*Tel.: #44-207-815-7970; fax: #44-207-815-7999.E-mail address: chaplimf@sbu.ac.uk (M.F. Chaplin).

12345678910111213141516171819202122232425262728293031323334353637383940414243444546474849505152535455565758

5960616263646566676869707172737475767778798081828384858687888990919293949596

Biochemistry and Molecular Biology Education 29 (2001) 54}59

Water: its importance to life

Martin F. Chaplin*School of Applied Science, South Bank University, 103 Borough Road, London SE1 0AA, UK

Abstract

Textbooks increasingly include material concerning the importance of water but this topic is often treated over-simplistically withinsu$cient attention being given to the central position of water in life processes. In this article, modern views of the fundamental rolethat water plays in biochemical function and process are summarized. The importance of water in the structures of nucleic acids andproteins is explained. � 2001 IUBMB. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Water; Structure; Hydrogen bonding; Proteins; Nucleic acids

1. Introduction

Although often perceived to be pretty ordinary, wateris the most remarkable substance. We wash in it, "sh in it,swim in it, drink it and cook with it, although probablynot all at the same time. We are about two-thirds waterand require water to live. Life as we know it could nothave evolved without water and dies without it.Droughts cause famines and #oods cause death anddisease. Because of its clear importance, water is the moststudied material on Earth. It comes as a surprise, there-fore, to "nd that it is so poorly understood, not only bypeople in general, but also by scientists working with iteveryday.Textbooks are including increasing amounts of mater-

ial concerning water. However, this material is usuallyconcentrated in one chapter and its importance is rarelyemphasized elsewhere. Chapters on proteins and nucleicacids, for example, often discuss structural and functionaldetails of these macromolecules with little prominencegiven to the pervasive e!ects of the surrounding water.Other areas, such as metabolism, often ignore the manyfunctions of water altogether. This lack of emphasis,evidenced in part from the textbooks' indexes, resultsfrom the multidisciplinary nature of the water literatureand the di$culty in bringing together the wide but oftenthinly-spread information available.Water seems, at "rst sight, to be a very simple molecu-

le, consisting of two hydrogen atoms attached to an

oxygen atom and indeed, few molecules are smaller. Itssize, however, belies the complexity of its properties, andthese properties seem to "t ideally into the requirementsfor carbon-based life as can no other molecule.Organisms consist mostly of liquid water, which per-

forms many functions and should never be consideredsimply as an inert diluent. Nevertheless, in spite of muchwork many of the properties of water are puzzling. It hasoften been stated that life depends on the anomalousproperties of water. In particular, the large heat capacityand high water content in organisms contribute to ther-mal regulation and prevent local temperature #uctu-ations. The high latent heat of evaporation givesresistance to dehydration and considerable evaporativecooling. Water is an excellent solvent due to its polarity,high dielectric constant and small size, particularly forpolar and ionic compounds and salts. Indeed its solva-tion properties are so impressive that it is di$cult toobtain really pure water. Water ionises and allows easyproton exchange between molecules, so contributing tothe richness of the ionic interactions in biology. Thestructuring of water around molecules allows them tosense and be sensed at a distance. The unique hydrationproperties of water towards biological macromolecules(particularly proteins and nucleic acids) to a large extentdetermine their three-dimensional structures, and hencetheir functions, in solution.

2. Structure

Water has the molecular formula H�O but the

hydrogen atoms are constantly exchanging due to

1470-8175/01/$20.00 � 2001 IUBMB. Published by Elsevier Science Ltd. All rights reserved.PII: S 1 4 7 0 - 8 1 7 5 ( 0 1 ) 0 0 0 1 7 - 0

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556

57585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111

protonation/deprotonation processes. Both acids andbases catalyse this process. Even when this proton ex-change is at its slowest (at pH 7), the average time thata water molecule (H

�O) exists between gaining or losing

a proton is only about a millisecond. As this brief periodis, however, much longer than the timescales encounteredduring investigations into water's hydrogen bonding orhydration properties, the water molecule is usuallytreated as a permanent structure.Water molecules are often described in school and

undergraduate textbooks as having four, approximatelytetrahedrally arranged, sp�-hybridized electron pairs,two of which are associated with hydrogen atoms plusthe two remaining lone pairs. Ab initio electron densitycalculations, however, do not con"rm the presence ofsigni"cant directed electron density where lone pairs areexpected. Although there is no apparent consensus ofopinion [1], such descriptions of substantial sp�-hybrid-ized lone pairs in the water molecules should perhaps beavoided [2]. In spite of this, the normal stereochemistryaround the oxygen atom in water is approximately tet-rahedral due to hydrogen bonding.

3. Hydrogen bonding

Hydrogen bonding occurs when an atom of hydrogenis attracted by rather strong forces to two atoms insteadof only one, so that it may be considered to be acting asa bond between the two atoms [3]. Typically this occursbetween oxygen and/or nitrogen atoms, but is also foundelsewhere, such as between #uorine atoms in HF�

�. In

water the hydrogen atom is covalently attached to theoxygen of a water molecule (bond enthalpy about470kJ/mol) but has an additional attraction (about23kJ/mol) to a neighbouring oxygen atom of anotherwater molecule. The hydrogen bond is part (about 90%)electrostatic and part (about 10%) covalent [4]. Thebond strength depends on its length and angle. However,small deviations from linearity in the bond angle (up to203) have a relatively minor e!ect [5]. The dependencyon bond length is very important and has been shown todecay exponentially with distance [6]. There isa trade-o! between the covalent and hydrogen bondstrengths: the stronger is the H2O hydrogen bond, theweaker is the O}H covalent bond, and the shorter is theO2O distance. If the hydrogen bond is substantiallybent then it follows that the bond strength is weaker andthe two water oxygen atoms will generally be furtherapart.The hydrogen bonding patterns are random in water

(and normal ice); for any water molecule chosen at ran-dom, there are equal chances (50%) that any hydrogenbond is located at each of the four sites around theoxygen. Water molecules surrounded by four hydrogenbonds tend to clump together forming clusters, for both

statistical [7] and energetic reasons. Hydrogen bondedchains (i.e. O}H2O}H2O) are cooperative; the break-age of the "rst bond is the hardest, then the next one isweakened, and so on. Thus unzipping may occur withcomplex macromolecules (e.g. nucleic acids) held to-gether by hydrogen bonding.The substantial cooperative strengthening of hydrogen

bonds in water is dependent on long-range interactions[8]. Breaking one bond weakens those around whereasmaking one bond strengthens those around and this,therefore, encourages the formation of larger clusters, forthe same average bond density. The hydrogen-bondedcluster size in water at 03C has been estimated to be 400[9]. A weakly hydrogen-bonding surface restricts thehydrogen-bonding potential of adjacent water so thatthese make fewer and weaker hydrogen bonds. As hydro-gen bonds strengthen each other in a cooperative man-ner, such weak bonding also persists over several layers.Conversely, strong hydrogen bonding will be evident atdistance.

4. Water clustering

Hydrogen bond lifetimes are 1}20ps whereas brokenbond lifetimes are about 0.1 ps. Broken bonds will prob-ably reform to give the original hydrogen bond, parti-cularly if the other surrounding hydrogen bonds are inplace. If not, breakage usually leads to rotation aroundremaining hydrogen bond(s), and not to translationaway. Bond breakage on the covalent side of the hydro-gen bond (dissociation) is a rare event, occurring onlytwice a day; i.e. only once for every 10��� times thehydrogen bond breaks.Hydrogen bonding carries information about solutes

and surfaces over signi"cant distances in liquid water.The e!ect is synergistic, directive and extensive, beingreinforced by additional polarization e!ects and the res-onant intermolecular transfer of O}H vibrational energy[10]. Reorientation of one molecule induces correspond-ing motions in the neighbours. Thus, solute moleculescan &sense' (e.g. a!ect each others' solubility) each other atdistances of several nanometers and surfaces may havee!ects extending to tens of nanometers [11].Water molecules form an in"nite hydrogen-bonded

network with localized and structured clustering. It hasbeen suggested that small clusters of four water molecu-les may come together to form relatively stable wateroctamers that may cluster further to form much largerwater clusters that are able to interlink and tessellatethroughout space (Fig. 1). Such clustering can dynam-ically form both open low density and condensed net-works. The clusters formed can interconvert betweenlower (&0.94 g/ml) and higher (&1 g/ml or greater)density forms by bending, but not necessarily breaking,some of the hydrogen bonds. As the temperature

M.F. Chaplin / Biochemistry and Molecular Biology Education 29 (2001) 54}59 55

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556

57585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111

Fig. 1. Water clusters. (a) A small but relatively stable octamer (H�O)

�, (b) twenty octamers may come together to form an open structure centred on

a water dodecahedron, (c) structure (b) may expand further to contain 280 water molecules forming an icosahedral cluster [12].

increases, the average cluster size, their integrity and theproportion in the low-density form all decrease. Thewater structuring [12] allows explanation of many of theanomalous properties of water including its temper-ature}density and pressure}viscosity behaviour, theradial distribution pattern, the presence of both pentam-ers and hexamers, the change in properties on supercool-ing and the solvation properties of ions, hydrophobicmolecules, carbohydrates and macromolecules.The presence of ions and macromolecular surfaces

a!ects the localized water clustering such that either thelow density or the condensed structures are favoured.For example, water next to a hydrophobic surface tendsto form clathrate-like surfaces that are central to thestructure of low-density clusters (see Fig. 1). Withouthydrogen bonding to the hydrophobic surface, suchclathrate surfaces have no "xed orientation relative to thesurface andmay easily slip (translate) sideways. There aredi!erent (equivalent) ways of describing what happens atsuch hydrophobic surfaces.

� A water molecule at a hydrophobic surface loses thehydrogen bond(s) that would have pointed towardsthat surface. Therefore the water molecules possessincreased enthalpy and compensate for this by doingpressure}volume work, i.e. the network expands toform low-density water with lower entropy.

� Water covers the hydrophobic surface with clathrate-like pentagons in partial dodecahedra, so avoiding theloss of most of the hydrogen bonds. This necessitatesan expanded low-density local structure. The forma-tion of clathrate structures maximizes the van derWaals contacts to the surface whilst retaining maximalhydrogen bonding.

As hydrogen bonding (through donation) is weakenedif one of the donor hydrogen bonds of water is hydrogenbonded to a stronger base than water, charged surfacegroups such as carboxylates and phosphates are expectedto give rise to a particularly weak hydrogen bonds in thenext shell, so encouraging a local collapse in the hydrogenbonded network. Near polyelectrolytes the osmolarity ishigh and water activity and chemical potential are low.The potential of water is partially increased by collapsingthe hydrogen bond network, so giving rise to higherdensity water (HDW). If the surface is highly charged, theHDW zone may reach out to several nanometers and thelocal density of the "rst hydration shell may be greaterthan 1.1 g/ml. The HDW zone is weakly hydrogenbonded, #uid and reactive, and accumulates small ca-tions, multivalent anions and hydrophobic solutes. Inorder to keep the potential of the water constant thewater surrounding this low potential HDW zone is re-duced in potential to match so producing a zone of lowerdensity water (LDW), which may also be extensive. Thesetwo zones (HDW) and (LDW) are unlikely to be sharplydistinguishable or perfectly formed, but the chemicalpotential of the water will be similar throughout showinga shallow gradient from the surface to the bulk. Vicinalwater, near the molecular surfaces but not surface hy-drated, has been found to have properties consistent withpartial conversion to low density water; e.g. reduceddensity (!3%) and raised speci"c heat (#25%), com-pressibility (#20}100%) and viscosity (#200}1100%).

5. Water and protein structure

Hydration is very important for the three-dimensionalstructure and activity of proteins. In solution, proteins

56 M.F. Chaplin / Biochemistry and Molecular Biology Education 29 (2001) 54}59

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556

57585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111

Fig. 2. A chain of 10 water molecules, linking the end of one �-helix(helix 9, 211}227) to the middle of another (helix 11, 272}285) is foundfrom the X-ray di!raction data of glucoamylase-471, a natural pro-teolytic fragment of Aspergillus awamori glucoamylase (data from theBrookhaven Protein Data Bank, structure 1GAH).

possess a conformational #exibility that encompassesa wide range of hydration states not seen in the crystal.There is a tension in the surface between LDW and itsassociated hydrophobic surfaces, and it is this that drivesthe constituent hydrophobic groups to form the hydro-phobic core. In addition, water acts as a lubricant, henceeasing the necessary hydrogen bonding changes. Watermolecules can bridge between the carbonyl oxygen atomsand amide protons of di!erent peptide links to catalysethe formation, and its reversal, of peptide hydrogenbonding. The internal molecular motions in proteins,necessary for biological activity, are very dependent onthe degree of plasticizing which the level of hydrationdetermines.The "rst hydration shell around proteins is ordered,

with high proton transfer rates. It is also 10}20% denserthan the bulk water. Using X-ray analysis of a number ofprotein crystals (which normally contain substantialamounts of water), this water shows a wide range ofnon-random hydrogen-bonding environments and ener-gies. Proteins possess a mixture of polar and non-polargroups. Water is most well ordered round the polargroups, where residence times are longer, than aroundnon-polar groups. Both types of group create order in thewater molecules surrounding them but their ability to dothis and the type of ordering produced are very di!erent.Polar groups are most capable of creating ordered hy-dration through hydrogen bonding and ionic interac-tions. This is energetically most favourable where there isno pre-existing order in the water that requires destruc-tion. The water is slow to exchange, showing the moreviscous dynamic behaviour of bulk (supercooled if below03C) liquid water, 253C colder [13]. Low-density water ispromoted [14] surrounding this dense hydration andpolyelectrolyte double layer. Non-polar groups promotelow-density clathrate structures [15] with greater rota-tional freedom [16] surrounded by denser water. It is nosurprise, therefore that the degree of hydrophobic hy-dration is correlated with the hydration of the polargroups. Under favourable conditions clathrate hydro-phobic hydration may exert pressure on non-polar C}Hbonds pushing them in, so contracting their bond lengthand increasing their vibrational frequency. This &push-ball' hydration [17] should, however, not be thought ofas hydrogen bonding even if the CH2OH

�distances

are suitably close.The water network around the protein links secondary

structures and so determines not only the "ne detail ofthe protein's structure but also explains how particularmolecular vibrations may be preferred (Fig. 2).Protein folding is driven by hydrophobic interactions,

due to the unfavourable entropy decrease caused byforming a large surface area of non-polar groups withwater. Consider a water molecule next to a surface towhich it cannot hydrogen bond. The incompatibility ofthis surface with the low-density water that forms over

such a surface [18] encourages the surface minimizationthat drives the proteins' tertiary structure formation.Compatible solutes (osmolytes), that stabilize this surfacelow-density water, will also stabilize the protein's struc-ture. Such osmolytes may compensate for the disruptinge!ects of high ionic concentrations in some naturalmicroorganisms. Typical amongst them is betaine,(CH

�)�N�CH

�COO�, a very soluble molecule with no

net charge, that favourably interacts with low-densitywater, due to its quaternary nitrogen group, withoutintroducing any locally disruptive micro-osmotic gradi-ents [18].Water is critical, not only for the correct folding of

proteins but also for the maintenance of this structure.The free energy change on folding or unfolding is due tothe combined e!ects of both protein folding/unfoldingand hydration changes. These compensate to such a largeextent that the free energy of stability of a typical proteinis only 40}90 kJ/mol; equivalent to a very few hydrogenbonds. There are both enthalpic and entropic contribu-tions to this free energy that change with temperatureand so give rise to heat denaturation and, in some cases,cold denaturation.Overall, protein stability depends on the balance be-

tween these enthalpic and entropic changes. For globularproteins, the �G of unfolding reaches a maximum at10}303C, decreasing both colder and hotter through zerowith the thermodynamic consequences of both cold andheat denaturation. The hydration of the internal polargroups is mainly responsible for cold denaturation astheir energy of hydration is greatest when cold. Thus, it isthe increased natural structuring of water at lower tem-peratures that causes cold destabilization of proteins insolution. Heat denaturation is primarily due to the in-creased entropic e!ects of the non-polar residues.

M.F. Chaplin / Biochemistry and Molecular Biology Education 29 (2001) 54}59 57

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556

57585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111

Fig. 3. DNA base pairs, showing the extent of hydration in the major and minor grooves.

6. Water and nucleic acid structure

Hydration is very important for the conformation andfunction of nucleic acids. B-DNA requires about 30%, byweight, water to maintain its native conformation in thecrystalline state; partial dehydration leading to denatura-tion. Hydration is greater and more strongly held aroundthe phosphate groups, due to their charged if ratherdi!use electron distribution, but more ordered and morepersistent around the bases with their more directionalhydrogen-bonding ability.Water molecules are held rela-tively strongly, with residence times for the "rst hy-dration shell being about 0.5}1 ns. Because of the regularrepeating structure of DNA, hydrating water is held ina cooperative manner along the double helix in both themajor and minor grooves (Fig. 3). The cooperative natureof this hydration aids both the zipping (annealing) andunzipping (unwinding) of the double helix.Nucleic acids have a number of groups that can hydro-

gen bond to water, with RNA having a greater extent ofhydration than DNA due to its extra oxygen atoms (i.e.ribose O2�) and unpaired base sites. In DNA, the basesare involved in hydrogen-bonded pairings. However,even these groups, except for the hydrogen-bonded ringnitrogen atoms (pyrimidine N3 and purine N1) are ca-pable of one further hydrogen-bonding link to waterwithin the major or minor grooves (see Fig. 3).There may be a spine of hydration running down the

bottom of the B-DNA minor groove particularly wherethere is an A"T duplex [19]. Water molecules hydrogen-bond by donating two hydrogen bonds, so bridging be-tween thymine 2-keto(s) and/ or adenine ring N3(s) insequential opposite strands (not base paired). This wateris fully hydrogen bonded by accepting two further hydro-

gen bonds from secondary hydration water. This hy-dration may occur regularly down the minor grooveconnecting strands but any cooperative e!ect is throughthe secondary hydration. These primary hydration watermolecules exchange slower than any other water hydrat-ing the DNA. Such a spine of hydration may be impor-tant in stabilizing the B-DNA [20]. The A"T basepairing produces the narrower minor groove and morepronounced spine of hydration, whereas the G,C basepairing produces a wider minor groove with more exten-sive hydration, due in part to the 50% greater hydrationsites. Such solvent interactions are key to the hydrationenvironment, and hence its recognition, around the nu-cleic acids and directly contributes to the DNA confor-mation; B-DNA, possessing higher phosphate hydration,less exposed sugar residues and smaller hydrophobicsurface, is stabilized at high water activity whereas A-DNA, with its shared inter-phosphate water bridges, ismore stable at low water activity.

7. Conclusions

Water should never be assumed to be just an inertdiluent in biochemical processes. It plays an intimatepart in determining the structure and reactions of macro-molecules and its own structuring can inform other pro-cesses over signi"cant distances. Water should be givengreater prominence in both research and teaching. Weshould always be alert to the central role that water playsin the rich diversity of biological processes

A more detailed description of the structure andproperties of water, including explanation of its anomal-ous properties, the Hofmeister series, interaction with

58 M.F. Chaplin / Biochemistry and Molecular Biology Education 29 (2001) 54}59

12345678910111213141516

171819202122232425262728293031

polysaccharides, Chime interactive "gures and many rel-evant references is given at http://www.sbu.ac.uk/water/.

References

[1] R.B. Martin, J. Chem. Education 65 (1988) 668}670.[2] M. Laing, J. Chem Education 64 (1987) 124}128.[3] L. Pauling, The Nature of the Chemical Bond, 2nd Edition,

Cornell University Press, New York, 1948.[4] E.D. Isaacs, A. Shukla, P.M. Platzman, D.R. Hamann, B. Barbiel-

lini, C.A. Tulk, J. Phys. Chem. Solids 61 (2000) 403}406.[5] C.N.R. Rao, Theory of hydrogen bonding in water, in: F. Franks

(Ed.), Water, a comprehensive treatise, Vol. 1, Plenum Press, NewYork, 1972, pp. 93}114.

[6] E. Espinosa, E. Molins, C. Lecomte, Chem. Phys. Lett. 285 (1998)170}173.

[7] H.E. Stanley, J. Teixeira, J. Chem. Phys. 73 (1980) 3404}3422.[8] M.I. Heggie, C.D. Latham, S.C.P. Maynard, R. Jones, Chem.

Phys. Lett. 249 (1996) 485}490.[9] W.A.P. Luck, J. Mol. Struct. 448 (1998) 131}142.[10] S. Woutersen, H.J. Bakker, Nature 402 (1999) 507}509.[11] D.P. Shelton, Chem. Phys. Lett. 325 (2000) 513}516.[12] M.F. Chaplin, Biophys. Chem. 83 (2000) 211}221.[13] M.-C. Bellissent-Funel, J. Mol. Liquids 84 (2000) 39}52.[14] G.W. Robinson, C.H. Cho, Biophys. J. 77 (1999) 3311}3318.[15] G.E. Walrafen, Y-C. Chu, Chem. Phys. 258 (2000) 427}446.[16] K.D. Collins, M.W. Washabaugh, Q. Rev. Biophys. 18 (1985)

323}422.[17] K. Mizuno, Y. Kimura, H. Morichika, Y. Nishimura, S. Shimada,

S. Maeda, S. Imafuji, T. Ochi, J. Mol. Liquids 85 (2000) 139}152.[18] P.M. Wiggins, Physica A 238 (1997) 113}128.[19] V.P. Denisov, G. CarlstroK m, K. Venu, B. Halle, J. Mol. Biol. 268

(1997) 118}136.[20] M. Feig, B.M. Pettitt, J. Mol. Biol. 286 (1999) 1075}1095.

M.F. Chaplin / Biochemistry and Molecular Biology Education 29 (2001) 54}59 59