Water as an Active Constituent in Cell Biology

Philip Ball*

Nature, 4-6 Crinan Street, London N1 9XW, U.K.

Received December 28, 2006

Contents1. Introduction 12. Water as a Liquid and Solvent 2

2.1. Water Structure 22.2. Small-Molecule Solutes: Hydrophiles 42.3. Small-Molecule Solutes: Hydrophobes 52.4. Large Hydrophobic Solutes and Surfaces 72.5. The Influence of Ions: Structure-Making and

Structure-Breaking9

2.6. Long-Range Hydrophobic Interactions and theRole of Bubbles

11

2.7. Hydrophilic Surfaces 133. The Aqueous Environment of the Cell 144. Protein Hydration: Nonspecific Effects 15

4.1. The Hydration Shell 154.2. Dynamics, Cooperativity, and the Glass

Transition17

5. Protein Hydration: Specific Roles of Water inStructure and Function

19

5.1. Secondary Structure 195.2. Protein−Protein Interactions 195.3. Mediation of Ligand Binding 205.4. Functional Tuning 225.5. Allostery 235.6. Hydrophobic Cavities 235.7. Electron Transfer 235.8. Involvement of Bound Water in Catalytic

Action24

5.9. Proton Wires 245.10. Function of Protein Channels 26

6. Water and Nucleic Acids 277. Conclusions 308. Acknowledgments 309. Note Added in Proof 30

10. Note Added after ASAP Publication 3011. References 30

1. IntroductionWhen Szent-Gyo¨rgyi called water the “matrix of life”,1

he was echoing an old sentiment. Paracelsus in the 16thcentury said that “water was the matrix of the world and ofall its creatures.”2 But Paracelsus’s notion of a matrixsanactive substance imbued with fecund, life-giving propertiesswas quite different from the picture that, until very recently,molecular biologists have tended to hold of water’s role inthe chemistry of life. Although acknowledging that liquid

water has some unusual and important physical and chemicalpropertiessits potency as a solvent, its ability to formhydrogen bonds, its amphoteric naturesbiologists have re-garded it essentially as the backdrop on which life’s molec-ular components are arrayed. It used to be common practice,for example, to perform computer simulations of biomol-ecules in a vacuum. Partly this was because the computa-tional intensity of simulating a polypeptide chain waschallenging even without accounting for solvent moleculestoo, but it also reflected the prevailing notion that water doeslittle more than temper or moderate the basic physicochem-ical interactions responsible for molecular biology. WhatGerstein and Levitt said 9 years ago remains true today:“When scientists publish models of biological molecules injournals, they usually draw their models in bright colors andplace them against a plain, black background”.3

Curiously, this neglect of water as an active componentof the cell went hand in hand with the assumption that lifecould not exist without it. That was basically an empiricalconclusion derived from our experience of life on Earth:environments without liquid water cannot sustain life, andspecial strategies are needed to cope with situations in which,because of extremes of either heat or cold, the liquid isscarce.4-6 The recent confirmation that there is at least oneworld rich in organic molecules on which rivers and perhapsshallow seas or bogs are filled with nonaqueous fluidstheliquid hydrocarbons of Titan7smight now bring some focus,even urgency, to the question of whether water is indeed a* E-mail: p.ball@nature.com.

Philip Ball is a science writer and a consultant editor for Nature, wherehe worked as an editor for physical sciences for more than 10 years. Heholds a Ph.D. in physics from the University of Bristol, where he workedon the statistical mechanics of phase transitions in the liquid state. Hisbook H2O: A Biography of Water (Weidenfeld & Nicolson, 1999) was asurvey of the current state of knowledge about the behavior of water insituations ranging from planetary geomorphology to cell biology. Hefrequently writes about aspects of water science for both the popular andthe technical media.

10.1021/cr068037a CCC: $71.00 © xxxx American Chemical SocietyPAGE EST: 34.6Published on Web 12/21/2007

unique and universal matrix of life, or whether on the con-trary it is just the one that happens to pertain on our planet.

Fundamental to that question is the role that water playsin sustaining the biochemistry of the cell. It has becomeincreasingly clear over the past 2 decades or so that water isnot simply “life’s solvent” but is indeed a matrix more akinto the one Paracelsus envisaged: a substance that activelyengages and interacts with biomolecules in complex, subtle,and essential ways. There is now good reason to regard the“active volume” of molecules such as proteins as extendingbeyond their formal boundary (the van der Waals surface,say), by virtue of the way they shape and manipulate theshell of water that surrounds them. Moreover, the structureand dynamics of this hydration shell seem to feed back ontothose aspects of the proteins themselves so that biologicalfunction depends on a delicate interplay between what wehave previously regarded as distinct entities: the moleculeand its environment. Many proteins make use of bound watermolecules as functional units, like snap-on tools, to mediateinteractions with other proteins or with substrate moleculesor to transport protons rapidly to locations buried inside theprotein.

Here I review the case for considering water to be aversatile, adaptive component of the cell that engages in awide range of biomolecular interactions. In order to providesome basis for assessing water’s often-alleged “uniqueness”to life, however, I shall try to highlight throughout this paperthe distinctions between generic and specific behaviors ofbiological water. That is to say, some of its roles andproperties may be expected from any small-molecule liquidsolvent. Others depend on water’s hydrogen-bonding capac-ity, but not in a way that could not obviously be fulfilledalso by other hydrogen-bonded liquids. But some of water’sbiochemical functions do indeed seem to be quite unique tothe H2O molecule. From an astrobiological perspective, thequestion is then whether we can regard these latter roles asoptional or essentialfor any form of life to be tenable.

2. Water as a Liquid and Solvent

2.1. Water StructureWater is not like other liquids, but neither is it wholly

different. The structure of a so-called simple liquidsone inwhich the molecules can be represented as particles thatinteract via some spherically symmetric potential functionscan be depicted in terms of a radial distribution function (rdf)g(r), which is related to the liquid densityF(r) around aparticle atr ) 0: F(r) ) Fbg(r), whereFb is the bulk density.The rdf for a simple liquid interacting through the Lennard-Jones potential (V ∼ σ/r12 - σ/r6, whereσ is the moleculardiameter) is not very different from that of a “hard-sphere”fluid, in which the particles experience no intermolecularforce until they touch, whereupon they act as infinitely hardspheres (V ) 0 (r > σ); V ) ∞ (r e σ)). In both cases, therdf is oscillatory, with a prominent first peak aroundr ) σand smaller subsequent peaks at separations close tor ) nσthat decay rapidly until the density reaches the bulk averagevalue (Figure 1). This implies that the oscillatory densityprofile is dominated by the steep repulsive core of theparticles and is related to the geometric aspects of a dense,random packing of particles. These repulsive interactionscreate short-ranged ordering in the liquid that the inclusionof an attractive potential (without which there is no liquid-gas transition) modifies only slightly.

Liquid water also has an oscillatory rdf. In this case asingle functiong(r) will not suffice to fully characterize theliquid structure, because there are two types of atom in themolecules: H and O. So one must define a series of partialrdfs gXY(r), which denote the probability of finding an atomY a distancer from the center of atomX. For example,gOO(r) indicates the radial distribution of oxygen atoms inother H2O molecules around any given molecule. The partialrdfs for water, determined by neutron scattering,8 are shownin Figure 2.

The structure is more complex than the oscillatory profilewith decaying amplitude found for simple fluids such as hard-sphere or Lennard-Jones systems or for real liquid argon;but the basic features look qualitatively similar. This localstructuring of liquid water has, however, a quite differentorigin. Whereas hard-sphere repulsion controls the short-range order of simple liquids, the structure seen in Figure 2is primarily due to the attractive interactions between watermolecules: the hydrogen bonds. These generate a peak ingOO(r) at a separation considerably greater than the mean“molecular diameter” (radius of gyration)σsthe peak is atabout 1.4σ. In other words, the molecules do not, on average,sit as “close” as do the particles of a simple liquid. They areheld apart by the hydrogen bonding, which imposes geo-metric constraints on the molecular positions: the hydrogenatom in an O‚‚‚H-O union sits, on average, roughly alongthe axis between the two oxygen atoms. In other words, thehydrogen bond is linear. If it is “bent”, the orbital overlap ispoorer and the bond is weaker. Thus, one might say thathydrogen bonding keeps the H2O molecules at “arm’slength”, preventing them from packing as closely as theywould in a simple liquid. The hydrogen bonds are direc-tional: they bind the water molecules into particular spatialorientations.

Figure 1. Typical radial distribution functions (rdfs) of simplefluids: (a) hard spheres; (b) Lennard-Jones potential.

Figure 2. Partial radial distribution functions (rdfs) for water at298 K. The first (off the scale) peak ingOH(r) is the intramolecularpeak. Data courtesy of Alan Soper, ISIS.

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The sp3 hybridized oxygen atom creates an essentiallytetrahedral coordination geometry, with each H2O moleculecoordinated to four others (Figure 3a). We should resist thetraditional notion of two “rabbit-ear” lone pairs on eachmolecule, however: the electron-density distribution showsonly a single, broad maximum on the “rear” of the oxygenatom. And since the molecules are mobile in the liquid,the four hydrogen-bonding sites are not necessarily fullyoccupied. Neutron-scattering measurements of the rdfs, alongwith computer simulations, have tended to suggest that thenumber of hydrogen-bonded nearest neighbors for watermolecules at room temperature averages about 3.5. RecentEXAFS (extended X-ray absorption fine structure) studiesby Wernet et al.9 challenged this picture, appearing to suggestthat only two nearest neighbors are, on average, actuallyhydrogen-bonded to the central molecule; but this interpreta-tion has been vigorously disputed.10-14

Simulations by Sciortino et al.15 posed a different challengeto the conventional picture of tetrahedral coordination vialinear hydrogen bonds, suggesting that the liquid statecontains many defects in the form of bifurcated bonds: asingle hydrogen coordinates to two oxygen atoms on differentmolecules (Figure 3b). Sciortino et al. conclude that thesebifurcated bonds in fact play a central role in the molecularmobility of the liquid state by lowering the Gibbs energybarrier to diffusion.

Nonetheless, it is clear that the tetrahedral hydrogen-bonded geometry is the reason for water’s well-knowndensity anomaly on freezing. The density of the solid stateis lower than that of the liquid because in crystalline ice theconstraints of hydrogen bonding are rigidly imposed: the

molecules form an essentially perfect tetrahedrally coordi-nated network, linking them into six-membered rings withmuch empty space between the molecules (Figure 4a). Whenthe lattice melts, the three-dimensional hydrogen-bondednetwork becomes more fluxional, distorted, and defective(Figure 4b), enabling molecules to approach one anothermore closely: the average distance between moleculesslightly decreases. Other anomalies of the liquid state, suchas the density maximum at 4°C and the putative existenceof a liquid-liquid phase transition in metastable, supercooledwater,16 can be considered to result from the competitionbetween these “icelike” and “disordered” configurations. Onemight say that as the temperature is lowered below 4°C,“icelike” configurations become predominant, leading to adecreasing density, whereas at higher temperatures thedecrease in density with increasing temperature is simply areflection of normal thermal expansion. The notion17,18 thatwater is best viewed as a two-phase mixture of distinct icelikeand liquid-like configurations, with fully formed and partlybroken hydrogen-bond networks, respectively, now seemsuntenable, however; rather, the distribution of hydrogen-bondgeometries and energies appears to be continuous.14

In pure liquid water, hydrogen bonds have an averagelifetime of about 1 ps, so although there exists an ex-tended, essentially infinite (percolating) dynamical three-dimensional network (Figure 4b), there are no long-livedicelike structures. Defects in this hydrogen-bonded networkowing to dangling OH bonds seem to be very short-lived(

For water’s role as a solvent, particularly in the cell, oneof the key questions is how this network structure is affectedby the introduction of a solute molecule or proximity to asurface. In each case, the structural response of liquid waterdepends on whether the foreign body is hydrophobic orhydrophilic. But in more or less all cases the issue has beencontentious and is still not well resolved.

2.2. Small-Molecule Solutes: Hydrophiles

Water is an extremely good solvent for ions. In part,this is a result of water’s high dielectric constant, whichscreens the ions’ Coulombic potential effectively and pre-vents the aggregation and crystallization of ions of oppo-site charge. By the same token, water is an efficient sol-vent for biomolecular polyelectrolytes such as DNA andproteins, shielding nearby charges on the backbone from oneanother.

As a polar species, the water molecule can engage infavorable Coulombic interactions with ions and other polarsolutes. According to the traditional picture, water moleculeswill solvate cations by orienting their oxygen atoms towardthe ion, whereas they will adopt the opposite configurationfor anions (Figure 5a). Such reorientation, however, perturbsthe hydrogen-bonded network, and the question is howreadily this disruption can be accommodated.

The key technique for investigating this issue is neutronscattering, particularly because of the large scattering crosssection of hydrogen and because of the facility for alteringthe various cross sections by isotopic substitution, enablingthe several water-water and ion-water partial rdfs to bedisentangled. Pioneering work by Enderby, Neilson, Soperand their co-workers20 has confirmed the expected picturein which anions such as chloride are coordinated to waterin the hydration shell via hydrogen atoms, such that theH-O‚‚‚Cl- bond is almost linear, whereas for cations likenickel the water molecules are oriented with the oxygenatoms facing toward the ions (Figure 5b). Recent XAS(X-ray absorption spectroscopy) studies of cation hydrationhave been interpreted as implying little or no geometricdistortion of “water structure” over long ranges,21,22althoughdivalent cations do seem to induce a redistribution of chargeamong water molecules in the solvation shell, leading tochanges in the X-ray absorption spectra.22 This very localview of the effects of ions on water structure is supportedby first-principles MD (molecular dynamics) simulations,which reveal only some degree of preferential orientation inthe first hydration shell for both monovalent and divalentions,23 and by femtosecond pump-probe spectroscopy,

which shows that ions have essentially no influence on therotational dynamics of water molecules beyond the firsthydration shell.24 It is important to note, moreover, thatneutron-scattering studies caution against too static a visionof hydration: there is clearly a lot of dynamic variation inthese structures.

While, as we shall see later, the hydration of hydrophilicregions of proteins has been investigated extensively, therehas been surprisingly little work to date on small, biologicallyrelevant hydrophiles. As a result, the “ground rules” forinterpreting the hydration of larger biomolecules have notreally been established. What little we do know about suchsimple “model” species, however, suggests that it may notbe possible either to generalize or to reduce the structuraland dynamic aspects of hydration to simplistic rules ofthumb. A neutron-scattering study of the hydration ofL-glutamic acid in alkaline solution25 suggested a significantdisruption of the water structure, with the average numberof water-water hydrogen bonds being reduced from 1.8 to1.4. But this was at high concentrations of both glutamicacid and NaOH (each 2 M), so it is not clear how the resultsmight relate to a physiological context. In contrast,L-prolineengages in strong hydrogen bonding with water but in amanner that seems barely to perturb the hydrogen-bondednetwork at all.26 Proline is an osmoprotectant, preventingprotein denaturation in the face of water stress. One possiblemechanism for this invokes the formation of a looselyassociated sheath of proline molecules around a protein to“chaperone” itssomething that appears feasible without anysignificant disruption of the protein’s hydration.

One of the best studied small hydrophiles is urea, whichcan be considered a mimic of a hydrophilic residue but isalso known for its tendency to denature proteins. A neutron-scattering study shows that the urea molecule can “substitute”quite readily for water in the hydrogen-bonded network: therdf of urea around water in a 1:4 solution looks remarkablylike that of water around water.27 Although urea has nearly3 times the molecular volume of water, the structure of liquidwater is sufficiently “open” that a urea molecule appears todisplace just two waters, offering up to eight hydrogen bondsin place of the displaced pair. The idea that urea “fits” rathereasily into the hydrogen-bonded structure of water has beensupported recently by dielectric spectroscopy.28 Yet despitethis apparently “easy” substitution, incorporating urea intothe network appears to disrupt it, creating a local compressionof the second hydration shell around water molecules in amanner similar to the effect of high pressure on the liquid.All the same, the orientational dynamics of the watermolecules seem largely unaffected even at urea concentra-tions high enough for all the water to be part of hydrationshells.29 Only one water molecule per urea, on average, hasa significantly slower reorientational time constant (about 6times that of bulk water), which can be rationalized accordingto a hydration structure in which one water is complexed tothe urea molecule via two hydrogen bonds.

These findings shed light on the action of urea as a proteindenaturant. Proposed reasons for this behavior have invokeddirect interactions of urea with the protein backbone, forexample, via hydrogen bonds30,31or electrostatic32 or hydro-phobic contacts,33 which act to swell the protein as aprecursor to denaturation, and indirect effects due to thedisruption of “water structure” by urea, making the hydro-phobic groups more readily solvated.34 The experimentalfindings for the effect of urea on water do not seem to support

Figure 5. Solvation of cations and anions. (a) The conventionalview. (b) Water orientations revealed by neutron scattering.

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the idea of significant disruption of the hydrogen-bondednetwork (although one might argue that the mimicking ofhigh pressure in the second hydration shell might encouragesomething akin to pressure-induced denaturation). Rather,it seems best to consider the nature of specific interactionsbetween the various solutes, instead of regarding the proteinas being solvated by “modified water”. Indeed, experimentsand thermodynamic analysis imply that in general a cosolventsuch as urea will be inhomogeneously partitioned around abiological macromoleculesurea tends to expel water froma protein surface, whereas sugars are preferentially excludedfrom the hydration sphere.35

We will see below that the same principlesconsideringlocal, direct interactions rather than global “ water structure”modificationssholds for hydrophobes too. In any event, onecan see that the overall picture of the effect of hydrophileson water structure is rather complex even for this relativelysimple species, demanding a detailed consideration of thestructure and dynamics of the hydration shell(s), and so itseems unwise to fall back on popular but simplistic notionsabout whether solutes such as urea “break” the structure ofthe hydrogen-bonded network36,37 (see below).

2.3. Small-Molecule Solutes: HydrophobesHydrophobic solutes in water experience a force that

causes them to aggregate. This hydrophobic interaction isresponsible for several important biological processes:38 forthe aggregation of amphiphilic lipids into bilayers, with theirhydrophobic tails “hidden” from water and their hydrophilicheads at the surface, for the burial of hydrophobic residuesin polypeptide chains that helps proteins to fold and to retaintheir compact forms, and for the aggregation of proteinsubunits into multi-subunit quaternary structures. There is atendency for even rather small hydrophobic moieties toclusterssomething of the sort seems to happen for methanoldissolved in water, leading to dynamic heterogeneity of themixture.39 The question is whether a single mechanism forthe hydrophobic interaction can account for all thesebehaviors, and if so, what is it?

We do not yet know how to answer either point. Part ofthe reason for that perhaps surprising ignorance may be thatthere has for some time existed an apparent explanation that

made such seemingly good sense that it has been hard todislodge and is still routinely cited in biochemistry textbooks.This is Kauzmann’s “entropic” origin of the hydrophobicattraction,40 which draws on the picture of hydrophobichydration posited in 1945 by Frank and Evans.41

The conventional story is as follows. In order to accom-modate a hydrophobic species, the hydrogen-bonded networkmust be disrupted to create a void. But this can be done insuch a way as to avoid the enthalpic penalty of losinghydrogen bonds, if the water molecules arrange themselvesaround the hydrophobe in a relatively ordered fashion (Figure6a). In the words of Frank and Evans, “when a rare gas atomor nonpolar molecule dissolves in water at room temperatureit modifies the water structure in the direction of greatercrystallinitysthe water, so to speak, builds a microscopiccage around it”.41

This is the “iceberg” model: the hydrophobe is encasedin an icelike shell of water. It has been often suggested42,43

that, based on a description of water structure due toPauling,44 a better model for this pseudocrystalline cage isthat found in gas hydrates, where small hydrophobic speciessuch as methane are enclosed in cavities made frompentagonal rings45 rather than the hexagonal rings of ice.(Hydration structures around the hydrophobic portions ofproteins are now commonly described in terms of pentagonalrings.) Kauzmann pointed out that the price of creating anyrelatively ordered structure of this sort to preserve theintegrity of the hydrogen-bonded network is that the rota-tional and translational freedom of the molecules in the cagewall are compromised: there is an entropy decrease. But iftwo “caged” hydrophobes were to come together, the“structured” water in the region between them is returnedto the bulk, leading to an entropy increase (Figure 6b). Thus,there is an entropically based force of attraction betweenthese solute particles.

The argument seems sound in principle, but the questionis whether hydrophobic hydration really has the “semicrys-talline” character proposed by Frank and Evans. To date,there is no good reason to suppose that it does, and someevidence indicating that it does not. Reviewing the existingdata on hydrophobic hydrationsboth structural and thermo-dynamic, and based on both experiment and computer

Figure 6. (a) “Iceberg” model of hydrophobic hydration. (b) Kauzmann’s explanation of the hydrophobic attraction.

Water as an Active Constituent in Cell Biology Chemical Reviews E

simulationsBlokzijl and Engberts46 concluded that there wasno reason to suppose that hydrogen bonding is enhanced inthe first hydration shell or that the favorable enthalpy ofhydrophobic hydration need be attributed to anything morethan normal van der Waals interactions between water anda nonpolar solute. Moreover, they found no evidence thathydration involved any enhancement in the ordering of watermolecules around the solute; rather, the hydrogen-bondednetwork seemed simply tomaintainits structure, in particularby orienting water molecules such that the O-H bonds aretangential to the solute surface. There is no inconsistencybetween the occurrence of such orientational effects and thelack of any enhancement in structure, since we must bear inmind that the hydrogen-bonded network introduces direc-tional preferences in local water orientation even in the bulk.The importance of attractive solute-water van der Waalsinteractions, relative to considerations of “water structure”and water-water interactions, was emphasized by Ashbaughand Paulaitis,47 whose Monte Carlo simulations show thatalthough water densities in the first hydration shells ofclusters of methane molecules are greater than those of thebulk, the corresponding density for hydrated hard spheres(lacking any attractive interactions) of the same size as themethane clusters decreases as the sphere’s radius increasesand ultimately falls below that of the bulk.

Blokzijl and Engberts suggested that aggregation ofhydrophobes results not from Kauzmann’s entropic mech-anism but from the increasing difficulty in accommodatinghydrophobes within the hydrogen-bonded network as theirconcentration in solution increases: “hydrophobic interac-tions”, they say, “are not so much a result of a structuralproperty of the hydrophobic hydration shell of apolarcompounds but rather reflect the limited capacity of liquidwater to accommodate the apolar solute and maintain itsoriginal network of hydrogen bonds.”46 From the perspectiveof whether there is anything “special” about water thatintroduces a hydrophobic interaction, therefore, the messageis mixed. Yes, the hydrogen-bonded network seems to beimportant, but not because it becomes more highly structuredby hydrophobes; rather, it is because this network is disruptedby too great an accumulation of cavities.

On the other hand, Lucas48 and Lee49,50have proposed thatthis disruption is not a function of the hydrogen-bondednetwork at all but stems merely from the small size of thewater molecules, which creates a high free-energy cost toopening up a cavity to accommodate a hydrophobesanargument based on scaled-particle theory,51 which Stillinger52

first adapted to the case of hydration. Hummer, Pratt, andtheir co-workers53-58 have argued that models based on thespontaneous formation of cavities through density fluctua-tions in liquid water can account quantitatively for thethermodynamics of small-hydrophobe solvationsa claimsupported by MD simulations.59 Yet Southall et al.60 assertthat neither water “structure” nor the small-size effect canby themselves fully explain several of the characteristics ofthe hydrophobic effect, such as its dependence on temper-ature and on solute shape.

While all of these studies help to decouple the real puzzlesof the hydrophobic effect from comfortable but outdatedexplanations, there is a tendency in discussions of thehydrophobic interaction to regard water asthe liquid ratherthana liquid: little comparison has been made with othersmall-molecule liquids, both associated and simple, perhapsbecause of the notion that the hydrophobic effect is clearly

biologically relevant, whereas such solvents are not. As aresult, we lack much basis for comparison in deciding howmuch of water’s behavior as a solvent for solvophobicparticles is unique (and thus perhaps due to its three-dimensional hydrogen-bonded network) and how much isgeneric to other, related liquids. Only when such questionsare answered can we expect to say much about water’ssupposed centrality to life.

Evidently, however, there is nothing unique to water aboutsolvophobic aggregation in general: the existence of reversemicelles in nonpolar solvents indicate as much. Morestrikingly, Huang et al.61 have shown that non-naturalpeptide-like molecules with N-linked rather than C-linkedside chains (N-substituted oligoglycines or “peptoids”62) willfold into well-defined secondary structures in acetonitrile inwhich the polar units (the carbonyl groups) are buried inthe interior (Figure 7): one apparently does not need a strongdegree of solvent structure in order for such packing to occur.It will be very interesting to explore the potential complexityof structure and function available to this and other non-aqueous pseudoprotein chemistry.

All the same, one can imagine there being scope for addedsubtlety in the interactions of a solvophobe and a structuredsolvent like water. Indeed, Hummer and co-workers53,54arguethat if cavity-opening fluctuations are the dominant factorin hydrophobic hydration, water’s unusual equation of statedistinguishes the nature of these effects from those that mightbe anticipated in other liquidssthere is then something“special” about water.

But how, if at all, does water structure manifest itself inthe hydration environment of hydrophobes? Obviously thebest way to deduce the effect of a hydrophobic moleculeplaced within water’s network is to measure it directly; andagain neutron scattering is the ideal probe. But that is nosimple matter, because archetypal small hydrophobes suchas methane and krypton are insufficiently soluble in waterto permit an easy detection of the hydration shells abovethe bulk water signal.

Nonetheless, de Jong et al.63 used neutron scattering todeduce that methane molecules in water are surrounded byabout 19 solvent molecules and that those in the firsthydration shell are tangentially oriented. The structure of ahydrophobic hydration shell has been most aggressivelypursued by Finney, Soper, and their co-workers. Filipponiet al.64 used EXAFS to show that krypton is hydrated by

Figure 7. Backbone folded structure of an N-substituted glycinenonamer peptoid in acetonitrile. The dashed green lines indicatehydrogen bonds. Reprinted with permission from ref 61. Copyright2006 American Chemical Society.

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around 20 water molecules. This hydration structure wasbarely altered by applying pressures of up to 1 kbar.65 Thisnot only throws into question some proposed explanationsfor pressure-induced protein denaturation in terms of hydra-tion changes but also cautions against too heavy a relianceon computer simulations for evaluating hydration structure,some of which66 predict significant pressure-induced changesin the hydration shell of methane.

Molecular dynamics simulations of krypton in aqueoussolution67,68show no sign of the clathrate-like hydration shellsinvoked in the classic iceberg model. That is borne outexperimentally by Bowron et al.,69 who used EXAFS tohighlight the differences between the hydration shell ofkrypton in solution and the clathrate cage of krypton in thecrystalline clathrate hydrate. The former is unambiguouslymore disordered, and the tangential orientation of watermolecules found in the hydrate is not rigorously main-tained in the liquid. Indeed, Finney70 points out that thefirst-neighbor water structure in the hydration shell is allbut unchanged from that in bulk water: “water in thisenvironment still thinks it is liquid water”. And ab initioMD simulations of the diffusion of small hydrophobes inwaterswhich is found experimentally to be anomalouslyfast71sindicate that, far from being icelike, the hydrationshell is unusually dynamic and loose.72 Even if theFrank-Evans iceberg model is no longer interpreted tooliterally in any case, these results give direct cause to doubtany such picture that places emphasis on enhanced “waterstructuring” around hydrophobes.

Another model small hydrophobe is benzene, sincearomatic groups are common in biological molecules. Butwhile phenyl groups are generally regarded as hydrophobic,it has been known for some time that theπ-electron systemcan act as a weak hydrogen-bond acceptor,73 creating strongorientational preferences in the bound water molecules.74 Theconverse is also possible: the lone pairs on the oxygen mayalso interact favorably withπ electrons. This balance appearsto be rather subtle, making it hard to generalize about howaromatic groups are likely to be hydrated. Allesch et al.75

find in first-principles MD simulations of benzene andhexafluorobenzene in water that both molecules act ashydrophobes equatorially but that the axial hydration struc-ture is very different: water molecules typically point towardthe ring hydrogen-first for benzene, but lone-pair-first forhexafluorobenzene.

A particularly attractive minimal model for investigatingthe role of hydration structure on hydrophobic interactionsin biological systems is provided by simple alcohols suchas methanol. Not only are they amphiphiles that mimic“shrunken lipids”, but the juxtaposition of nonpolar groupsand polar, hydrogen-bonding groups shares to that extent thecharacter of a typical portion of a protein’s peptide chain.Moreover, they provide an opportunity to study hydrophobichydration without the problems of low solubility. Dixit etal.76 find that a concentrated solution of methanol (7:3methanol/water ratio) is imperfectly mixed and highlystructured: water clusters bridge hydroxyl groups on thealcohol to form hydrogen-bonded chains that thread througha “fluid” of methyl groups. Most of the water molecules,however, cluster into groups of 2-20, in which their localstructure is surprisingly bulklike, again challenging any ideaof iceberg-like ordering. The segregation of methanol andwater even persists at high dilution of the alcohol:77 in a1:19 mixture of water and methanol, more than 80% of the

alcohol molecules are clustered into groups of 3-8. Again,here neutron scattering shows no evidence for structureenhancement in the hydration shells of the nonpolar portionsof the methanol molecules.78

What, then,is the origin of this clustering effect? Dixit etal.77 propose that it might indeed have an entropic origin,but subtly so. They find that the hydration shells of the watermolecules themselves are slightly altered by the presence ofmethanol (at a 1:19 concentration, most of the watermolecules lie within the hydration shell of at least onemethanol): the second hydration shell becomes slightlycompressed, and the correlations between second-neighborwaters slightly sharpened, leading to a small reduction inentropy. This small decrease in freedom of the watermolecules could promote aggregation of the methanols.“Perhaps it is here”, Finney et al.79 conclude, “in thewater’ssecondshell rather than thealcohol’s first hydration shell,that the entropic driving force for the hydrophobic interactionis to be found”. If so, this is a small effect, and by no meansobvious, and of unproven generalitysso it might be arguedthat explanations based on general factors such as cavityformation53-58 have a stronger appeal, not least because theyare more intuitive. But in any event, these direct structuralprobes seem to have diminished any argument for the classic“enhanced structuring” model of the hydrophobic interactionalong the lines defined by Kauzmann.

2.4. Large Hydrophobic Solutes and SurfacesAlthough small hydrophobic species can be accommodated

in the hydrogen-bonded network of liquid water withoutmuch perturbation of the network, large hydrophobes, forexample the hydrophobic surfaces of proteins, are anothermatter. An attractive interaction evidently exists betweensuch species too, being responsible for protein foldingand aggregation. Does this have the same origin as theforce that causes small hydrophobes to cluster? That is notobvious.

Close to an extended hydrophobic surface, it is geo-metrically impossible for the network to maintain its integrity.It has been proposed that this can even lead to “drying” ofthe interface43sthe formation of a very thin layer of vaporseparating the liquid from the surface. In a highly stimulatingcontribution, Lum, Chandler, and Weeks (LCW)80 argue thatthis difference between small and large hydrophobes shouldlead to qualitatively different behavior, with a crossoverlength scale somewhere in the region of 1 nmsabout thevan der Waals diameter of anR-helix. This idea derives fromthe notion, proposed by Wallqvist and Berne,81 that whentwo hydrophobic surfaces come in close proximity, watercan withdraw from between them (Figure 8) and that the

Figure 8. Hydrophobic attraction in the model of Lum et al.80The hydrophobic surfaces are surrounded by a thin layer of vapor.At some critical separationDc, there is a collective drying transitionin the space between the surfaces.

Water as an Active Constituent in Cell Biology Chemical Reviews G

resulting imbalance in pressure would cause the two surfacesto attract. In effect, the confined water undergoes “capillaryevaporation”. This kind of drying transition has been seenin simulations of hydrophobic plates in water when theseparation between them falls to just a few molecularlayers.81-83

Simulations of hard-sphere solvation by Rajamani et al.59

support the LCW picture, indicating that the thermodynamicsof hydration is entropically controlled for small hydrophobesbut enthalpic for larger ones. This is precisely what onewould expect if the former case depends on density fluctua-tions and cavity formation while the latter involves theformation of a liquid-vapor interface and thus introducesthe surface tension into the hydration Gibbs energy. Thiscrossover of hydration mechanisms is accompanied by achange in the variation of hydration Gibbs energy∆G withsolute size: ∆G is a linear function of solute volume forsmall sizes but becomes closer to being proportional to solutesurface area for larger sizes. Rajamani et al. find that thecrossover occurs for hard-sphere solute radii of the order ofa few angstroms under ambient conditions but that this sizescale can be “tuned” either by altering thermodynamicparameters (for example, placing the water under negativepressure) or by adding other solutes. Ethanol, for example,decreases the crossover length scale to molecular dimensions.Ashbaugh and Pratt58 show that this picture of a crossoverfrom entropically to enthalpically dominated hydrophobichydration as a function of particle size can be rationalizedby applying scaled-particle theory52 to a thermodynamicanalysis of cavity formation.

But does hydrophobic collapse induced by cooperativedewetting really play any role in the association of hydro-phobic macromolecules, for example, in protein folding andaggregation? Here the picture remains unclear, although itseems fair to say that the LCW model looks increasinglyunlikely to provide a general description of macromolecularhydrophobic interactions. Simulations by ten Wolde andChandler84 suggest that a hydrophobic polymer acquires acompact conformation in water via a process resembling afirst-order phase transition in which the rate-limiting step isthe nucleation of a sufficiently large vapor bubblestheclassical mechanism of heterogeneous nucleation. But simu-lations of protein folding show a more complex situation.Berne’s group found that collapse of the two-domain enzymeBphC, which breaks down polychlorinated biphenyls, showedno sign of a sharp dewetting transition as the domains cametogether.85 Here the drying seen between hydrophobic plates82

seems to be suppressed by attractive interactions betweenthe protein and water: dewetting was recovered when theelectrostatic protein-water forces were turned off and wasstronger still in the absence of attractive van der Waals forces.Thus, it seems that the inevitable presence of such interac-tions in proteins complicates the simple picture obtained fromhydrophobic surfaces. Consistent with this view, MacCullumet al. found that simulations of the dimeric association ofboth polyalanine and polyleucine (A20 and L20) R-helicesshowed no dewetting between the chains until it was inducedsterically by a mere insufficiency of space for a watermonolayer.86

On the other hand, Berne’s group found a first-order-likedewetting transition in simulations of the association of themelittin tetramer, a small polypeptide found in honeybeevenom.87 But single mutations of three hydrophobic isoleu-cine residues to less hydrophobic ones were sufficient to

suppress the dewetting. Is melittin a rarity, even a uniquecase, or might other proteins also exhibit dewetting? Berne’sgroup has performed a survey of the protein data bank tosearch for other structures that might show similar behavior.88

They find that dewetting is indeed rather rare but does happenin several other cases: they identify two two-domainproteins, six dimers, and three tetramers that behave this way.It seems that any significant number of polar residues in thehydrophobic core (which is common) is generally enoughto suppress dewetting. Using the same tools, Berne’s groupfinds preliminary evidence that dewetting may also some-times play a role in ligand binding.

These results suggest that even if the LCW mechanismcan operate in the collapse of some proteins, nonetheless itis extremely sensitive both to the precise chemical nature ofthe protein domains involved and perhaps to the geometryof association: melittin subunit association forms a tubelikeenclosed space, whereas that for BphC is slablike.

Choudhury and Pettitt have attempted to clarify theseissues by returning to the case of two planar, nanoscopichydrophobic plates.89,90 They find89 that the existence orabsence of a wetting layer between the plates at separationsof less than about 1 nm depends on a fine balance betweenthe plate-water interaction energy, the hydrogen-bondingenergy, and the plate size. For example, graphite-like platesmeasuring 11× 12 Å2 undergo a steric dewetting transitionfor separation below about 6.8 Å for a Lennard-Jonesinteraction potential, but dewetting occurs at about 10 Å fora purely repulsive plate-water interaction. This LCW-likebehavior vanishes, however, if the plates are smaller.

This finding seems consistent with the view provided bydensity functional calculations on confined simple liquids,91

which show that, although unfavorable liquid-surfaceintermolecular interactions (relative to liquid-liquid interac-tions) can counteract the usual enhancement of liquid densityclose to a wall owing to packing effects, and can even leadto a depletion in average density here, it takes a ratherextreme set of interaction parameters to induce capillaryevaporation in the manner of the LCW model. In otherwords, the bulk liquid-solid contact angle has to be verylow, and it is far from clear that water in contact with atypical hydrophobic surface (an alkyl-covered surface, say,let alone the hydrophobic surface of a protein) representssuch an extreme case. Choudhury and Pettitt concur90 thatin general, “chemically reasonable” estimates of the plate-water interaction strength lead to a microscopically wet stateand not to plate association triggered by a dewettingtransition. That conclusion is supported by MD simulationsof Bresme and Wynveen, who have studied the effect oninteractions between two hydrophobic solutes of varying theirpolarizability.92 The solute polarizability has a strong influ-ence on the water contact angle, and a drying transitionoccurs only for rather extreme conditions (outside the rangeof permittivities typical for proteins) in which the contactangle is close to 180°. Otherwise a fluid layer remainsbetween the solute surfaces, albeit with a density significantlylower than that of the bulk liquid. This situation is neverthe-less associated with strong hydrophobic forces, showing thatcomplete drying is not essential to promote an attractiveinteraction.

This perspective leads to the more general question of howwater behaves and is structured close to asinglehydrophobicsurface. Although, as Choudhury and Pettitt point out,90

drying in the interplate region may be a cooperative

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phenomenon and thus does not necessarily demand dryingof the isolated surfaces themselves, nonetheless the natureof the interface between water and a hydrophobic surface isthe precondition, one might say, for any discussion ofpotential dewetting by confinement. Moreover, this questionis central to the more general and much-disputed issue ofthe hydrophobic hydration of proteins.

Experiments have tended to give a rather confused andcontradictory picture of this situation,93 although a consensusnow seems to be emerging. X-ray reflectivity measure-ments94-96 suggest that, although there is a depletion in liquiddensity adjacent to alkane monolayers, it is far less pro-nounced than what would be observed for complete dryingand happens only within a few molecular diameters of thesurface. Measurements of water density adjacent to acrystalline paraffin monolayer floating on the surface of watersuggest that the depletion region extends about 1.5 nm intothe liquid phase and that it corresponds to a deficit of aboutone water molecule for every 25-30 Å2 of the paraffinsurface.94 On the other hand, MD simulations in the samestudy indicated the formation of a very thin (about 1 Å) layerof “vacuum” between the water phase and the surfacessomething beyond the resolution of the experiment itself.This latter result might be considered broadly consistent withthe simulations of Pertsin et al.,97 which indicated only avery small reduction in water density close to an alkylatedsurface. Other MD simulations suggest that, contrary to whatwould be expected in the presence of a vapor-like film, thereis significant penetration of water molecules into a layer oftetheredn-C18 chains.98

A more recent high-resolution X-ray reflectivity study95

corroborates the existence of a “hydrophobic gap” for amonolayer ofn-C18 chains on silica but suggests that itextends no further than 1-6 Å from the surface and that itcorresponds to an integrated density deficit of 1.1 Å g cm-3.Similarly, Poynor et al.96 find a density deficit of more than40% extending about 2-4 Å from the surface.

In contrast, some neutron reflectivity studies of the waterdensity adjacent to a self-assembled monolayer of alkyl-thiols99,100 have apparently indicated a density depletionextending for several nanometers. But recent results makethis now seem unlikely. Doshi et al.101 found a reduceddensity extending only 1 nm or less from the surface, thedistance depending on the amount and chemical nature ofdissolved gases (see below; at this stage the influence ofdissolved gases is by no means clearsthe smaller hydro-phobic gap reported by Mezger et al.95 was unaffected by awide range of such gases). And Maccarini et al. report adepletion layer of no more than about 2 Å.102

From a theoretical perspective, even if hydrophobicsurfaces do not induce anything like complete drying, theexistence of a depletion layer extending over distances of2-5 nm would be very perplexing, since there is no obviousphysical interaction in the system that could introduce sucha length scale. That recent results seem to be converging ona depletion layer with a thickness of the same order of thewater molecule itself is therefore reassuring.

It is important to know whether similar effects are seenfor other liquids so that one might elucidate the role (if any)of water’s hydrogen-bonded network. Maccarini et al.102 dofind that depletion layers of a similar order, that is, just afew angstroms thick, appear to be present at the interface ofhydrophilic surfaces and nonpolar liquids, showing that we

should be wary here of attributing anything “special” towater.

How water dynamics might be affected within a hydro-phobic depletion layer is another matter, which has receivedlittle attention. Dokter et al.103 found that nanodroplets inreverse micelles, where the interface is not hydrophobic butis thought nonetheless to have decreased hydrogen bonding,have slow orientational dynamics and relatively immobilewater molecules in the interfacial layer. Jensen et al. alsofound retarded dynamics in simulations of water next to ahydrophobic surface.104 As we will see below, a significantchange in rotational dynamics in this region could haveimplications for the hydrophobic interaction itself.

Any change in the nature of “water structure” close to ahydrophobic surface can be expected to alter its solvatingcharacteristics, leading to the possibility of segregation ofsmall solutes such as ions at or away from such interfaces.105

But it seems likely that this view is putting the cart beforethe horse. There is now strong evidence that some ions doindeed segregate preferentially at, or away from, the air-water interface.106-112 Yet this is not because the water is“different” there, but because there is an intrinsic thermo-dynamic driving force for this segregation, and if anythingwe might expect an excess of ions to alter the properties ofthe solvent rather than vice versa. Since one might expectthe interface of water with a hydrophobic surface to mimicin many respects that with air, this inhomogeneity of solutesat a surface could have significant implications for thesolvation of proteins, as we will see below.

The same applies to the finding that hydronium ions seemto have a preference for the water surface.113-118 This result,which was predicted theoretically113,114,118and confirmed bythermodynamic analysis,111 by surface spectroscopy,114,116,117

and by deuterium exchange at the surface of ice nanocrys-tals,118 apparently has a rather different origin from thesurface segregation of other ions. H3O+ may form three donorhydrogen bonds to neighboring water molecules, but becausemost of the positive charge resides on the oxygen atom, itcan no longer act as a good hydrogen-bond acceptor. Indeed,this makes the oxygen somewhat hydrophobic, so thathydronium acts as an amphiphile.113,115 Both that and thereduced hydrogen-bond capacity encourage the surfaceaccumulation of hydronium, oriented with the oxygen atomoutermost. It has been estimated113,118that this effect shiftsthe surface pH of pure water to around 4.8 or less (althoughthe applicability of this bulk parameter on a localized scaleis not entirely clear). As much the same behavior might beexpected at hydrophobic surfaces, this finding could havesignificant implications for biomolecular hydration that haveyet to be investigated; for example, one might expect to seea shift in the dissociation of protonatable residues close tohydrophobic patches and perhaps even a stabilization ofhydrophobic species by a kind of surfactant behavior ofhydronium.

2.5. The Influence of Ions: Structure-Making andStructure-Breaking

The coexistence of ions and hydrophobes in aqueoussolution has some puzzling consequences. Hofmeister notedin 1888 that some salts tend to precipitate albumin fromsolution (salting-out), whereas others enhance its solubility(salting-in).119,120The Hofmeister series ranks ions in orderof their “salting-out” tendency for proteins:

Water as an Active Constituent in Cell Biology Chemical Reviews I

Similar sequences are observed for the solubility of am-phiphiles.

The traditional explanation for the Hofmeister seriesintroduces another of the tenacious myths of hydration: theconcept of “structure-making” and “structure-breaking” ions.The basic idea is that large, low-charge ions such as I- andNH4+ disrupt “water structure”sthey are structure-breakersswhereas small or highly charged ions such as F- and Mg2+

are structure-makers, imposing order on the hydrogen-bondednetwork. Then salting-out and salting-in of proteins areexplained on the basis of entropic changes induced in theirhydration shells by the addition of ions or of a reduction inthe strength of hydrogen bonding of water moleculescomplexed to dissolved ions. The classical hypothesis is thatsalting-out arises from a competition for solvation betweenthe salt and the protein, in which an ion’s ability to sequesterwaters of solvation is somehow connected to its effect onwater structure. Thus, the structure-making effect of smallor highly charged ions depletes proteins of hydration waterand causes precipitation.

Yet there is little consensussindeed, sometimes outrightcontradictionsabout what structure-making and -breakingactually entails. For example, one suggestion holds thatalthough an ion always induces ordering of the watermolecules in its first hydration shell, because of the tendencyfor the molecules to orient themselves either oxygen-first orhydrogen-first, subsequent layers in the hydration sphere maybecome more or less ordered as a result, depending on thecommensurability with the hydrogen-bonded network. More-over, although some interpret “structure-making” as anenhancement in the ordering of the hydrogen-bonded net-work, Franks121 insists that the local structure enforced bysmall and/or highly charged ions is not commensurate withthe tetrahedral pattern of bulk water and so may disrupt that.

Such ideas have been extended to produce “two-state”models of liquid water in which different solutes areclassified in terms of their ability to enhance or degrade theshort-range order in the liquid: they are called eitherkosmotropes or chaotropes, respectively. Kosmotropes, byincreasing the local order, render the water more icelike, witha consequent decrease in its ability to dissolve electrolytes.Chaotropes promote a denser, more “liquid-like” waterstructure. Thus, chaotropes and kosmotropes may set upgradients in chemical potential and solute concentration: ahypothesis that has been advanced to account for a range ofcell functions extending well beyond the original Hofmeistereffects.105

Monte Carlo simulations using a simple two-dimensionalmodel of ion hydration, in which water molecules arerepresented as disks interacting via a Lennard-Jones potentialon which directional hydrogen-bonding interactions aresuperimposed, present an opposite picture.122 That is to say,small ions with high charge densities act as kosmotropesinsofar as they enhance the first peak of the rdfssbut thisincrease in density, although in some sense representing anenhancement the local liquid structure, is produced by thestronger electrostatic interactions, at the cost ofbreakinghydrogen bonds. The hydration environments of large ions

(chaotropes), meanwhile, are more hydrogen-bonded. Thus,the peculiarity of water, whereby enhanced ordering of thehydrogen-bonded network leads to a decrease in density,seems to have introduced further confusion even about what“structure-making” and “structure-breaking” imply. One hasto concur with Franks,121 who says that “much has beenwritten about structure-making and structure-breaking, andmuch of it is misleading.”

The fact is that all of the conceptual scaffolding of“structure-making” and “structure-breaking” has been erectedwithout any real evidence from experimental studies of thestructure of electrolyte solutions that significant changes tothe bulk hydrogen-bonded network of water really do occur.Indeed, as we saw in section 2.2, the available evidence isto the contrary: simple ions seem to have little or no effecton “water structure”, at least beyond the first hydration shell.

So instead of trying to understand the Hofmeister serieson the basis of “global” changes in solvent structure inducedby ionic solutes, it seems far more logical to consider theeffects that these ions have on the local hydration of proteinresidues or other hydrophobes. The fact is that ions do not,in general, simply disperse homogeneously throughout thesolution so as to create a kind of “mean-field” solvent forother large solutes such as macromolecules; rather, as notedabove, many ions tend to segregate preferentially at eitherhydrophilic or hydrophobic surfaces.106-112,123It now seemslikely that Hofmeister effects must be understood in termsof these specific and often rather subtle interactions betweenions and proteins or other biomolecules.

For example, although the ability of so-called chaotropicanions to inactivate enzymes has been rationalized on thebasis of their tendency to disrupt water structure and, inconsequence, enzyme structure, Ninham and co-workers124,125

have shown that the effect of adding sodium salts of variousanions to buffer solutions of lipase enzyme is to alter theenzymatic activity systematically in a manner that cannotbe explained this way but, rather, according to the specificinteractions of the anions with the enzyme surface. AndSachs and Woolf126 found that large anions seem to pene-trate deeply into lipid bilayers, becoming partially strippedof their hydration shells as they do so. Thus, whereas chlorideions penetrate to within 17.5 Å of the center of palmitoyl-oleoylphosphatidylcholine bilayers, anions with a van derWaals radius 37.5% greater reach to within 12.5 Å of thecenter. This can help to explain how membrane structuremight be disrupted by different ions again without any needto invoke “chaotropic” disruption of the general waterstructure in the hydration of the lipid head groups.

So far as the original Hofmeister effectsthe ion-specificchanges in solubility of proteinssis concerned, the phenom-enon is now generally interpreted in terms of the tendencyof ions of different size and charge to modify the hydro-phobic interaction. Direct probing of the effect of ions onhydration structure of organic solutes has been conductedonly for simple model systems such as alcohols, usingneutron scattering.127-129 These, as indicated previously, aremodel amphiphiles, in which the polar hydroxy group canengage in hydrogen bonding while the nonpolar alkyl tailmodels the hydrophobic residues of a protein. Adding sodiumchloride to solutions oft-butyl alcohol alters the hydrationenvironment of the alcohol molecules due to a directinteraction with chloride ions. At the concentrations studied(alcohol/water ratios of 1:50), in the absence of salt thealcohol molecules engage in “head-to-head” contacts between

H2PO4- > SO4

2- > F- > Cl- > Br- >

NO3- > I- > ClO4

- > SCN-

Mg2+ > Li+ > Na+ ∼ K+ > NH4+

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the nonpolart-butyl regions: a classic instance of hydro-phobic clustering. But for NaCl/water ratios of 1:100, abouthalf of the chloride ions form salt bridges between the polarOH groups of two alcohol molecules. The other chloridesremain fully hydrated, as do the sodium ions. (Althoughmany of the cations lie close to the nonpolar regions of thealcohol molecules, they do not significantly perturb thehydration structures here: there is no sign of any generalizedstructure-making or structure-breaking in the alcohol hydra-tion layer caused by sodium.) Thus, the interactions ofalcohol molecules change dramatically: from nonpolar-nonpolar to polar-polar, bridged by chloride (Figure 9). This,of course, exposes the hydrophobic regions to the solvent,and it would be expected to make the alcohol less favorablydisposed to the aqueous environment.

Zangi and Berne have considered the more generalquestion of how in ions interact with small hydrophobicparticles.130 In simulations of hydrophobic Lennard-Jonesparticles of diameter 0.5 nm, they found that ions with highcharge density (q) produce salting-out, inducing strongerhydrophobic interactions that promote particle aggregation.But low-q ions could have either a salting-out or a salting-in effect, depending on their concentration (low or high,respectively). These effects were related to preferentialabsorption or exclusion of the ions at the particle surfaces,but not in any simple, monotonic fashion. High-q ions tendedto be depleted at the surface of the hydrophobic particleclusters, but are tightly bound to water elsewhere, therebydecreasing the number of water molecules available forsolvating the particles. Low-q ions are absorbed preferentiallyat the particle surfaces, and at high ionic concentrations thiscan lead to salting-in in a subtle way: the hydrophobicparticles form clusters surrounded by ions, a micelle-likearrangement that keeps the aggregates stably dispersed(Figure 10a). At lower concentrations, the ions are unableto solubilize aggregates in this way but can nonetheless stillact as pseudosurfactants that stabilize the interface formedon phase separation and precipitation of large aggregates(salting-out). Such a concentration-dependent switch froman enhancement to a reduction of hydrophobic associationof t-butyl alcohol in aqueous solution induced by urea hasbeen observed in NMR studies,131 and Zangi and Bernesuggest that this polar molecule may be acting in the samemanner as a low-q ion.

The interaction of ions and protein molecules is, however,perhaps often better viewed as comparable to that betweenthe electrolyte and asurface. As noted, a hydrophobic surfacemight be expected to induce effects similar to those of theair-water interface, particularly if there is water depletion,or indeed complete drying, at the interface as suggestedabove. Traditionally, ions have been considered to beexcluded from the air-water interface because electrolytesincrease surface tension. But recent studies show that thepicture is not so simple.106-112 For example, Jungwirth andco-workers108,109find that although this picture may hold forhard (nonpolarizable) ions such as sodium and fluoride, largesoft ions such as iodide (and to a lesser extent, bromide andchloride) may accumulate preferentially at the surface. Atthe same time, these latter ions seem to be depleted in the

subsurface layer, where there is an enhanced concentrationof cations.

Zangi et al. have considered what the analogous partition-ing of ions at the surfaces of nanoscale hydrophobic platesmight do to the hydrophobic interaction between them.132

Again, changes in the strength of this interaction appear tobe related to adsorption or exclusion of ions at the interface.High-q ions are once again excluded (Figure 10b), but theconsequent salting-out is here identified as an entropic effectrelated to the formation of tight ion-hydration complexesand thus a decrease in configurational entropy of all of thespecies in solution. Medium-q ions induce salting-in becauseof a different entropic effect: they are adsorbed by the plates,and their expulsion into solution when the plates associateleads to a reduction in water entropy owing to the formationof hydration complexes. But low-charge-density ions causesalting-in enthalpically, since they bind to the surfaces andlower the surface tension of the plate-water interface, amechanism analogous to the surface stabilization of largeaggregates of small hydrophobic particles.130 These resultsimply that Hofmeister effects may have a different origin,and thus a different character, for small and large hydro-phobic particles: whereas in the former case there is anincrease in hydrophobic aggregation for both high-q andlow-q but not medium-q ions (except at high concentrations),for hydrophobic plates the relationship is monotonic, withan increasing tendency toward salting-in as the ion chargedensity decreases. But in both cases the mechanism issomewhat subtle and dependent on the direct ion-hydro-phobe interaction, and need not (indeed, should not) invokethe deus ex machina of “water structure”.

2.6. Long-Range Hydrophobic Interactions andthe Role of Bubbles

As though this picture were not complicated enough, thereseems to be a further type of hydrophobic interaction. Inthe early 1980s, measurements using the surface-forceapparatus (SFA) revealed that there is an attractive interactionbetween hydrophobic surfaces that seems to extend over verylong distances, exceeding the range of the normal hydro-phobic interaction.133,134 This puzzling observation wasanticipated by the measurements of Blake and Kitchener in1972,135 who inferred the action of such a force by lookingat the rupture of a water film at a hydrophobic surface as itwas approached by an air bubble. This attraction is measur-able even at separations of about 300 nmsseveral thousandmolecular diameters.136 What could be the source of such along-ranged interaction? Speculations that it might be con-nected to some kind of extreme ordering or structuring ofliquid water between the two surfaces137 never quite shruggedoff an air of implausibility. It has suggested that correlatedcharge or dipole fluctuations on the two surfaces might leadto a long-ranged electrostatic interaction, in a manner thatmakes no direct appeal to water structure per se.138-140

Recently, Despa and Berry141 have refined this perspectiveby considering the effects of the apparent orientationalordering of water molecules at hydrophobic surfaces46,104andthe concomitant retardation of relaxation dynamics.142 Theysuggest that the resulting slow reorientation of water dipolesat the surface will give rise to strong coupling and correla-tions between them, creating a polarization field that willinduce a dipole at the hydrophobic surface. In this view, thatthe long-range attraction between two such surfaces is thuselectrostatic, arising from induced dipole-dipole interactions.

Figure 9. Chloride bridging oft-butyl alcohol in solution.

Water as an Active Constituent in Cell Biology Chemical Reviews K

Interestingly, Despa and Berry suggest that the orientationalordering of water at a hydrophobic surface provides avindication of Frank and Evans’ “iceberg” model41sacontrast with what Blokzijl and Engberts46 concluded fromthe same basic observation, indicating how little consensusthere is about what precisely is implied by “water structure”in this context.

An alternative explanation for the long-range hydrophobicattraction invokes the formation of submicroscopic bubblesbetween the surfaces, whereupon the meniscus pulls themtogether.136,143Such bubbles are hard to visualize directlysthey would be too small to be seen in optical microscopysand moreover it was not clear how the highly curvedinterfaces could be viable, since they would generate a highinternal gas pressure (via the Laplace equation) that shouldlead to bubble dissolution.

Nonetheless, there is now some evidence that such bubblesmay be formed. Using high-resolution optical microscopy,Carambassis et al.144 saw bubbles about 1µm in diameter inwater in contact with a glass surface coated with fluorinatedalkylsilanes. They observed jumps to contact between thesurface and a similarly coated glass microsphere as it wasbrought toward the surface on the tip of an atomic forcemicroscope (AFM). These jumps occurred at differentseparationsstypically 20-200 nmsin different experimentalruns, suggesting the abrupt appearance of bubbles of varioussizes. Tyrrell and Attard145,146have also imaged submicro-scopic bubbles, about 100 nm in radius and flattened againstthe surface, in AFM studies of hydrophobic surfaces in water.This flattening might explain why the bubbles are not

ruptured by the high pressures that would be inferred if theywere assumed to be spherical with a radius equal to the jump-to-contact distance as the surface is approached by anotherhydrophobic object. More recently, such bubbles were seenalso by Simonsen et al.147 and Zhang et al.148 using the AFM.The latter report flat gas bubbles about 5-80 nm thick and4 µm across that remain stable at such a hydrophobicinterface for over 1 h. But the bubbles form only when aparticular protocol is followed for introducing the gas layer(carbon dioxide): in other words, the presence of the gasphase depends on the previous history of the interface.

A possible objection remains, however, that the bubblesimaged this way might be nucleated by the AFM probe tipitself, rather than pre-existing. Doshi et al.101 argue that adynamically fluctuating water density depletion owing to (orat any rate enhanced by) the adsorption of dissolved gasesat a hydrophobic surface149 could act to help nucleate bubblesheterogeneously when two such surfaces are brought to-gether, as suggested in ref 99, rather than there being anystable bubbles already present at such surfaces.

Thus, it has been proposed that there may be distinctregimes for the interaction between hydrophobic surfaces:a long-ranged attraction created by bridging bubbles (eitherpre-existing or nucleated as the surfaces come together) anda medium-ranged interaction felt at separations of less than20 nm or so where the attraction is of the same type as thatinvolved in protein aggregation and foldingswhatever thatmight entail. If the latter is due (at least for nanometer lengthscales) to the capillary-evaporation mechanism of Lum etal.,80 any such distinction is at risk of becoming blurred: the

Figure 10. (a) Distribution of ions around hydrophobic (Lennard-Jones) particles in water. The hydrophobes are yellow, positive ions arered, and negative ions are blue. Low-q ions (left) are adsorbed preferentially at the particle surfaces, leading to micelle-like clusters ofhydrophobic particles surrounded by ions, which prevents further aggregation and precipitation. High-q ions (right) tend to be depleted atthe particle surfaces, which again leads to the formation of clusters. In the intermediate-q case (center) there is neither adsorption nordepletion, and the hydrophobes remain individually dispersed. Reprinted with permission from ref 130. Copyright 2006 American ChemicalSociety. (b) The distribution of high-q (|q| ) 1.00 e) ions around hydrophobic (LJ) plates at varying plate separationsd. The ions arepreferentially excluded at the surfaces and in the intervening water film, which retreats in a drying transition at aroundd ) 0.96 nm.Reprinted with permission from ref 132. Copyright 2007 American Chemical Society.

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force is then “bubble-driven” in both cases. But it isimportant to maintain the distinction between “bubbleformation” induced by the solvent’s liquid-gas equilibrium(cavitation/drying/capillary evaporation) and that induced bydissolved gas. It is strictly the latter that has been proposedas a mechanism for the long-ranged hydrophobic interaction.

If bridging bubbles are truly responsible for the long-ranged interaction, one would then expect the removal ofdissolved gas from the liquid to influence the effect.Degassing does indeed seem to decrease the range andmagnitude of the attraction150-152 and, consistent with thateffect, to increase nanoparticle adsorption on surfaces153 andto enhance the stability of colloids154,155salthough it is hardto differentiate between bulk and surface effects here.150

Doshi et al.101 found that removal of dissolved gasesdecreased the width of the depletion layer observed byneutron reflectivity, which could make the nucleation ofbubbles less likely. None of this is inconsistent with theobservation of Meyer et al.156 that, although deaeration alteredthe force curves between two hydrophobic surfaces in theSFA for separations greater than about 25 nm, the short-ranged “jump-in” behavior was essentially identical foraerated and partially deaerated solutionsssupporting the ideathat there are indeed two distinct attractive hydrophobicmechanisms involved. It is only fair to conclude, however,that we are still not sure how either of them operates.

Indeed, Meyer et al.157,158have proposed that the principalsource of the long-ranged hydrophobic interaction may haveyet another origin. This force has generally been observedto operate between two surfaces rendered hydrophobic bymonolayer coatings of surfactants: in the SFA, these filmsare typically adsorbed onto sheets of mica. AFM images ofa mica surface coated with the cationic surfactant dimeth-yldioctadecylammonium bromide (DODAB), however, showthat once immersed in water, the monolayer becomes patchyon a scale of about 100 nm. The film delaminates and formsbilayer patches separated by bare mica (Figure 11). Meyeret al. argue that as the two surfaces are brought together,bilayer patches will migrate by a rolling mechanism to bringthem opposite bare patches on the opposing surface. Thisplaces regions of opposite charge facing one anothersthe

cationic head groups of DODAB against the negativelycharged mica surfacesgiving rise to an electrostatic attrac-tion. In this picture, then, there is no real “hydrophobicinteraction” at all: the long range of the attraction is duesimply to the Coulombic force.

Bubble coalescence should be influenced by a long-rangedinteraction between hydrophobic surfaces. It appears to besuppressed by ions,159but in a selective manner: certain com-binations of anions and cations have this effect, whereasothers do not.160 This is deeply perplexing, and there is noknown explanation for it. Since salts in general decrease thesurface tension of water, they would be expected to reducecoalescence; indeed, this has been proposed as the explana-tion for the foaminess of seawater relative to pure water.But the fact that some electrolytes apparently do not havethis effect is truly strange. Craig et al.160 suggested thatcoalescence might be somehow mediated by the long-rangedhydrophobic attraction, which salts might modify in an ion-specific way related to Hofmeister effects. But if this attrac-tion is itself to arise from bubble formation or coalescence,then the argument becomes circular, and one might insteadelect to invert the argument and explain the reduction of thelong-ranged attraction in the presence of salts such as KBrand MgSO4 by the salt effect on bubble coalescence.161,162

The phenomenon provides another reminder of how poorlyunderstood the influence of salts is on water structure andbehavior. Nonetheless, Craig et al.160 propose that thissuppression of bubble coalescence might be physiologicallyuseful, in that the coincidence of the salt concentration formaximum suppression and the concentration in blood sug-gests a role in the avoidance of decompression sickness.

2.7. Hydrophilic SurfacesWe must note with some resignation that the interactions

between two hydrophilic surfaces are equally mired inuncertainties and controversy. Measurements with the SFAhave suggested that there is a monotonically repulsiveinteractions between such surfaces.163-165 But van der Waalsinteraction between surfaces would be attractive, and so onceagain “structuring effects” unique to water are among theexplanations proposed to account for the difference.166-168

Israelachvili and Wennerstro¨m dispute that idea,169 arguingthat in fact the hydration force between two hydrophilicsurfaces is indeed either attractive or, because of the layeringeffects experienced by any liquid close to a sufficientlysmooth solid surface, oscillatory. They suggest that the steeprepulsion often measured between hydrophilic particles andsurfaces at small separations is instead due to the charac-teristics of the surfaces themselves, for example, an entropiceffect caused by increasing confinement of mobile surfacegroups such as silicic acid protrusions on the surface of silica,or the constraints imposed on the fluctuations of bilayermembranes. “As a suspending medium”, they argue, “watershould be seen as an ordinary liquid”.169

In the cell, this situation is commonly encountered whentwo bilayer membranes come into close proximity, sand-wiching a layer of water between the sheets of hydrophilichead groups. A repulsive force is indeed experienced by thebilayers when they are 1-3 nm apart.170,171 Whatever itsorigin, this force is clearly of fundamental importance to themembrane dynamics. Simulations suggest that water mol-ecules within 1 nm of the bilayer surface might haveenhanced orientational order,172,173and Cheng et al.174 haveconfirmed this picture experimentally by using coherent anti-

Figure 11. Patchy structure of surfactant films on mica, as revealedby AFM (a), has been explained in terms of the delamination andfolding over of the monolayer to form bilayers separated by baremica (b). (a) Reprinted with permission from ref 157. Copyright2005 National Academy of Sciences.

Water as an Active Constituent in Cell Biology Chemical Reviews M

Stokes Raman scattering (CARS) microscopy to study waterat lipid bilayer surfaces. They found that these superficialwater molecules are preferentially oriented with their dipolesopposed to those of the lipids (Figure 12) and that they aremore weakly hydrogen-bonded than in the bulk. It is temptingto conclude that in this instance water is not such an“ordinary” solvent after all. But one should not leap too farwith that inference. No one should expect water adjacent toa surface to be bulklike: even simple liquids are layered inthat circumstance by packing effects, and indeed models thatinclude laterally ordered surfaces predict such lateral orderingof the liquid too.175 Both densification induced by molecularpacking and the lateral ordering due to surface structuresuniversal effects expected for any liquidswould be expectedto disrupt the hydrogen bonding in the water layer. So hereas elsewhere, before concluding that a change in “waterstructure” is a consequence of its unique hydrogen-bondednetwork, we must remember to ask not just whether thatstructure is different from that in the bulk but whether thedifferences go beyond those we might expect from the theoryof simpler fluids.

3. The Aqueous Environment of the CellIn that same spirit, whatever else we do and do not know

about water structure and hydration, experience with simpleliquids shows that we cannot expect much if any of the waterin the cell to behave as it does in the bulk. The cytoplasmtypically contains up to 400 g L-1 of macromolecules, whichmay occupy 5-40% of the total volume of the cell,176 andas a result the cell is extremely crowded (Figure 13):macromolecules are typically separated by only 1-2 nm.Such narrow confinement would be expected to alter thestructure of any liquid; for water in hydrophilic pores (Vycorglass, where the surface is covered with OH groups that mayengage in hydrogen bonding), the hydrogen-bonded networkappears to be significantly perturbed at these scales,177 andthe average coordination number is reduced from about 3.6in the bulk to about 2.2. If such a structural change iswidespread in the cytoplasm, the implications for hydrationand hydrophobic association (for example, in terms of the

free-energy costs of displacing bound water) could beprofound, indeed rather fearsomely so.

Moreover, molecular crowding means that diffusion ratesare considerably lowerstypically by a factor of 3-8 in themitochondrion and endoplasmic reticulumsthan in the bulk.NMR studies suggest that in fact confinement of water evenwithin spaces several hundreds of nanometers wide can lowerthe molecular mobility of water significantly below that ofthe bulk,178whereas force-microscopy experiments reveal thatwater confined between two hydrophilic surfaces less thanabout 2 nm apart has a viscosity several orders of magnitudegreater than that of the bulk, apparently owing to greatertetrahedral “ordering” of the liquid and (in this case) a largenumber of hydrogen bonds to the surfaces.179,180 There isevidence181 that crowding in the cytoplasm may make watermolecular motions subdiffusive: the typical timet for amolecule to travel a distancel scales not as the Browniant∼ l2 but as t ∼ l2/R, where R ) 0.74. This means thatmolecules take longer to reach their “target” but then stayin its vicinity for longer.

It seems nature may put this crowding to good effect:since processes that reduce the crush are entropicallyfavorable, both the native stability and the refolding rate ofglobular proteins seem to be enhanced in a crowdedenvironment relative to dilute solutions.182 In addition, someprotein functions may be optimized to the dynamics of thecrowded environment in which they will operate.183

As well as being crowded, it seems likely that thecytoplasm is inhomogeneous: there is increasing evidencethat even soluble proteins in such concentrated solution formrelatively long-lived clusters. Small-angle X-ray and neutronscattering shows that lysozyme forms clusters of about 3-10molecules at volume fractions of between 0.05 and 0.2,184

whereas light scattering from concentrated solution ofbacterial lumazine synthase reveals metastable clusters withlifetimes of around 10 s and a mean radius of about 350 nm(the individual molecules are about 15.6 nm in diameter).185

If water in the cell is evidentlynot like bulk water, thequestion that remains unanswered is whether those differ-ences are sufficiently pronounced to matter. Pretty much allextremes of opinion, and everything in between, can be foundrepresented in the literature. Despite the existence of a well-developed thermodynamic theory of small chemical sys-tems,186 biochemistry texts have tended to employ bulkdescriptions of the thermodynamics and kinetics of thecellular environmentsand generally to surprisingly goodeffect. On the other hand, Pollack has argued that thecytoplasm is like a gel (without the macromolecular networkthat makes ordinary gels cohere), which maintains itsintegrity if relatively large sections of the cell membraneare removed.187,188It has been claimed that the proton NMRrelaxation times of water in cells differ from that in the bulkand, moreover, that the relaxation becomes more “bulklike”in diseased, such as cancerous, cells.189,190 (The recentobservation that most of the cell water in the halophilic DeadSea extremophileHaloarcula marismortuihas a translationaldiffusion coefficient more than 2 orders of magnitude lowerthan that of bulk water191 seems to be exceptional andsomehow due to the high salt concentration, although thisremains as yet unexplained.) The notion often advanced hereis that the cell somehow “tames” bulk water and therebyrenders it “biophilic”. This superficially appealing idea tendsto blur together the many different things that can happento water close to ions and surfaces and in confined spaces

Figure 12. Orientational ordering of water molecules between lipidbilayers, as proposed in ref 174.

N Chemical Reviews Ball

within a cellssome of them perhaps specific to water, somegeneric to any associated liquid, and others to any liquid. Ihave discussed some of the general phenomena that mightbe involved in this putative “taming” (one should really saymodification) of water. Now I shall look at some of thespecific roles that solvation (in general) and hydration (inparticular) play in the structure and behavior of biologicalmacromolecules.

4. Protein Hydration: Nonspecific Effects

4.1. The Hydration ShellThe pioneering early studies of protein secondary structure

by Pauling192,193 have, perhaps understandably, somethingof the in vacuo mentality about them insofar as they stressintra- and intermolecular hydrogen bonding in the polypep-tide chains without much consideration of the role of thesolvent. Thus Pauling’s iconicR-helices andâ-sheets areheld together by hydrogen bonds between polar residues, andthe apparent implication is that there is an enthalpic penaltyto breaking up these structures, without consideration of thehydrogen bonds that can then form between the peptideresidues and water. That picture would actually argue foran enhanced stability of these secondary structural motifs innonpolar solvents, where there would be no such competitionfor hydrogen bonding from the solvent molecules. Indeed,completely unsolvated polyalanine oligomers in the gas phaseremain helical up to at least 450°C, where the peptide isalmost entirely dissociated: the noncovalent interactionsexceed the strength of a covalent bond.194 The role of water,then, appears to be not to stabilize theR-helix but to loosenit and allow flexibility of the peptide chain.

What Pauling’s model lacked, of course, is the existenceof hydrophobic interactions between the nonpolar protein

residuessa feature proposed by Langmuir195 and promotedby Bernal196 in the late 1930s. Crudely speaking, this notioninvokes the burial of hydrophobic residues in the proteininterior. It is now generally accepted that protein folding isdriven primarily by a balance between these two factorssintramolecular hydrogen bonding and hydrophobic inter-actionssalthough there is no consensus on precisely wherethe balance lies. Certainly it is an oversimplification to pres-ent globular proteins as a kind of “polymer micelle” that iswholly hydrophobic inside and wholly hydrophilic on thesurface: the two types of residue are in any case not suffi-ciently segregated along the chain to make such a separationpossible. It is true that on average 83% of nonpolar sidechains are buried in the interior of folded proteinssbut soare 63% of polar side chains and 54% of charged sidechains.197 And as we shall see in section 5.6, it seems unwiseto assume that the hydrophobic interior of a folded proteinis “dry”.

We have seen that the precise mechanism of the attractionbetween hydrophobic groups in water remains imperfectlyunderstood. And it is as well to point out now, beforeexpanding on it below, that any attempt to understand proteinfunction cannot rely on statics (structure) alone but mustconsider also the dynamical behavior. With those provisos,what can be said about the role of water in producing thecharacteristic folded structures of proteins in general? Andhow does water hydrate and decorate those three-dimensionalforms?

A key point is that it does not seem sufficient toincorporate water into protein structure prediction merely viasome heuristic potential that acknowledges the existence ofhydrophobic interactions. This approach has been commonin attempts to simulate protein folding, because an explicit,atomistic representation of the solvent in MD simulations

Figure 13. Cytoplasm is a crowded environment, as revealed in this scale drawing. For clarity, the small molecules are drawn only in theupper right corner. Reprinted with permission from D. Goodsell,The Machinery of Life. Copyright 1993 Springer-Verlag.

Water as an Active Constituent in Cell Biology Chemical Reviews O

remains computationally very expensive, restricting thetimescales and the conformational space that can be accessed.But atomistic simulations of the folding of the SH3 proteindomain have revealed that the process may depend on arather gradual, molecule-by-molecule expulsion of waterfrom the collapsed interior.198-200 These studies characterizefolding as a two-stage process: first, collapse to a near-nativestructure that retains a partially hydrated hydrophobic core,followed by slower expulsion of the residual water. Thiswater might play the part of a lubricant to enable thehydrophobic core to find its optimally packed state. More-over, some water molecules typically remain in the core,hydrogen-bonded to the peptide backbone. NMR evidencesuggests that the SH3 domain of the drk protein ofDroso-phila indeed may adopt a collapsed but loosely structuredconformation.201,202This challenges the picture presented byLum et al.80 and ten Wolde and Chandler84 of an abrupt,collective drying transition as the water is confined betweenbasically hydrophobic surfaces.

Papoian and co-workers203,204 have modified a typicalHamiltonian used for protein structure prediction to incor-porate the possibility of water-mediated interactions betweenresidues. This allows for the formation of relatively long-ranged (6.5-9.5 Å) connections between hydrophilic partsof the folding chain through bridging water moleculesssomething that is not permitted by the “dry” Hamiltonian.Such water bridges can be “squeezed dry” in the later stagesof the folding process: the water acts as a temporary, looseglue that holds the folded chain together until it is ready forfinal compactionsin effect constraining the conformationalfre