PROBING THE PROTEIN-BOUND WATER WITH OTHER SMALL MOLECULES USING NEUTRON SMALL ANGLE SCATTERING

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PROBING THE PROTEIN-BOUND WATER WITH OTHER SMALL MOLECULES USING NEUTRON

SMALL ANGLE SCATTERING

M. Lehmann

To cite this version:

M. Lehmann. PROBING THE PROTEIN-BOUND WATER WITH OTHER SMALL MOLECULES USING NEUTRON SMALL ANGLE SCATTERING. Journal de Physique Colloques, 1984, 45 (C7), pp.C7-235-C7-239. �10.1051/jphyscol:1984726�. �jpa-00224291�

J O U R N A L DE PHYSIQUE

Colloque C7, supplCment au n09, Tome 45, septembre 1984 page C7-235

PROBING THE PROTEIN-BOUND WATER WITH OTHER SMALL MOLECULES USING NEUTRON SMALL ANGLE SCATTERING

M.S. Lehmann

I n s t i t u t Laue-Langevin, 156 X , 38042 GrenobZe Cedex, France

La diffusion neutronique A bas angle sur les proteines paparneet ribonuclease-A a mis en evidence une grande difference entre l'interaction des macromolecules avec ethanol ou dimethyl- sulfoxide et glycerol. Avec le glycerol la proteine paralt garder une couche d'eau presque complhte, en accord avec les mesures thermodynamiques. Par contre, les mesures utilisant 1' ethanol montrent une grande accessibilite A la surface de proteines de cette mol6cule. L a difference entre les compartiments de ces molecules e s t discut6e, ainsi que llimportance de 18eau pour la stabilitd sttuczt:urelle de la protbine.

Neutron small angle measurements on proteins (Papain and Ribonuclease -A) in mixed solvents of ethanol/water, dimethyl sulfoxide/water and glycerol/water have been used to estimate the region near the macromolecule that only contains water. For glycerol quite a large region of 'bound" water is observed. This

is in agreement with thermodynamic measurements. For ethanol and dimethyl sulfoxide the region excluding the solute is small. The observations are used to speculate on the bound water required for a protein to retain its form and to function.

Neutron SmaLlAnale Scattering

A plenitude of techniques have been used to study the interactions between biological macromoles and their solvents, leading to a multitude of results which at first sight seem contradictory. For water, for example, the number of molecules reported to be distinctly bound to the the macromolecule or in other ways different from the common solvent, ranges from a few per macro- molecule up to more than one layer. An extensive analysis of the results /1/ show as expected these to be strongly dependent on the technique employed. Before reporting the results of small angle scattering studies on protein bound water we must therefore first clarify exactly what we measure using this technique.

In a coherent neutron diffraction experiment at low resolution (from solutions) the signal reflects the difference in scattering density between the molecule and the same volume occupied by the solvent continuum. For a single particle we can therefore write the amplitude of the scattered radiation as

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1984726

C7-236 JOURNAL DE PHYSIQUE

-, Q is the scattering Vector with length 4~sine/A, where 8 is half the scattering angle, and r is the position of atom i. The apparent scattering length mi is bi-v.Ps, where b. is the normal neutron scattering length, v. is tAe volume an& pS is the scatter ing density of the contikuum.

For small Q the scattered intensity can be approximated by /2/

1(Q) = F~(Q) = I(O) exp(-RGQ/3)

where I(0) , the forward scattering, is given as

with V being the volume of the macromolecule. RG is the radius of gyration defined as

The ri is _to the center of the mass defined from E(bi - PsVi)r = 0.

The main experimental variable is P . Due to the large-difference between the s~atterinq~tength for hydrogen (-0.37 x 10 cm) and deuterium (0.67 X 10 cm) the solvent scattering (mainly H O/ 0) can be varied from zero to approximately twice the s6at?gring density of the protein. This has been exhaustively used /3,4/ to analyse biological material in solution employing both RG and the linear relationship fory1(0). The results have been inter- preted in terms of molecular shapes, where the molecule is defined as the part of the solution which has a scattering density diffe- rent from that of the solvent.

For the case of protein bound water we must therefore in the interpretation exclude this water from the molecule, as it is unlikely that its scattering density is much different from the rest of the solvent, as long as this mainly consists of water. If however we introduce a sufficient amount of other solvent molecules this changes. Consider for example a protein in a solu- tion of D20. Ps is then about twice the scattering density of the molecule. We now add (CH )2S0, and P will drop. However, some region around the molecuqe might only contain D 2 0 and as this will now have a scattering different from P , we must include this as part of the molecule. In this kind of exgeriments we therefore obtain information about the regions around the molecule which are

inaccessible to the probe molecule added. The linear relationship for I(0) as a function of p will have a different slope and

intercept than in H20/D20,

and'^^

As we add more and more probes the inaccessible regions might very well change, and the I(Q) is not any longer a linear function of

Eventually, the protein might unfold. We shall in the g~ilowing only be concerned with limited amounts of solute, and assume the region of bound water to be constant.

Similar results can be obtained in studying three component systems - protein, water, small organic solute molecule - in terms

of thermodynamic var iahles / 5 / . In this case interaction para- meters are obtained /6,7/ indicating whether there is preferential binding of water or of the organic molecule to the protein. As these parameters are thermodynamic variables, an interpretation in structural terms is not possible, but a comparison with the small angle scattering results is very useful.

Fx~erimental Results

Measurements have been carried out on papain and ribonuclease-A.

Both molecular structures are known /8,9/. The small angle scattering instrument Dl1 of the Institut Laue-Langevin was employed. Details of the experiments are given elsewhere /10,11/.

The probe molecule was in all cases added to the D20 buffer solution of the macromolecule, and in order not to perturb the level of deuteration in the macromolecule and thereby change its scattering power all labile protons in the probe had been replaced by deuterium. Three kinds of probes were used, (CH )2S0, CH3CH20D and CH20D CHODCH20D. P igure 1 shows the example

02

l/l(0) = Cbi

-

For both molecules the behaviour of the solute molecule is the same, indicating that the solute excluded volume is much less than one layer of water on the surface. In all about 70 % of the surface is equally accessible to the solute and the water /lo/.

Similar results were found for ribonuclease and ethanol /11/.

Using glycerol at pH = 7.2, however, the preferred hydration was 0.23

*

Similar conclusions were reached from observations of the radius of gyration. In ethanol and dimethylsulfoxide changes were small, whereas a reduction of R was found when adding glycerol. This

shows that water was found bound on the surface. G

JOURNAL DE PHYSIQUE

Discuss ion

In a three component system there are three main intermolecular interactions to consider. For glycerol we observe that there is no contact between the probe and the macromolecule. This is probably related to the three hydrogen bond donors in glycerol. It is most unlikely that many parts of the protein surface will accept all three. Therefore some of the hydrogen bonds from glycerol must involve water, and this water must in turn as well participate in some hydrogen bond formation as donor. It must therefore either donate further hydrogen bonds to the protein, or to the solvent.

This is done in an environment constrained by the rigidity of the macromolecule, so in all binding of glycerol to the protein does probably not occur easily.

Ethanol does contact the surface, and following the above argu- ments this would arise from it having only one hydrogen bond donor / acceptor group, and only one hydrophobic group. The question is then which end of the ethanol is in function. The hydrogen bond donation / acceptation capacity is probably comparable to water. Very little is thus gained by replacement, and whenever well defined water structures exist it is unlikely that ethanol would form a better network of hydrogen bonds than water.

The interaction of the CH3 and of ethanol with water is considered to be unfavourable /13/, and the solubility of molecules like ethanol in water is partly due to removal of the hydrophobic part by self-aggregation /14/. If macromolecules are present, similar interactions might occur between CH3 and hydrophobic parts of the molecule. The amount of ethanol - accessible surface might thus be a measure of the size of the hydrophobic part of the surface. This

is supported by the measurements with dimethyl-sulfoxide, which give results similar to ethanol. Dimethyl-sulfoxide does only have one hydrogen bond acceptor group, and the common action of the two probe is therefore most likely to be related to CH3. Recently, the

interaction of ethanol with crystalline triclinic lysozyme was studied (M.S. Lehmann, S.A. Mason and G.J. McIntyre, in preparation), and 15 possible locat ions of ethanol on the surf ace were identified. They were all in regions of hydrophobic nature, and it was obvious from an inspection of the protein surface that a good part of it might accept hydrophobic groups.

None of the macromolecules studied undergoes measurable structural changes for the solute concentrations used. It is therefore reasonable to conclude that only a limited mound of the surface water is needed to retain the structure of the macromolecule. The macromolecular function might however not be retained, as the probe molecules will cover the surface, and prevent the necessary

intermolecular recognitions to take place. Ethanol denaturation might therefore be inhibition of activity by a blocking a good part of the hydrophobic patches of the molecule.

REPEIiENCES

1- FINNEY J.L., GOODFEmOW J.M. and POOLE P.L., Structural Molecular Biology (1982), 387. Eds. D.B. Davies, W. Saenger and S.S. Danyluk. Plenum Press, London.

2- GUINIER A. and FOURNET G., Small Angle Scattering of X-Rays (1955). Wiley, New York.

3- STUHRMANN H.B., Proceedings of the Brookhaven Symposium in Biology no. 27 (1976), IV-3.

4- JACROT B., Repr. PrOgr. Phy8. _34 (1976), 911.

5- EISENBERG H., Q. Rev. Biophys. && (1981), 141.

6- CASASSA E.F. and EISENBERG H., Advan. Protein Chem. (1964), 287.

7- INOUE H. and TIMASHEFF S.N., Biopolymers U (1972), 737.

8- DRENTH J., JANSONIUS J.N., KOEKOEK R. and WOLTHERS B.G., Adv.

Protein Chem. 2 (1971), 78.

9- WLODAWER A. and SJOLIN L., Proc. Natl. Acad. Sci. USA 18 (1981) 2853.

10- LEHMANN M.S. and ZACCAI G., Biophysics of Water (1982), 134.

Eds. F. Franks and S. Mathias. John Wiley and Sons.

11- LEHMANN M.S. and ZACCAI G., Biochemistry (1984) In press.

12- GEKKO K. and TIMASHEFF S.N., Biochemistry 20 (1981), 4667.

13- HVIDT Aa. Ann. Rev. Biophys. Bioeng. L.2 (1983), 1.

14- TANFOR C., The Hydrophobic Effect : Formation of Micelles and Biological Membranes (1980), 1. Wiley, New York.