Solvent effects on the structure, dynamics and activity of lysozyme

Transcription

1 Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci., Vol. 8, Nos 1 & 2, August 1985, pp Printed in India. Solvent effects on the structure, dynamics and activity of lysozyme J. L. FINNEY and P. L. POOLE Crystallography Department, Birkbeck College, Malet Street, London WC1E 7HX, England Abstract. Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupley et al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0 2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for the Raman detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function. Keywords. Protein hydration; protein structure; hydration of lysozyme; activity of lysozyme; solvent effects on lysozyme. Introduction In attempting to study water in biological systems, there are many difficulties. For example, the effects are small at the molecular level, relating to subtle changes, or small perturbations, of the structure and/or dynamics of water in different environments. Though these perturbations are small for each water molecule, for a complete assembly they may aggregate to relatively large effects. For example, so-called hydrophobic effects derive from small perturbations close to apolar groups, while hydrogen bonding effects may relate to small differences in strengths of different hydrogen bonds (Finney et al., 1980; Finney 1984). Secondly, most biological processes occur in excess water, of which only a very small fraction is likely to be perturbed. Thus, useful experimental techniques should ideally be able to measure a small effect on a small fraction of the water present. Such techniques are not easy to find; in practice, we usually have to make do with a poor signal to noise ratio when making measurements of these small perturbations. One possible way of ameliorating this problem is to remove most of the water in the system. At first sight, such a partially dried system may seem to be of only limited biological relevance. However, there is increasing evidence to show that the totality of the solvent is not necessary for either the stability (or perhaps more correctly metastability?) or the activity of a protein. Moreover, crystallographic work has shown that most of the water can be replaced by other solvents, as in cryosolvent work at low 25

2 26 Finney and Poole temperatures, or the replacement by alcohol in low angle neutron scattering experiments (Lehmann, 1984). We discuss here one particularly interesting low solvent content system, namely the dehydration, or controlled rehydration, of lysozyme. Rupley has shown (Rupley et al., 1980) that enzyme activity remains even after most of the water has been removed, and the enzyme-water system is in the solid phase. He has also argued that there is no structural change during dehydration. In contrast, using a variety of techniques, mainly spectroscopic, we argue that both structural and dynamic changes occur on rehydrating the previously dried protein, and that these changes which are solvent mediated are necessary for the protein to regain its activity. Lysozyme activity and monolayer coverage Figure 1 shows schematically the activity of hen egg-white lysozyme as a function of water content for three diffferent ph values, measured by Rupley et al. (1980). Also marked are several water content values of interest from other experiments. We note immediately that activity is zero at hydrations less than about 0 2 g water/g protein. Above this water content, activity rises, at a rate which depends on the ph. We note also that the critical hydration is very low, being well below the water content of about g/g at which lysozyme goes into solution. Two other markers are given in Figure 1. Lysozyme activity as a function of hydration at three different ph values (Rupley et al., 1980). The hydration values corresponding to polar monolayer and total monolayer coverage are shown, together with the hydration at which the protein goes into solution.

3 Solvent effects on lysozyme structure 27 the figure. The first, at about 0 3 g/g, corresponds to the non-freezing fraction of water as measured by Golton (Golton, 1980; Finney et al., 1982) using NMR, IR and DSC techniques. Arguments based on available exposed surface area suggest this may correspond to saturation by water of all available surface polar sites, although we should bear in mind that the calculations leading to this conclusion were performed on the static molecular coordinates as determined from X-ray crystallography. A second marker denotes the water content at which further coverage by water of apolar groups on the molecular surface results in complete coverage of the protein by water molecules. This figure of about 0 5 g/g is based on the area assumed covered by a single water molecule at normal density (Golton 1980; Finney et al., 1982). Thus, enzyme activity is seen to commence well below even that water content at which all polar groups are interacting with water, and well below monolayer coverage. We are thus led to ask questions such as the following. (a) What molecular events occur on rehydrating the dried protein? (b) What changes if any occur to the protein as a result of these hydration interactions, and how might these relate to the onset of activity? Obvious possible answers relate to changes in both structure and/or dynamics, and a discussion of these possibilities forms the major part of this paper. Hydration events Using a direct difference infrared method (for which special techniques had to be developed (Poole and Finney, 1982)), Poole has followed the various hydration events that occur on rehydrating previously dried lysozyme (Poole and Finney, 1982, 1984; Finney et al., 1982). The samples are films, prepared by slowly drying down under vacuum and P 2 O 5 a previously prepared concentrated solution. The structure of these films had previously been examined by low angle neutron scattering, the results of which were interpreted in terms of a uniform swelling of a random close-packed assembly of lysozyme molecules (Golton, 1980; Finney et al., 1982). The mean intermolecular separation of the lysozyme molecules (d) remained constant at the contact distance d = d c at hydrations below about 0 3 g/g (the nonfreezing fraction, and polar monolayer coverage value). Above this water content, d increased approximately linearly with hydration as the molecules moved apart until, at about g/g, the film collapsed, yielding a concentrated solution. At this point, the mean intermolecular separation distance was equivalent to about three water molecules, implying that the swollen glass can maintain its structural integrity through this number of intervening water molecules. Examining electrostatic effects on the strength of this intermolecular interaction through ph changes would be of considerable interest. By following the perturbation of characteristic IR vibrations in the protein glass sample as a function of hydration, Poole concluded the following (Poole and Finney, 1984; Finney and Poole, 1984). First, at low hydrations, a proton redistribution takes place as the acid (and by implication the basic) groups ionise and revert to their normal pk order. This (and also the following charged group hydration) occurs rapidly and non-cooperatively; it is complete by about 0 10 g/g. The peptide NH, CO, and sidechain polar groups rehydrate more slowly and cooperatively (cooperativity coefficients is

4 28 Finney and Poole about 2). The hydration profiles of these three processes are similar, though they occur at different rates (with respect to water addition). Of interest from our viewpoint is the completion of polar sidechain hydration by the commencement of activity, while the main chain NH hydration is almost complete. Peptide CO hydration is incomplete at the critical hydration value. Structural changes In principle, structural changes in solution can be probed by following the behaviour of structure-sensitive bands in the Raman spectrum. Yu and Jo (1973) have already shown that there are Raman spectral differences between dry lysozyme powder and lysozyme in solution. It is therefore clearly of interest to follow the behaviour of structurally sensitive bands through the sequential hydration process, to see how such changes may relate to the commencement of enzyme activity. Figure 2 shows traces of the lysozyme Raman spectrum in solution and at three lower Figure 2. Lysozyme Raman spectra in solution and at three lower values of hydration.

5 Solvent effects on lysozyme structure 29 hydrations. Spectra were taken using the green Kr + laser line at nm, using both glass pellet and powder samples (Poole and Finney 1983). We see clear changes in the spectra. For example, as hydration is lowered, the aromatic band at about 1620 cm 1 splits into a doublet. The 1361 cm 1 band, assigned to a buried tryptophan, shows a significant fall in intensity; as this band is thought to be particularly conformationally sensitive, there is thus some indication of a change in the tryptophan environment. Changes are also evident in the 1554 cm 1 band; this again is assigned to a tryptophan. Changes in the S-S band at about 508 cm 1 are also seen (not shown in figure). The behaviour of the amide III band (at about 1260 cm 1 ) is complex. The band itself is complex, being made up of bands assigned to α-helical ( cm 1 ), β-sheet ( cm 1 ), and random coil structure ( cm 1 ), and the profile changes can be interpreted in terms of the changing contributions of these secondary structure elements to the overall shape of the band. Figure 3 shows the behaviour of three Raman spectroscopic parameters as a function of hydration. In all three cases, there are clear changes in the parameter concerned. Moreover, the changes are all completed i.e. have reached their solution values by about 0 2 g/g. This is of particular interest on two counts. First, only about 0 2 g/g of water appears to be necessary for the structure, as characterised by Raman, to return to Figure 3. Behaviour of conformation-related Raman parameters as a function of hydration. (a) is the normalised (with respect to the CH vibration at 1448 cm 1 ) intensity of the 1361 cm 1 tryptophan vibration. (b) is the average shift of the frequency of amide I and III, while (c) shows average half band widths of the amide I and III bands.

6 30 Finney and Poole that seen in solution. The remainder of the water appears to play no further structural part. Secondly, activity commences as soon as the changes have gone to completion on increasing hydration. It is therefore tempting to speculate that these structural changes are necessary for the protein activation. In addition, we might note that these changes do not commence until the charged group ionisation has effectively gone to completion. We cannot say categorically, from the evidence at hand, what these structural changes are precisely. There is clearly some change in the environment of the buried tryptophan (1361 cm 1 ), which indicates some structural change. The change in disulphide intensity implies a further structural shift. The changes in the composite amide III band are of particular interest, however, as the components of the band relate to the relative contributions of α-helix, β-sheet, and random coil. A detailed analysis of this band (Poole and Finney 1983) suggests that, on rehydration, there is a reordering of the helix at the expense of random coil, the (small) β-sheet component remaining essentially unchanged. We thus might propose a model in which, on removal of water, a distortion of the helix occurs, which is removed on water readdition. Such an interpretation is consistent with recent work which suggests that water interactions with main chain groups within a helix may be important in determining the structural details of the helix itself (in particular its distortion from ideal) (Blundell et al., 1983; Finney 1978). Flexibility and dynamic changes For any structural changes to occur, there must be a certain degree of molecular mobility. Moreover, for activity, we would expect a degree of flexibility to be necessary. We therefore discuss now probes of molecular flexibility as hydration is increased. The steps in an exchange in-exchange out NMR experiment are detailed in table 1. Table 1. The NMR exchange experiment.

7 Solvent effects on lysozyme structure 31 The essentials of this technique are to replace all the easily-exchanged protons with deuterium, and then to study the rate of back exchange of protons on exposure to environments of different relative humidities of H 2 O. We restrict ourselves to the amide hydrogens, and figure 4 shows typical spectra as a function of water content of the rehydrating protein. The NH proton peak clearly increases with hydration; the behaviour of the residual deuterated amide as a function of hydration is summarised in figure 5. Although two methods are used to estimate residual deuterated amide in figure 5, the trends in the two sets of results are similar. At low hydration, little back-exchange of protons occurs; in fact, static accessibility calculations show that about 22 % of amide protons are surface exposed as seen by crystallography, implying that at very low hydration only these foully exposed deuterons can exchange. As hydration increases, more amide hydrogens exchange (region I), but this increase stops between about g/g. There follows in fact a plateau region (region II) in which increasing hydration fails to induce further significant hydrogen exchange. At higher hydration, Figure 4. Proton NMR spectra (a) deuterated, and after exposure to (H 2 O) relative humidities of (b) g/g and (c) g/g. The growth of the proton NH peak is evident.

8 32 Finney and Poole Figure 5. Behaviour of the exchangeability of hydrogens as a function of water content. Curve 1 relates to peak area measurements using weighing of the proton resonance peak, while curve 2 results from the integrated traces. the curve begins a further downward trend, corresponding to a further increase in exchange (region III). A possible and attractive interpretation of this data can be made in terms of molecular flexibility. As hydration is increased, there is a change in dynamics above 0 07 g/g which enables further hydrogen exchange with solvent to occur. Interestingly, this change in mobility commences at about the hydration at which ionisation of charged groups is nearing completion. Moreover, this discontinuity in the dynamics occurs before the onset of the structural shifts indicated by the Raman work. We might thus suggest that these flexibility changes are necessary before the molecule can undergo the structural changes which seem to be required before activity can recommence. Unfortunately, this interpretation of the NMR data in terms of dynamics is somewhat indirect; it would be preferable to obtain direct information to confirm the proposal. Recent inelastic neutron scattering data does in fact confirm this interpretation (Poole, 1984; Poole et al., in preparation). In this experiment, inelastic neutron scattering measurements were made on the IN5 spectrometer at the Institut Laue- Langevin, Grenoble, France. Two samples were used. The first was a powder sample hydrated previously to 0 20 g/g D 2 O (equivalent to 0 18 g/g H 2 O). This was sealed in a modified standard powder sample holder, and spectra taken over a 24 h period. Dry air was then passed slowly through this sample to lower the water content over a 12 h period; a further 24 h data collection followed on this dry sample, whose hydration was finally measured to be 0 07 g/g D 2 O (0 06 g/g H 2 O). Figure 6 shows the resultant dry minus wet difference spectrum. The central peak

9 Solvent effects on lysozyme structure 33 Figure 6. Dry wet inelastic neutron scattering spectrum. Note the increase in the elastic component, and the decrease in the near-inelastic region as the protein is dried down. corresponds to the elastic region, which is clearly enhanced in the dry sample. In addition, there is a reduction in the region at about 15 cm 1, implying, as hydration is reduced, a decrease in the quasielastic and inelastic regions. Both these kinds of behaviour are consistent with the NMR interpretation: between 0 06 and 0 18 g/g, there is an increase in molecular flexibility. Before finally summarising the results discussed here, we might pause to consider the nature of this change in dynamic behaviour as water is removed. First, that the globular structure does not totally denature on removing most of the water should not be considered as surprising. When considering the effect of solvent on protein stability, we normally are working within a solution framework, and we are concerned with the free energy difference between the native structure in solvent and the unfolded conformation also in solvent. In both cases, extensive protein-solvent hydrogen bonding will occur, and the overall stability will result from a fine balance of forces which will give a small stabilisation (only about kt in typical cases) to the folded conformation (Finney et al., 1980; Finney, 1984). The case we are discussing here is very different. To unfold, even partially, the protein must break a considerable number of intramolecular interactions (probably mainly hydrogen bonding and van der Waals) which, because of the absence of solvent, would be neither satisfied, nor compensated in any other way (which in solution would be through solvent interaction) in the unfolded state. Thus, the compact state we would expect to be more stable than a partially unfolded state under the low solvent conditions. Clearly, we would expect structural readjustments to occur to partly compensate for the disruption of local force balance caused by water removal, but we have no reason to expect the protein myself to unfold. In some ways, this structural relaxation behaviour might be compared with the changes that occur on passing through a glass transition, although whether our globular, almost dry protein should be

10 34 Finney and Poole thought of as a glass (and therefore having a global minimum difference from the structure we find) is not clear. Conclusions Dry lysozyme, although probably not structurally very different from lysozyme in solution, shows some structural shifts from the solution structure. The Raman measurements reveal such structural shifts near disulphide bridges, buried tryptophans, and in secondary structure. These latter changes are interpreted in terms of a reordering of the helical region as water is added. These changes are not initiated until the completion of a dynamic change, which occurs above about 0 07 g water/g protein. This flexibility change is completed before the onset of, and hence may be necessary for, these structural shifts. In turn, the flexibility changes are not complete until enough water has been added to facilitate side-chain ionisation, and the recovery of the normal pk order. This side chain ionsiation (and consequent hydration) seems to dominate the low hydration behaviour. Related measurements by Rupley et al. (1980, 1983), and Bone and Pethig (1982) also indicate dynamic changes as a function of hydration. These structural and dynamic changes go to completion before enzyme activity recommences. The final model is summarised in figure 7, which shows the regions in which various Figure 7. Various hydration events, and how they may relate to the onset of lysozyme activity as water content is increased.

11 Solvent effects on lysozyme structure 35 changes occur in relation to the critical hydration. At the low hydration end, the primary process is one of proton redistribution and acid hydration. Little else seems to happen to the protein until this process has effectively reached completion. Interestingly, the amount of water necessary here corresponds to that amount which low angle neutron scattering studies suggest is not displaced by addition of alcohol (Lehmann, 1984). At this point, a change in flexibility occurs, allowing enhanced access of exchanging solvent hydrogens to the amide groups that are, according to the static X-rays structure, buried. Once the flexibility change has occurred, a number of (probably small) structural shifts take place: these go to completion before the enzyme regains its activity at about 0 2 g/g. At this hydration, even though the Raman spectroscopic parameters investigated have reached their solution values, yet our protein is not yet foully hydrated even as far as the exposed polar surface groups are concerned. We should stress in conclusion that the details of this reactivation process remain to be understood. We can follow the hydration events that occur, e.g. by difference IR (Poole and Finney, 1984), and also some of the hydration-induced changes in the protein myself. The precise way in which the solvent is participating, however, remains unclear. Acknowledgements P.L.P. thanks the SERC for a research studentship. J.L.F. thanks The Royal Society, IUPAB, and the Symposium organisers for financial support which made his attendance at the Symposium possible. References Blundell, T., Barlow, D., Borkakoti, N. and Thornton, J. (1983) Nature (London), 306, 281. Bone, S. and Pethig, R. (1982) J. Mol. Biol., 157, 571. Finney, J. L. (1978) J. Mol. Biol., 119, 415. Finney, J. L., Gellatly, B. J., Golton, I. C. and Goodfellow, J. M. (1980) Biophys. J., 32, 17. Finney, J. L., Goodfellow, J. M. and Poole, P. L. (1982) in Structural Molecular Biology (eds D. Β. Davies, W. Saenger and S. S. Danyluk), (New York: Plenum) p Finney, J. L. (1984) J. de Phys. (Paris), 45, (Colloque C7, Suppl. no. 9), C Finney, J. L. and Poole, P. L. (1984) Comments Mol. Cellular Biophys., 2, 129. Golton, I. C. (1980) An Experimental and Theoretical Study of the Interaction Between Water and a Globular Protein, Ph.D. thesis, University of London. Lehmann, Μ. S. (1984) J. de Phys. (Paris), 45 (Colloque C7, Suppl. no. 9), C Poole, P. L. and Finney, J. L. (1982) J. Phys. (E), 15, Poole, P. L. and Finney, J. L. (1983) Int. J. Biol. Macromols., 5, 308. Poole, P. L. and Finney, J. L. (1984) Biopolymers, 23, Poole, P. L. (1984) J. de Phys, (Paris), 45, (Colloque C7, Suppl. no. 9) C Rupley, J. Α., Yang, P. Η. and Tollin, G. (1980) in Water in Polymers (ed. S. P. Rowland) (American Chemical Society) 127, 111. Rupley, J. Α., Gratton, Ε. and Careri, G. (1983) Trends Biochem. Sci., 8, 18. Yu, N-T. and Jo, Β. Η. (1973) Arch. Biochem. Biophys., 156, 469.