Physica 136B (1986) 260-264 North-Holland, Amsterdam

NEUTRON SCATTERING STUDIES OF HYDRATION OF MOLECULES OF BIOLOGICAL IMPORTANCE

J. TURNER 1, J.L. FINNEY 1, J.P. B O U Q U I E R E 1, G.W. NEILSON 2, S. CUMMINGS 2 and J. BOUILLOT 3'* aDepartment of Crystallography, Birkbeck College, London WC1 E 7HX, UK 2H. H. Wills Physics Laboratory, University of Bristol, Bristol BS8 1 TL, UK 3Institut Laue-Langevin, 156 X, 38042 Grenoble, France

The isotopic difference method of neutron diffraction has been used to study aqueous solutions of 2 molal urea, formamide and acetamide, to obtain information on the hydration structure around the polar amide group. In each case the difference between the coherent differential cross-sections for solutions containing natural abundance nitrogen and nitrogen-15 has been determined and Fourier transformed to give the atomic distribution function with reference to nitrogen. Although the difference measurements obained from formamide and acetamide are of less good quality than that from urea, the results in the intramolecular distance range are in agreement with the known molecular structures. The intermolecular structures, assumed to be due to solute-water correlations at this concentration, give water-coordination numbers consistent with the results of computer simulation studies.

1. Introduction

The hydration of the amide (CONH2) group has received wide attention as a model for the hydration of the peptide group in proteins. The present work describes the application of the first difference method of neutron diffraction [1], using nitrogen isotope substitution, to the study of amide group hydration in aqueous solution. The method has previously been used to study nitrate and ammonium ion hydration [2, 3]; in this work we attempt to extend it to the study of molecular hydration.

2. Experimental procedure

Experiments were performed using 2.0 molal u r e a ( C O ( N D 2 ) 2 ) , 1.9 molal formamide (HCOND2) and 1.6 molal acetamide (CD3COND2). In each case two isotopically dis- tinct solutions in D 2 0 w e r e prepared, containing natural abundance nitrogen and nitrogen-15. Dif- fraction measurements were made on the urea

*Present address: Reactor Radiation Division, National Bureau of Standards, Gaithersburg MD 20899, USA.

solutions using the D4B diffractometer (ILL) and on the formamide and acetamide solutions using the D2 diffractometer (ILL). The data were corrected for multiple scattering and absorption and put on a scale of barns by reference to a vanadium standard, and the difference between the distinct coherent differential cross-sections of the two solutions AN(k ) was determined [1].

3. Results

The Fourier transform of the difference, GN(r), is the atomic distribution function with reference to any nitrogen atom and is given in terms of the pair correlation functions gN~ (r) by

GN(r) = E AN~(gNa(r) -- l ) , (1) ot

where r is the interatomic distance and the sub- scripts denote atom types a in the solution. The values of the coefficients A N~ and the atomic concentration of nitrogen, CN, are given in table I for the three experiments.

GN(r ) is expected to show peaks corresponding to solute structure and to short-range inter- molecular structure, assumed to be due to the

0378-4363/86/03.50 © Elsevier Science Publishers B.V. (North-Holland Physics Publishing Division)

J. Turner et al. I Hydration of biological molecules 261

0 . 0 3

0 . 0 2

AN(k) 0.01

0 . 0

- 0 . 0 1

- 0 . 0 2

J ~

t ,,J ._ .¢". " ~ , .

0 . 0

!

4 . 0

Fig. 1. First order difference for 2.0 molal urea in D20.

k (.~_1) I I I I I

8 . 0 1 2 . 0 1 6 . 0

The units of AN(k ) are barns steradian 1 nucleus 1.

solvent at 2 molal concentration. The differences AN(k ) obtained and the Fourier transform of AN(k ) for urea are shown in figs. 1-4. Table II gives the interpretation of the observed peaks in GN(r ) in each case. The intramolecular regions are compared with the known solute structures [4-7].

In the case of urea, the theoretical intramolecu-

lar differential cross-section [8] was calculated, and was used to separate the intramolecular and intermolecular contributions to the experimental difference by an iterative method [9]. The result- ing intramolecular function was fitted to the molecular model for urea by a least-squares minimization with an R factor of 0.18.

0 . 0 6

0 . 0 4

(r) N

0 . 0 2

0 . 0

GN(O)

- 0 . 0 2

- 0 . 0 4

• °

~2 . . . . . . . . . r

• •

IQ g I 61I 0 0 0 @ODI~IIQ

I0 9Q ~qO QOI6 00m QI00 IIQQ 0 Oelg I

iI e

IQ 0 Q 00

QOI0010~I 0

I l I ! | I I l I

0 . 0 1 .0 2 . 0 3 . 0 4 . 0 5 . 0

Fig. 2. GN(r ), the Fourier transform of the first-order difference, for 2.0 molal urea in D20.

262

0 . 0 3 0

J. Turner et al. / Hydration of biological molecules

AN(k) 0 . 0 1 5

0 . 0

- 0 . 0 1 5

- 0 , 0 3 0

?..

g

" % " ~ - ' ~ ' ' ' ' ' ~ ' ' ' ¢ ~ 4 ~ " .,,- ~..r-.,-,,~- ~ " ' ~ " ~ - " " " ~ " ~ ' ¢ ' . . ~

k

I I I 1 I I I I I I I I I I

0 . 0 5 . 0 1 0 . 0

Fig. 3. First order difference for 1.9 molal formamide in D20 . The units are as in fig. 1.

Table I Sample parameters for deuterated amide solutions in D : O

1 5 , 0

Solute molality c N A r~o A No ANc A Nr~ A N H GN(0) (millibarns)*

CO(ND2) 2 2.0 0.024 5.91 2.48 0.11 0.26 - 8 . 7 6 H C O N D 2 1.9 0.012 2.88 1.26 0.05 0.06 - 0 . 0 3 - 4 . 2 3 CD3COND 2 1.6 0.010 2.40 1.00 0,07 0.04 - 3 . 5 2

*1 barn = 10 -24 cm 2.

0 , 0 0 8

A (k) N

0 . 0 0 4

0 , 0

- 0 . 0 0 4

t

~., ,.

• . , • ' . . .

. . , o . .

. . . ,

• , . . . ~ : .. ' , , : . : , , : . . . ; ' , . . . ' , , : : , " . ' . . . • . : "

'.. , .,:,, . . . . . ~ " , • . , . . . . , .¢ . . : : , . . . , , . , ,

'ol- i . - , ' • . . ' °

k

I | | ~ | i i I i i i

0 . 0 5 . 0 1 0 . 0

Fig. 4. First order difference for 1.6 molal acetamide in D20 . The units are as in fig. 1.

1 5 . 0

J. Turner et al. / Hydration of biological molecules

Table II Interpretation of GN(r) for amide solutions

263

Peak position Assignment of peak

(~) atom pair separation (A)

Coordination number

measured predicted

Urea 2.0 m (ref. 4)

0.95 2 x ND 1.00 2.2 / 3.1(1) 1.38 NC 1.35 0.9 l 2.27 NO, NN, ND 2.29, 2.30, 2.50 2.8(2) 2.67 NO (water) 1.1(2) 2.55 - 4.00 ND 3.21

N-water 7.1(5)D20 1 8 + 0.5 D20*

(ref. 10)

Formamide 1.9 m (ref. 5, 6) 0.90 2 x ND 1.00 1.5 2 1.35 NC 1.33 0.6 1 2.04 NH 2.06 0.7 1 2.31 NO 2.25 1.0 1 2.50 - 4.25 N-water I0.1 D20 10 D20

(ref. 11)

6.6 DzO

Acetamide 1.6 m (ref. 7) 1.20 2 x ND 1.00 2.2 1.80 NC 1.35 0.5 2.35 NO, NC(Me), 2.29, 2.41 3.3

ND(Me) 2.65 - 4.20 2 x ND(Me)

N-water

*Calculated from results given in ref. 10; error quoted is the estimated uncertainty in our calculation.

The agreement of the experimental results with the solute molecular models is less good for formamide and acetamide than for urea. A known problem in the formamide data was a small inequality in light-water content between the two samples, while the statistical accuracy of the acetamide difference is obviously less good than that obtained f rom the two previous experi- ments.

In the intermolecular distance range, integr- ation of Or~(r ) gives about 7 water molecules within approximately 4 A of the solute nitrogen atom for urea and acetamide, and about 10 in the case of formamide.

in the vicinity of the nitrogen a tom is consistent with the results of computer simulation studies of urea and formamide solutions [10, 11]. In the case of urea, where the intramolecular agreement is good, it should be possible to interpret the inter- molecular (water) region in more detail. How- ever, there are difficulties in doing this because the peaks are not well defined, although the 2.7 ,~ peak may perhaps be assigned to an oxygen a tom of water. For this reason we aim to repeat the measurements in a solvent of different H 2 0 / D 2 0 composit ion to investigate the possibility of separating the N . . O and N . . D contributions in the solvent region.

4. Discussion Acknowledgements

The results show that the method has given the solute structures in agreement with the models while the total number of water molecules found

We would like to thank the Institut L a u e - Langevin for the use of facilities for, and expert assistance with, the carrying out of the experi-

264 J. Turner et al. / Hydration of biological molecules

ments, and the Science and Engineering Research Council for finance. J.T. would like to acknow- ledge an S.E.R.C. studentship. We would also like to thank Professor J.E. Enderby for discus- sions and for stimulating the original idea to work on urea.

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