrimidine nucleotides and a less pronounced contribution from this conformation in adenosine 2': 3'monophosphate. EXPERIMENTAL PROCEDURE Proton spectra were run on a Bruker WH-90DS spectrometer operating at 90 MHz in the Fourier transform mode. Proton shifts are given in p.p.m. from the sodium salt of 3-(trimethylsily1)-propane sulphonic acid, which was also used to monitor changes in relaxation times arising from outer sphere complexation. The longitudinal relaxation times, TI, were determined with a 180"-~-90"pulse sequence and TZ fromline-width measurements [12]. Lanthanide nitrate solutions were prepared by dissolving lanthanide oxides (99.99 %) in nitric acid. Lanthanide and substrate solutions were dissolved in 99.8% 'H20, adjusted to pH 2.5 with 'HC1 and lyophililized three times. Experiments were carried out at 28 "C. The calculated shifts and internuclear distances were obtained from careful model building using Dreiding models corresponding to known bond lengths from crystal structure data [4,5]. The ribose conformation was then studied in all its predicted forms [I, 21. The metal - oxygen bond distance used was 0.23 nm found from studies on 3': 5' cyclic phosphates

338

Conformation of Adenosine 2’: 3’-Monophospbate by NMR

previously 19,10], which again proved to give the best fit of the data. The reported data are corrected for the small shifts obtained by titration with diamagnetic lanthanum nitrate. RESULTS AND DISCUSSION The incremental addition of 5 I\/I dysprosium nitrate solution to a 20 mM solution of adenosine 2‘: 3‘-monophosphate (Fig. 1) induces shifts in the resonances (Fig. 2). In addition to the information derived from these shifts it provides a method of separating the resonances to allow for accurate measurements of the relaxation times. A suitable partially shifted spectrum is shown in Fig.3. Under conditions of axial symmetry and if the origin of the shifts is pseudo-contact the shift results can be related to the conformation of the lanthanide . substrate complex [13]. The shifts extrapolated back to zero metal ion concentration are required and these are shown in Fig.4 and Table 1 relative to the shift of H-2‘. The data could be further complicated by intramolecular stacking and the presence of more than one metal-binding site. Although purine bases are known to stack in aqueous solution [14,15] experiments at 5 mM substrate concentration gave very similar data indicating that stacking is not significant. The addition of similar concentrations of lanthanides to a solution of adenosine did not induce significants shifts indicating that only the phosphate group is involved in binding. The relative shifts for different lanthanides are similar indicating that the complex possesses axial symmetry and that the shifts are pseudo-contact in origin. Experiments with europium gave rather poor results arising from a low shift-to-broadening ratio. The average of the results obtained in experiments with dysprosium, praesodymiuin and holmium were used in subsequent calculations. The magnitude of the pseudo-contact interaction in terms of r , the metal-ion - observed-proton internuclear distance, 8, the angle between the magnetic anisotropy axis and the vector joining the metal ion and the observed nucleus has been given by La Mar

in the case of axial symmetry where D is a constant during the experiment. From the near equivalence of the shifts of H-2’ and H-3’ this magnetic axis must bisect the 0 - P - 0 angle. In studies on adenosine 3‘:5‘-monophosphate it was found to bisect the 0 - P - 0 angle and lie in the plane of those three atoms [8,9]. The possible conformations of the ribose rings have been discussed in detail previously [1,2]. The

i”’

Fig. 1. Adenosine 2‘ :3 ‘-monophosphate

phosphate ring imposes very much less restraint upon the ribose pucker than in the 3‘: 5‘ cyclic nucleotides. Computing the shifts for the known possible ribose conformations shows that no single conformation fits the data but that a rapid equilibrium between two or more conformations must be present. Although minor contributions from other conformations cannot be excluded the best fit for the H-l’, H-2’. H-3’, and H-4’ shifts is found with a mixture of 2’-endo(3’exo) and 3‘-endo(2’-exo). The computed shifts for three weighted averages are shown in Table 1. Included is the computed shift average for the exo cyclic 5’ protons assuming the rotamer populations predicted previously 121. We have not attempted to determine this independently and the poor fit may arise from using incorrect populations. The coupling constant study was made at pH 6.8 and no data are available for the populations at pH 2.5. Thus the best fit is found with 55 & 5 % 2’-endo(3‘-exo). No significant improvement in the fit of the data was found by moving the magnetic axis out of the 0 - P - 0 plane and this is supported by the results on the base protons. Relaxation Studies

Gadolinium(II1) induces no measurable shifts in the proton resonances but markedly affects the relaxation times. Under conditions of fast chemical exchange the change in the relaxation times are reIated by the Solomon [17] and Bloembergen [18] equations to the metal-ion - observed-proton internuclear distance, r, by T TIM, (l/TzM)being proportional to F 6 . The conditions under which this holds have been discussed previously [l2]. The resonances of the free substrate are insufficiently resolved to allow accurate measurements of the relaxation times and a sample containing 0.15 M

339

G. V. Fazakerley and M. A. Wolfe H-4' H-1

H-5'

H-8

0 H -2

0.05

0

Fig. 2. Shifts

of

0.15

0.10

0 20

(W

[D?']

proton resonances against dysprosium concentrution (adenosine 2' :3'-monophosphate 20 m M )

1

a

9

7

6 Shift (p.p.rn.)

Fig.3. Specrruni ofudenosine 2':3'-monophosphutr 120 rnMJ in the presence

1.o-Xxx

x x x

V

of 150 mM

"

"

A

5

4

Pr3'

V

X

H-3'

H-4: H-1

0 -0.1

0

0.05

0.10 [Dy3'1 (W

Fig.4. Proton shifts relative to H-2' ugainst Dy3' concentration

0 .15

0

.m

340

Conformation of Adenosine 2' :3'-Monophosphate by N M R

Table 1. Shgt ratios relative to H-2 f o r protons

€3-8

Lanthanide

DY Pr Ho Average €11 -

--

-- -

0.18 0.18 0 17 0.18 0 29 - _ _

of

adenosine 2':3'-monophnsphate (0.02 M ) ,for difTer.ent lanthanides

H-2

H-1'

H-2'

H-3'

H-4'

H-5'/H-5"

0.02 - 0.05 - 0.02 - 0.03 - 0.02

0.81 0.86 0.73 0.80 0.93

1.oo 1.oo 1 .oo 1.oo 1.oo _-

1.01 1.04

0.82 0.90 0.72 0.81 0.95 -_

0.28 0.31 0.26 0.28 0.36 ___

-

_ _

1.oo

1.02 111 ~-

Computed shifts

-

30% 2'-endo" 55 yu2'-end0 70 2'-endo

x

~~~~

~

~

0.94 1.02 1.06

1.00 1.oo 1.oo

0.76 0.88 0.96

0.83 0.80 0.77

0.39h 0.41 0.42b

~~

Corresponding to a mixture of 30% 2' endo (3' exo) and 70"/, 3' endo (2' exo). Calculated using the rotamer populations from [2], 60 "/, gg, 34 "/, gt, and 6 % tg

a

Table 2. Relu.ration data of the protons of adenosine 2' :3'-nionophosphate (0.02 M ) with gudoliniumiIII) ielutive to H-2' Parameter

H-8

H-2

H-1'

H-2'

H-3'

H-4'

H-5'tH-5"

MI -

0.196 7.87 0,202 7.83

0.158 8.16 0.202 7.83

2.10 5.30 1.19 5.83

1.oo 6.00 1.oo 6.00

1.36 5.70 0.95 6.05

0.88 6.13 0.60 6.53

0.15 8.21 0.12 8.54

5.60 5.56 5.5

6.00 6.00 6.00

5.90 5.88 5.85

5.90 6.02 6.17

7.69b 7.76 7.85

(7-1

Calculated distance ratios

TZM)I ~

Calculated distance ratios Measured ratios for:

60 "/, 2'-endo" 70 2'-endo 80 Z'-endo

x

Determination of the solution conformation of adenosein 2':3'-monophosphate by nuclear magnetic resonance with lanthanide probes.

Eur. J. Biochem. 74, 337-341 (1977) Determination of the Solution Conformation of Adenosine 2' : 3'-Monophosphate by Nuclear Magnetic Resonance with...
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