TIBS 17 - NOVEMBER

1992

6 Nicholson, D. W., Hergersberg, C. and Neupert, W. (1988) J. Biol. Chem. 263, 19034-19042 7 Dumont, M. E., Cardillo, T. S., Hayes, M. K. and Sherman, F. (1991) Mol. Cell. Biol. 11, 5487-5496 8 Hakvoort, T. B. M., Sprinkle, J. R. and Margoliash, E. (1990) Proc. Natl Acad. Sci. USA 87, 4996-5000 9 Hartl, F-U. and Neupert, W. (1990) Science 247, 930-938 10 Lill, R. et al. (1992) EMBO J. 11, 449-456 11 Hurt, E. C. and van Loon, A. P. G. M. (1986) Trends Biochem. Sci. 11, 204-206 12 van Loon, A. P. G. M. and Schatz, G. (1987) EMBO J. 6, 2441-2448 13 Glick, B. S. et al. (1992) Cell 69, 809-822 14 Hartl, F-U., Ostermann, J., Guiard, B. and Neupert, W. (1987) Cell 51, 1027-1037 15 Wickner, W., Driessen, A. J. M. and Hartl, F-U. (1991) Annu. Rev. Biochem. 60, 101-124 16 van Loon, A. P. G. M., Br~ndli, A. W. and

IMPRESSIVE PROGRESS towards an understanding of the forces that stabilize the folded structures of globular proteins has been described in recent reviews in T I B S ~ and elsewhere 2,3. For most of the past 30 years it has been commonly supposed that the hydrophobic interaction makes a prominent contribution to the stability of the native form, as proposed by Kauzmann4. However, it has recently repeatedly been claimed that the contrary is true ~ . That no consensus now exists is demonstrated by the fact that two highly respected reviewers have examined the same body of data and have drawn opposite conclusions 2,3. This review summarizes the way in Which these divergent opinions arose and scrutinizes some of the arguments for reversing Kauzmann's hypothesis, especially those based on observations of relative stabilities of proteins of differing hydrophobicity. After some deliberation, it then appears that all available data are quite consistent with the conclusion that structural reorganization of water adjacent to non-polar groups opposes unfolding, as originally proposed.

Protein unfolding and dissolution of liquid .... hydrocarbons in water Protein denaturation resembles the formation of aqueous solutions of simple hydrocarbons in that both involve N. Muller is at the Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.

© 1992,ElsevierSciencePublishers, (UK)

Schatz, G. (1986) Cell 44, 801-812 17 Hurt, E. C., Pesold-Hurt, B. and Schatz, G. (1984) EMBO J. 3, 3149-3156 18 Hwang, S. T., Wachter, C. and Schatz, G. (1991) J. Biol. Chem. 266, 21083-21089 19 Ostermann, J., Horwich, A. L., Neupert, W. and Hartl, F-U. (1989) Nature 341, 125-130 20 Smagula, C. and Douglas, M. G. (1988) J. Biol. Chem. 263, 6783-6790 21 Li, J-M. and Shore, G. C. (1992) Science 256, 1815-1817 22 Trumpower, B. L. (1990) Microbiol. Rev. 54,

101-129 23 Cheng, M. Y. et al. (1989) Nature 337,

620-625 24 Hartl, F-U. et al. (1986) Cell 47,939-951 25 Hendrick, J. P., Hodges, P. E. and Rosenberg, L. E. (1989) Proc. Natl Acad. Sci. USA 86,

4056-4060 26 Isaya, G., Kalousek, F., Fento0, W. A. and Rosenberg, L. E. (1991) J. Cell Biol, 113,

65-76

27 Mahlke, K. et al. (1990) Eur. J. Biochem. 192,

551-555 28 Koll, H. et al. (1992) Ceil 68, 1163-1175 29 Wachter, C., Schatz, G. and Glick, B.S. EMBO J.

(in press) 30 Behrens, M., Michaelis, G. and Pratje, E. (1991) Mol. Gen. Genet. 228, 167-176 31 Pfaller, R. et al. (1988) J. Cell Biol. 107,

2483-2490 32 Schneider, H. et al. (1991) Science 254,

1659-1662 33 Hase, T., MOiler, U., Riezman, H. and Schatz, G. (1984) EMBO J. 3, 3157-3164 34 S6llner, T. et al. (1990) Cell 62,107-115 35 Mitoma, J. and Ito, A. (1992) J. Biochem. 111,

20-24 36 Zhuang, Z. and McCauley, R. (1989) J. Biol. Chem. 264, 14594-14597 37 Miller, B. R. and Cumsky, M. G. (1991) J. Cell Biol. 112, 833-841 38 van Loon, A. P. G. M. et al. (1984) EMBO J. 3,

1039-1043

Does hydrophobichydration destabilize protein native structures?

Water in the immediate vicinity of a non-polar solute has characteristically low entropy and high heat capacity at 25°C. Common opinion has been that the insolubility of such species is caused by thermodynamic changes associated with the formation of these layers of abnormal water, 'hydrophobic hydration'. Recently, however, it has been proposed instead that hydrophobic hydration favors solution of hydrocarbons, or hydrocarbon sidechains, in water and therefore promotes protein unfolding. It is argued here that available data do not convincingly support this hypothesis.

transfer of non-polar groups from a nonaqueous to an aqueous environment4,5,7,9. This is characteristically accompanied by a large increase in the heat capacitY, proportional to the surface area of the hydrocarbon solute 1° or, for a protein, to the non-polar area exposed to water upon unfolding u. I t is generally interpreted as a manifestation of special structural or thermodynamic properties of water that is in contact with a non-polar group. Formation of this special kind of water is called hydrophobic hydration, and the resulting thermodynamic changes, here labelled with the subscript hph, constitute the hydrophobic effect. Observation of hydrocarbon solutions provides an opportunity to study this effect with minimal

interference from other interactions. Transfer of an alkane molecule from the pure liquid (1) to a water solution (w) is opposed by a large increase in the unitary Gibbs energy4, AG0(I-+w)

=

AH0(1-+w)

= - RTlnX,

-

TAS0(1--+w) (1)

where X is the mole fraction in the saturated solution, AH° is the enthalpy change and AS° is the entropy change. Near room temperature, the dominant term is TAS° (1-+w), which is large and negative. It has been reported that for an assortment of hydrocarbons the transfer enthalpies and entropies at any (Kelvin) temperature are well represented by the following empirical equations9:

459

TIBS 17 - NOVEMBER 1992

For cyclohexane, a typical solute that has ACp,hp h = 360Jmol-1K -1 (Ref. 9), one finds AH°vdw(l~w) = 32.8 kJmol-l, a value where ACp.hph : d[AH°(1-*w)]/dT, almost equal to the enthalpy of vapormeasured at room temperature. (ACp is ization. This is rather disturbing. If corthe heat capacity change at constant rect, it would signify that van der Waals interactions produce only a negligible pressure.) By convention, the entropy change is contribution to the enthalpy of transfer assumed to arise from the hydrophobic of a hydrocarbon from the vapor phase effect alone, allowing one to write to an aqueous solution 7, which seems AS°(l~w) : AS°ph . However, the en- hard to believe. Before exploring other consequences thalpy change may include a contribution from ordinary van der Waals of accepting eqn 5, it should be meninteractions (superscript vdW) that tioned that eqns 2, 3 and 5 all depend on would remain even if the hydrophobic the implicit assumption that ACp,hp h is independent of the temperature. Of effect could be eliminated. Then course this is not consistent with the AH°(I~w) : AH°ph + AH°vdw(l~w) (4) proposal that ACp,hpharises from iceberg melting and that eventually, at T~, none and AH~,p, cannot be evaluated from remain to be melted. Experimental data and model calculations suggest that AH°(l-*w) unless AH°~w(l~w) is known. d(ACp,hph)/dT is nearly zero at about room temperature, but the heat capacity Origin of the opposinginterpretations The root of the disagreement be- change does decrease appreciably on tween the traditional view and what has heating above 330K (and perhaps on been called the new view of hy- strongly cooling as well, though this is drophobic effects 12 is that AH°vdW(1--~w) hard to verify because of the limited cannot be directly measured, and quite liquid range of water) 7,12,16.When this is different assumptions have been taken into account, the estimated value of T~ is seen to need upward revision, adopted for dealing with it: In essence, the traditional approach perhaps from 386K to about 413K, and is to say that if an arbitrary assumption then it has been assumed that AH~ph is required, it may as well be the sim- also vanishes at this higher tempera0 4 4, w) = 0. tureL For present purposes, this corplest possible one, i.e. AHvdw(1 Since the process in question transfers rection is relatively unimportant, and the solute species from one liquid equations derived with fixed ACp,hph phase to another, this may in fact rep- (that is, representing what has been resent a reasonably good first approxi- called 'linear' extrapolation 17) are retained. mation. The two treatments of the van der The new view is based on a very different approach. It attributes the nega- Waals enthalpy contribution have protive entropy change found at room tem- foundly different implications for the perature to the formation of 'more Gibbs energy change produced by structured' water, sometimes called hydrophobic hydration. Looking first at 'iceberg' formation, although the sup- the traditional approach, eqns 1-3 give posed structure may not closely resemble that of ice 13d4. The large ACp,,ph AG°lph = ACp,hph{T- 295 + Tln(386/T)]. (6) is seen as a consequence of the absorption of energy associated with progress- This yields large, positive values at all ive melting of the icebergs on heating. attainable temperatures. The effect is Since eqn 3 shows that AS°ph is zero at primarily entropic near 298K, as noted 386K, it has been supposed that at this above, but becomes mainly enthalpic on temperature, T~, the melting of icebergs heating towards 386K. The Gibbs energy is complete. Then AHhp h should also change is maximal at T~, as thermovanish at T~, and indeed water should dynamics requires. It then progressively be just a normal polar solvent at and decreases on cooling, which is often above this temperature ~5. Accordingly, regarded as paradoxical: if the hydrothe temperature-independent van der phobic effect depends on iceberg formaWaals contribution may bd evaluated tion, why should AG°ph be smaller when one would expect most icebergs (near by noting that at 386K 273K) than at higher temperatures, where they are supposedly largely melted? kH°dw(1-+w) = AH°(I~w) The new view offers a way to avoid = ACp,hph(386- 295). (5) this paradox. If AH°ph a n d AS0hph both AH°(l~w) = ACp,hph(T- 295) (2) AS°(I~w) = ACp,hphln(T/386), (3)

460

vanish at T~, then of course so must AG°ph. But this does not cancel the requirement that the Gibbs energy change must be maximal when the entropy change is zero. Thus it has been inferred that the maximal value of AG0hphis precisely zero and that its values at lower temperatures are increasingly negative, though small because of enthalpy-entropy compensation 5,7. Since the preceding discussion implies AG°(I~w) = AG°ph + AH°aw(l~w), (7) then at 298K there is a transfer Gibbs energy composed of a small, negative, hydrophobic term that is quite overwhelmed by the large, positive AH°vdW ( l ~ w ) given by eqn 5. The advantage of this approach is that the hydrophobic effect now becomes 'stronger' as the postulated degree of iceberg formation increases. A part of the price that must be paid for this is the need to accept a negative sign for AG~lph. This is the basis of the proposal that hydrophobic hydration assists transfer of non-polar solutes into the aqueous medium and unfolding of globular proteins 5 8.~5. The conjecture that the hydrophobic effect-melts away when solutions are heated to T~ involves several other difficulties 12. The most serious of these is that experimental results and model calculations both indicate that although ACp.hph diminishes on heating, it is still far from zero at T~. This raises two troublesome questions. First, how is the residual ACp.hph to be explained, since it has been assumed that no icebergs remain unmelted, and second, the textbook equations ACp = d(AH)/dT = Td (AS)/dT require that if AH°ph and AS°ph indeed both vanish at T~, then both must 'become increasingly positive at still higher temperatures. Since the iceberg paradigm does not offer even a clue as to how this could be explained, what is one to do?

Modified hydration-shell H-bondmodel The obstacles confronted on trying to work out consistently and quantitatively the implications of the iceberg hypothesis are so vexing that a totally different analysis of hydrophobic hydration appears to be needed. The modified hydration-shell hydrogen bond (H-bond) model was introduced in an attempt to meet this need 12,~8.Its basic premise, described below, is that it is possible to account for ACp,hph without employing the iceberg concept.

TIBS 17 - NOVEMBER 1992

The heat capacity of pure, liquid water is itself so large that a sizeable fraction of it, perhaps 50%, must be ascribed to the breaking of more and more H-bonds as the temperature rises. If H-bond breakage is considered as an equilibrium process, with enthalpy and entropy changes AH° and AS° and equilibrium constant K = exp(-kJ-I°/RT + &S°/R), the heat capacity contribution is Ch = (AH°)2K/RT2(1 + K) 2.

(8)

In effect, breaking H-bonds provides an energy-storage mechanism 2. The experimental data show that this mechanism is much more effective for hydration-shell than for bulk water. Introducing a solute converts some bulk H-bonds (subscript b) to hydration-shell bonds (subscript hs). Each population consists of a mixture of broken and intact bonds, and each is assumed to have its own AH°, AS° and K. The resulting heat capacity change is

ACp,hph = n[(AH°h~)2Khs/RT2(i + Kh~)2 (AH°)2Kb/RT2(1 + Kb)2]. (9) where n is the number of H-bonds connecting water molecules both resident in the hydration shell. When the shell contains N molecules, n = 3N/2. Realistic results can be obtained with this equation only if AH°~ > AH° and Khs > Kb, which requires aS°h~ > AS°. That is, hydration-shell H-bonds are found to be stronger enthalpically, but owing to the entropy term a larger fraction of them is broken at any given temperature, compared with bulk water. This means that it is misleading to call the effect of a non-polar solute 'structure making'. The hydration shell is not like an iceberg, which supposedly has stronger H-bonds than bulk water and also a greater degree of bonding. This conclusion makes it possible to rationalize otherwise anomalous proton nuclear magnetic resonance (NMR) chemical shifts for water that contains solutes with non-polar groups TM. The model does not require that cooling must strengthen the overall hydrophobic effect, although cooling does increase the fraction of intact H-bonds for both bulk and hydration-shell water 12. Equations for calculating AH°ph, ASl°lph and AGOph with this model have been published, along with some numerical results obtained using a provisional set of parameters that characterizes the two classes of H-bonds '2. It is found that ACp.hph is indeed temperature depen-

dent, but also that the calculated values of AG°ph are only slightly different from those acquired with the approximations 19 AH°lph= ACp,hph.298(T- 307)

(10)

kS°ph = ACp,hph,2981n(T/389), (11) where only the value calculated for ACp,hph at room temperature is used. These are remarkably similar to eqns 2 and 3, and, if they are combined with eqns 2 and 4 they yield AH°vdW(1-+w) : ACp,hph,298(307-295). (12) For cyclohexane (see above, following eqn 5), this gives AH°vdw(1-+w) = 4.3 kJ mo1-1. This is intermediate between the values required by the traditional and the new view, discussed above, but much closer to the former and, in the author's opinion, more credible than either. This model certainly does not call for AH°ph and AS°ph to vanish at the same temperature 12. The Gibbs energy change is obtainable from eqns 10 and 11. It is always positive and is maximal at a T; of 389K. This is in harmony with the traditional conclusion that the hydrophobic effect favors removal of nonpolar groups from water. Modest readjustment of the model parameters does not change this result, nor does use of exact calculations rather than eqns 10 and 11.

a measure of the hydrophobicity of its interior 3,8. Then it is usually found that the more hydrophobic protein has the higher maximal stability temperature, the lesser maximal stability and the greater readiness to unfold upon cooling below room temperature ('cold denaturation') 3,8. It thus appears that, other things being equal, increasing hydrophobicity implies reduced stability, in agreement with the new view. However, it will be seen that it is easy to be misled unless the apparently innocent phrase, 'other things being equal', is given very careful consideration. Very recent work offers an entirely different interpretation of these findings by showing that they virtually coincide with the expected behavior of hypothetical proteins that are assumed to be stabilized by hydrophobic interactions (N. Muller, submitted). A very simple model is used, in essence a combination of Baldwin's liquid hydrocarbon model 9 and the modified hydration-shell H-bond model 12. Baldwin's model involves assuming that the specific enthalpy and entropy of unfolding are the sums of hydrophobic (hph) and residual (res) terms, that is AH°unf= AH0hph+ AHO~es

(13)

AS°unf = AS0hph + AS°~es .

(14)

The hydrophobic terms are evaluated using eqns 10 and 11, replacing the heat capacity change that appears there by AC~.unf, still regarded as a measure Trends in protein unfolding thermodynamics of the hydrophobicity. This requires In spite of the difficulties of the new assuming that the heat capacity change view, it has continued to attract propo- originates entirely from the hydronents, because a collection of measure- phobic effect and therefore that the rements of globular protein stabilities sug- sidual terms are independent of temgests a prima-facie case in its favor3. This perature 9. It has been stressed recently involves two main lines of reasoning. that sidechains on the interior of a folded First, the specific enthalpies of protein are more tightly packed together unfolding (enthalpy per amino acid than molecules in an organic liquid residue, symbol AH°unL) for an assort- phase. As a result, they should experiment of proteins range from about 0-3 ence stronger van der Waals interkJmo1-1 at ambient temperature, but on actions, which may enhance the overall extrapolation to T~ they converge very stability of the native form2°,21. This nearly to a common value of 6.25 kJ effect is quite independent of any mo1-1 (Refs 5,19). One explanation is special behavior of water adjacent to that the enthalpy includes a hydro- non-polar groups. Since t h e hydrophobic contribution that vanishes at T~, phobic effect i s regarded here as a as postulated in the new view, which manifestation of this special behavior, then leaves a residual term that, for it seems consistent to suppose that any some unknown reason, is the same for such unusual van der Waals interacdiverse proteins: tions should be included with the other Second, stabilities for pairs or groups contributions that give rise to AWes. It is of proteins have been compared, taking thus still appropriate to use data for the specific heat capacity change for liquid-to-water transfer of non-polar denaturation, AC~,,,f, for each species as solutes to obtain AH°ph and AS0hph.

461

TIBS 1 7 -

36o

340

300

(c)

J

/

[ 280

I

I

I

1

I

I

I

45

50

55

60

65

70

75

~

ACp u~f (J mol K -~)

Figure I Calculated results for model proteins with T m = 355K, ASO*res = 18 J mol 1K-l, and variable ACp,~nf. Curve (a): cold denaturation temperatures; the plotted quantity is (To + 100). Curve (b): maximal stability temperatures, T~; the filled circles are experimental values based on data from Ref, 5. Curve (c): specific residual unfolding enthalpies, AHO*res' Curve (d): maximal stabilities, AG°m,x.

Each protein has its own characteristic values of AH°resand AC~,unf,but it is assumed that all have AS°~e~ = 18 J mol-aK-L This assures that at T~ = 389K the specific unfolding entropies will converge to a common value of 18 J mol-~K-1, as observed for real proteins at 386K. A proposed explanation is that the dominant contribution to each AS°~s is a conformational entropy change that is not far from Rln8 for a typical amino acid residue 9,~9. Any protein has an array of significant properties, any of which may be singled out for attention. In addition to AH°~,, AS°~e~ and ACp,unf, the list may include: Tm, the ordinary denaturation midpoint temperature; To, the cold denaturation temperature; T~ (not the same as T~), the temperature where AS°unf is zero, commonly taken as the maximal stability temperature17; AG°m~, the maximal stability, equal to the value of AG%.f at %; Th, the temperature where AH°unfiS zero and where the function AG%nf/Tis maximal. It is well known, but apparently occasionally forgotten, that only three of the quantities in this list cala function as independent variables, This follows from the fact that, providin~g AC~,unfis constant, the stability curve of any protein is completely determined when just three quantities are specified ~7. An illustration is the equation ~

462

AG°;n, = AH°unf,Tm(l - T/T~) + Z~Cp,unf[(T

--

T~) - Tln(T/T.~)] (15)

NOVEMBER 1992

mol-~K-1, a range that likewise corresponds with experimental findings5,8. Equations for predicting other relevant variables are very easily derived and are being published elsewhere together with selected numerical results (N. Muller, submitted). Figure 1 shows calculated values of To, Ts, AH°~es and AG°m~xas functions of ACp,unf, with Tm = 355K and AS°~os = 18 J mol-lK-l. It is instructive to re-examine the data that purportedly support the new view in light of these results. The maximal stability temperature [curve (b)] rises smoothly from about 260K to 306K as the hydrophobicity increases. Experimental values (calculated from the data in Table I of Ref. 5) are shown as filled circles in Fig. 1 and define a curve parallel to and only slightly below the model curve, As AC~..nfincreases, the maximal stability fails [curve (d)], but this does not mean that hydrophobicity in itself destabilizes the native form. Instead, there is actually an appreciable increase in AG°hph,but this is swamped by a very large and unavoidable concomitant decrease in AH°~e~[curve (c)]. Thus it turns out that in practice 'increasing the hydrophobicity, other things being equal' usually means that Tm is not allowed to change, but then it is not permissible to stipulate that

In working with the model proteins, AS°:~, T~ and AC~.u,fwere taken as the independent variables, to be adjusted with a view to making them as similar to real prbteins as possible (N. Muller, submitted). 6.0 With AS°~ already fixed as described above, two 5.0 degrees of freedom remain. 4.0 Nature seems to show a preference for proteins 3.0 with maximal stabilities in a fairly narrow range 17, and because of the inter2.0 (a) connectedness of the variables this implies a simi1.0 larly narrow range for Tm (N. Muller, submitted). In 0 fact, at their pH of maximal stability, most globu, , , , lar proteins have denaturation temperatures ~9 T(K) that differ from 355K by, at most, about 25K (7%). This suggests holding Tm Figure 2 fixed at 355K, or at one of Calculated temperature dependence of the specific a set of trial values not unfolding enthalpies, AHO*unf,with Trn and ASO*res as in very different from this, Fig,. 1. Curve (a): AC~,unf = 45 J mol-lK-1. Curve (b): * I 1 * while allowing ACp,unf to A(~punf = 55 J mol- K-. Curve (c): ACpuof = 65 J * mol-'1 K-.1 Curve (d): ACp,un r = 75 J roof 1 K- 1.'. vary between 45 and 75 J

TIBS 17 - NOVEMBER 1992 AH°;es shall also be kept unchanged! This is why, for two proteins having the same Tin, the more hydrophobic must be less stable even though AG%ph > 0. However, more complete calculations 19 show that if they have significantly different values of Tm then the more hydrophobic protein may also be the more stable, and this is confirmed by experimental results for cytochrome c and chymotrypsin 5. The dependence of AH°;es o n AC;,un f has another important corollary. Equations 10 and 13 give • AH%nf : AH°;es+ ACp,unt(T- 307), (16) showing that the unfolding enthalpy increases linearly on heating. The model results show that increasing AC~,unf decreases the intercept but increases the slope, so that lines corresponding to different hydrophobicities will tend to converge. This is illustrated in Fig. 2, where it is seen that at 298K, values of AH°unlrange from 2.4 to - 0.3 kJ mo1-1, again correlating well with experimental results 5, while at 387.5K, or 1.5K below T~, they converge to a common value of 6.39 kJ mo1-1, very near the experimental value of 6.25 kJ mo1-1 mentioned above. This would appear surprising if it had not recently been s h o w n 22 that when AH°unf is linearly extrapolated to T~ the resulting value must always be slightly greater than the

THE TRANSMEMBRANE EXPORT of intracellular metabolites is a critically important cellular function. Recent studies of the multidrug resistance phenotype of tumor cells have led to the discovery of 'P-glycoprotein', a 170 kDa plasma membrane glycoprotein that mediates the efflux of certain anticancer drugs from cells 1. The overexpression of this export pump in tumor cells has been found to be closely related to the multidrug resistance phenotype. There is now evidence for the existence of another, distinct, type of ATPdependent export pump that transports a variety of glutathione S-conjugates out T. Ishikawa is at the Section of Experimental

Therapeutics, Department of Experimental Pediatrics, The Universityof Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA. © 1992,ElsevierSciencePublishers,

product TmAS°~es.For the proteins represented in Fig. 1, this product has the value 6.39 kJ mo1-1. More comprehensive calculations, including model proteins with somewhat different denaturation temperatures, still show such convergence; however, it is somewhat less precise (N. Muller, submitted). The majority of cold denaturation temperatures (Fig. la) are far below the freezing point; it should be noted that the quantity actually plotted is (Tc + 100), in order to conserve space. Again, the values are realistic, since cold unfolding is usually not observable unless the solution can b~ supercooled or the stability of the Protein is first reduced by pH adjustment or addition of denaturant 6. Moreover, the calculated Tc rises strikingly as ACp,unlincreases, which nicely accounts for the observation that the most hydrophobic proteins most readily unfold on cooling3. To summarize, the behavior of the model proteins shows trends that agree not only qualitatively but almost quantitatively with those found in the laboratory. It cannot be claimed that the model is thereby shown to be correct, since any model, is ipso facto, an oversimplification, and therefore, 'correct model' is a contradiction in terms. However, it seems probable that the model, like a good cartoon, captures important features of the reality it is meant to portray, and that it is correct insofar as it

shows that there is no compelling reason to accept the conclusion that hydrophobic hydration promotes unfolding. References 1 Pace, C. N. (1990) Trends Biochem. Sci. 14, 291-294 2 Dill, K. A. (1990) Biochemistry29, 7133-7155 3 Creighton, T. E. (1991) Curr. Op. Struct. Biol. 1, 5-16 4 Kauzmann, W. (1959) Adv. Prot. Chem. 14, 1-63 5 Privalov, P. L. and Gill, S. J. (1988) Adv. Prot. Chem. 3 9 , 1 9 1 - 2 3 4 6 Privalov, P. L. (1989) Annu. Rev. Biophys.~ Biophys. Chem. 18, 47-69 7 Privalov, P. L. and Gill, S. J. (1989) Pure Appl. Chem. 61, 1097-1104 8 Murphy, K. P., Privalov, P. L. and Gill, S. J. (1990) Science 247, 559-561 9 Baldwin, R. L. (1986) Prec. Natl Acad. Sci. USA 83, 8069-8972 10 Gill, S. J. (1988) J. Chem. Thermodynam. 20, 1361-1382 11 Livingstone, J. R., Spolar, R. S. and Record, M. T., Jr (1991) Biochemistry 30, 4237-4244 12 Muller, N. (1990) Acc. Chem. Res. 23, 23-28 13 Frank, H. S. and Evans, M. W. (1945) J. Chem. Phys. 13, 507-532 14 Frank, H. S. and Wen, W-Y. (1957) Discuss. Faraday Soc. 24, 133-140 15 Shinoda, K. (1977) J. Phys. Chem. 81, 1300-1302 16 Gill, S. J., Dec, S. F., Olofsson, G. and Wads6, I. (1985) J. Phys. Chem. 89, 3758-3761 17 Becktel, W. J. and Schellman, J. A. (1987) Biopolymers 26, 1859-1877 18 Muller, N. (1988) J. Sol. Chem. 17,661-672 19 Privalov, P. (1979) Adv. Prot. Chem. 33, 167-241 20 Richards, F. M. (1977) Annu. Rev. Biephys. Bioeng. 6, 151-176 21 Murphy, K. P. and Gill, S. J. (1991) J. MoI. Biol. 222, 699-709 22 Baldwin, R. L. and Muller, N. (1992) Prec. Natl Acad. Sci. USA 89, 7110-7113

The ATP-dependentglutathione S-conjugate export pump

The ATP-dependent glutathione S-conjugate export pump (GS-X pump) plays a physiologically important role as a member of the 'phase II1' system in xenobiotic metabolism as well as in the release of biologically active endogenous substances from cells. In addition, this export pump is potentially involved in the modulation of the antiproliferative action of certain antitumor agents.

of cells. The fact that such an export pump exists is becoming increasingly important, since evidence showing that the glutathione conjugation reaction is a key defense mechanism in the detoxification of electrophilic-reactive corn-

pounds derived from both exogenous and endogenous sources continues to accumulate 3,4. Glutathione S-conjugates, generated from the conjugation reaction, are actively exported out of cells. This process is a critical step, not only

463