ELSEVIER Biochemistry and Molecular Biology Education 29 (2001) 54-59

BIOCHEMISTRY and

MOLECULAR BIOLOGY EDUCATION

www .elsevier.corn/locate/bambed

Water: its importance to life Martin F. Chaplin"

School of Applied Science, South Bank University, 103 Borough Road, London SEI 0AA. UK

Abstract

Textbooks increasingly include material concerning the importance of water but this topic is often treated over-simplistically with insufficient attention being given to the central position of water in life processes. In this article, modern views of the fundamental role that water plays in biochemical function and process are summarized. The importance of water in the structures of nucleic acids and proteins is explained. (0 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, water is the most remarkable substance. We wash in it, fish in it, swim in it, drink it and cook with it, although probably not all at the same time. We are about two-thirds water and require water to live. Life as we know it could not have evolved without water and dies without it. Droughts cause famines and floods cause death and disease. Because of its clear importance, water is the most studied material on Earth. It comes as a surprise, there- fore, to find that it is so poorly understood, not only by people in general, but also by scientists working with it everyday.

Textbooks are including increasing amounts of mater- ial concerning water. However, this material is usually concentrated in one chapter and its importance is rarely emphasized elsewhere. Chapters on proteins and nucleic acids, for example, often discuss structural and functional details of these macromolecules with little prominence given to the pervasive effects of the surrounding water. Other areas, such as metabolism, often ignore the many functions of water altogether. This lack of emphasis, evidenced in part from the textbooks' indexes, results from the multidisciplinary nature of the water literature and the difficulty in bringing together the wide but often thinly-spread information available.

Water seems, at first sight, to be a very simple molecu- le, consisting of two hydrogen atoms attached to an

oxygen atom and indeed, few molecules are smaller. Its size, however, belies the complexity of its properties, and these properties seem to fit ideally into the requirements for carbon-based life as can no other molecule.

Organisms consist mostly of liquid water, which per- forms many functions and should never be considered simply as an inert diluent. Nevertheless, in spite of much work many of the properties of water are puzzling. It has often been stated that life depends on the anomalous properties of water. In particular, the large heat capacity and high water content in organisms contribute to ther- mal regulation and prevent local temperature fluctu- ations. The high latent heat of evaporation gives resistance to dehydration and considerable evaporative cooling. Water is an excellent solvent due to its polarity, high dielectric constant and small size, particularly for polar and ionic compounds and salts. Indeed its solva- tion properties are so impressive that it is difficult to obtain really pure water. Water ionises and allows easy proton exchange between molecules, so contributing to the richness of the ionic interactions in biology. The structuring of water around molecules allows them to sense and be sensed at a distance. The unique hydration properties of water towards biological macromolecules (particularly proteins and nucleic acids) to a large extent determine their three-dimensional structures, and hence their functions, in solution.

2. Structure

*Td: + 44-207-815-7970; fax: + 44-207-815-7999. E-nioil address: chaplimf@sbu.ac.uk (M.F. Chaplin).

Water has the molecular formula H 2 0 but the hydrogen atoms are constantly exchanging due to

1470-X 175/01/$20.00 0 2001 IUBMB. Published by Elsevier Science Ltd. All rights reserved. PII: S 1 4 7 0 - 8 1 7 5 ( 0 1 ) O O O 1 7 - 0

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

protonation/deprotonation processes. Both acids and bases catalyse this process. Even when this proton ex- change is at its slowest (at pH 7), the average time that a water molecule (H,O) exists between gaining or losing a proton is only about a millisecond. As this brief period is, however, much longer than the timescales encountered during investigations into water's hydrogen bonding or hydration properties, the water molecule is usually treated as a permanent structure.

Water molecules are often described in school and undergraduate textbooks as having four, approximately tetrahedrally arranged, sp3-hybridized electron pairs, two of which are associated with hydrogen atoms plus the two remaining lone pairs. Ab initio electron density calculations, however, do not confirm the presence of significant directed electron density where lone pairs are expected. Although there is no apparent consensus of opinion [ 11, such descriptions of substantial sp3-hybrid- ized lone pairs in the water molecules should perhaps be avoided "1. In spite of this, the normal stereochemistry around the oxygen atom in water is approximately tet- rahedral due to hydrogen bonding.

3. Hydrogen bonding

Hydrogen bonding occurs when an atom of hydrogen is attracted by rather strong forces to two atoms instead of only one, so that it may be considered to be acting as a bond between the two atoms [3]. Typically this occurs between oxygen and/or nitrogen atoms, but is also found elsewhere, such as between fluorine atoms in HF,. In water the hydrogen atom is covalently attached to the oxygen of a water molecule (bond enthalpy about 470 kJ/mol) but has an additional attraction (about 23 kJ/mol) to a neighbouring oxygen atom of another water molecule. The hydrogen bond is part (about 90%) electrostatic and part (about 10%) covalent [4]. The bond strength depends on its length and angle. However, small deviations from linearity in the bond angle (up to 20") have a relatively minor effect [5]. The dependency on bond length is very important and has been shown to decay exponentially with distance [6]. There is a trade-off between the covalent and hydrogen bond strengths: the stronger is the H - - - O hydrogen bond, the weaker is the 0-H covalent bond, and the shorter is the 0 ... 0 distance. If the hydrogen bond is substantially bent then it follows that the bond strength is weaker and the two water oxygen atoms will generally be further apart.

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 hydrogen bond is located at each of the four sites around the oxygen. Water molecules surrounded by four hydrogen bonds tend to clump together forming clusters, for both

statistical [7] and energetic reasons. Hydrogen bonded chains (i.e. 0 -H ... 0-H ... 0) are cooperative; the break- age of the first bond is the hardest, then the next one is weakened, and so on. Thus unzipping may occur with complex 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 [S]. Breaking one bond weakens those around whereas making one bond strengthens those around and this, therefore, encourages the formation of larger clusters, for the same average bond density. The hydrogen-bonded cluster size in water at 0°C has been estimated to be 400 [9]. A weakly hydrogen-bonding surface restricts the hydrogen-bonding potential of adjacent water so that these 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 at distance.

4. Water clustering

Hydrogen bond lifetimes are 1-20 ps whereas broken bond 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 in place. If not, breakage usually leads to rotation around remaining hydrogen bond@), and not to translation away. Bond breakage on the covalent side of the hydro- gen bond (dissociation) is a rare event, occurring only twice a day; i.e. only once for every times the hydrogen bond breaks.

Hydrogen bonding carries information about solutes and surfaces over significant distances in liquid water. The effect is synergistic, directive and extensive, being reinforced by additional polarization effects and the res- onant intermolecular transfer of 0 - H vibrational energy [lo]. Reorientation of one molecule induces correspond- ing motions in the neighbours. Thus, solute molecules can 'sense' (e.g. affect each others' solubility) each other at distances of several nanometers and surfaces may have effects extending to tens of nanometers [ll].

Water molecules form an infinite hydrogen-bonded network with localized and structured clustering. It has been suggested that small clusters of four water molecu- les may come together to form relatively stable water octamers that may cluster further to form much larger water clusters that are able to interlink and tessellate throughout space (Fig. 1). Such clustering can dynam- ically form both open low density and condensed net- works. The clusters formed can interconvert between lower ( - 0.94g/ml) and higher ( - 1 g/ml or greater) density forms by bending, but not necessarily breaking, some of the hydrogen bonds. As the temperature

56 M.F. Chaplin J Biochernisty and Molecular Biology Education 29 (2001) 54-59

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 the proportion in the low-density form all decrease. The water structuring [12] allows explanation of many of the anomalous properties of water including its temper- at ure-density and pressure-viscosity behaviour, the radial distribution pattern, the presence of both pentam- ers and hexamers, the change in properties on supercool- ing and the solvation properties of ions, hydrophobic molecules, carbohydrates and macromolecules.

The presence of ions and macromolecular surfaces affects the localized water clustering such that either the low density or the condensed structures are favoured. For example, water next to a hydrophobic surface tends to form clathrate-like surfaces that are central to the structure of low-density clusters (see Fig. 1). Without hydrogen bonding to the hydrophobic surface, such clathrate surfaces have no fixed orientation relative to the surface and may easily slip (translate) sideways. There are different (equivalent) ways of describing what happens at such hydrophobic surfaces.

0 A water molecule at a hydrophobic surface loses the hydrogen bond(s) that would have pointed towards that surface. Therefore the water molecules possess increased enthalpy and compensate for this by doing pressure-volume work, i.e. the network -expands to form low-density water with lower entropy.

0 Water covers the hydrophobic surface with clathrate- like pentagons in partial dodecahedra, so avoiding the loss of most of the hydrogen bonds. This necessitates an expanded low-density local structure. The forma- tion of clathrate structures maximizes the van der

As hydrogen bonding (through donation) is weakened if one of the donor hydrogen bonds of water is hydrogen bonded to a stronger base than water, charged surface groups such as carboxylates and phosphates are expected to give rise to a particularly weak hydrogen bonds in the next shell, so encouraging a local collapse in the hydrogen bonded network. Near polyelectrolytes the osmolarity is high and water activity and chemical potential are low. The potential of water is partially increased by collapsing the hydrogen bond network, so giving rise to higher density water (HDW). If the surface is highly charged, the HDW zone may reach out to several nanometers and the local density of the first hydration shell may be greater than l.lg/ml. The HDW zone is weakly hydrogen bonded, fluid and reactive, and accumulates small ca- tions, multivalent anions and hydrophobic solutes. In order to keep the potential of the water constant the water surrounding this low potential HDW zone is re- duced in potential to match so producing a zone of lower density water (LDW), which may also be extensive. These two zones (HDW) and (LDW) are unlikely to be sharply distinguishable or perfectly formed, but the chemical potential of the water will be similar throughout showing a shallow gradient from the surface to the bulk. Vicinal water, near the molecular surfaces but not surface hy- drated, has been found to have properties consistent with partial conversion to low density water; e.g. reduced density ( - 3%) and raised specific heat ( + 25%), com- pressibility ( + 20-100%) and viscosity ( + 200-1 100%).

5. Water and protein structure

Waals contacts to the surface whilst retaining maximal hydrogen bonding.

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

M.F. Chaplin 1 Biochemistry and Molecular BiologV Education 29 (2001) 54-59 57

possess ;I conformational flexibility that encompasses a wide range of hydration states not seen in the crystal. There is a tension in the surface between LDW and its associated hydrophobic surfaces, and it is this that drives the constituent hydrophobic groups to form the hydro- phobic core. In addition, water acts as a lubricant, hence easing the necessary hydrogen bonding changes. Water molecules can bridge between the carbonyl oxygen atoms and amide protons of different peptide links to catalyse the formation, and its reversal, of peptide hydrogen bonding. The internal molecular motions in proteins, necessary for biological activity, are very dependent on the degree of plasticizing which the level of hydration determines.

The first hydration shell around proteins is ordered, with high proton transfer rates. It is also 10-20% denser than the bulk water. Using X-ray analysis of a number of protein crystals (which normally contain substantial amounts of water), this water shows a wide range of non-random hydrogen-bonding environments and ener- gies. Proteins possess a mixture of polar and non-polar groups. Water is most well ordered round the polar groups. where residence times are longer, than around non-polar groups. Both types of group create order in the water molecules surrounding them but their ability to do this and the type of ordering produced are very different. Polar groups are most capable of creating ordered hy- dration through hydrogen bonding and ionic interac- tions. This is energetically most favourable where there is no pre-existing order in the water that requires destruc- tion. The water is slow to exchange, showing the more viscous dynamic behaviour of bulk (supercooled if below 0°C) liquid water, 25°C colder [13]. Low-density water is promoted [ 141 surrounding this dense hydration and polyelectrolyte double layer. Non-polar groups promote low-density clathrate structures [ 151 with greater rota- tional freedom [ 161 surrounded by denser water. It is no surprise, therefore that the degree of hydrophobic hy- dration is correlated with the hydration of the polar groups. Under favourable conditions clathrate hydro- phobic hydration may exert pressure on non-polar C-H bonds pushing them in, so contracting their bond length and increasing their vibrational frequency. This ‘push- ball’ hydration [17] should, however, not be thought of as hydrogen bonding even if the CH ... OH2 distances are suitably close.

The water network around the protein links secondary structures and so determines not only the fine-detail of the protein’s structure but also explains how particular moleculnr vibrations may be preferred (Fig. 2).

Protein folding is driven by hydrophobic interactions, due to the unfavourable entropy decrease caused by forming a large surface area of non-polar groups with water. Consider a water molecule next to a surface to which it cannot hydrogen bond. The incompatibility of this surface with the low-density water that forms over

Fig. 2. A chain of 10 water molecules, linking the end of one a-helix (helix 9, 21 1-227) to the middle of another (helix 11, 272-285) is found from the X-ray diffraction data of glucoamylase-471, a natural pro- teolytic fragment of Aspergillus awamori glucoamylase (data from the Brookhaven Protein Data Bank, structure 1GAH).

such a surface [ 181 encourages the surface minimization that drives the proteins’ tertiary structure formation. Compatible solutes (osmolytes), that stabilize this surface low-density water, will also stabilize the protein’s struc- ture. Such osmolytes may compensate for the disrupting effects of high ionic concentrations in some natural microorganisms. Typical amongst them is betaine, (CH3)3N+CH2COO-, a very soluble molecule with no net charge, that favourably interacts with low-density water, due to its quaternary nitrogen group, without introducing 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 to the combined effects of both protein folding/unfolding and hydration changes. These compensate to such a large extent that the free energy of stability of a typical protein is only 40-90 kJ/mol; equivalent to a very few hydrogen bonds. There are both enthalpic and entropic contribu- tions to this free energy that change with temperature and 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 globular proteins, the AG of unfolding reaches a maximum at 10-30°C, decreasing both colder and hotter through zero with the thermodynamic consequences of both cold and heat denaturation. The hydration of the internal polar groups is mainly responsible for cold denaturation as their energy of hydration is greatest when cold. Thus, it is the increased natural structuring of water at lower tem- peratures that causes cold destabilization of proteins in solution. Heat denaturation is primarily due to the in- creased entropic effects of the non-polar residues.

58 M.F. Chapiin J Biochemistty and Molecular Biology Education 29 (2001) 54-59

I I I I i '

/ ..

, I Minor groove \oHH

/ H-0

H H O \ H

Fig. 3. DNA base pairs, showing the extent

6. Water and nucleic acid structure

Hydration is very important for the conformation and function of nucleic acids. B-DNA requires about 30%, by weight, water to maintain its native conformation in the crystalline state; partial dehydration leading to denatura- tion. Hydration is greater and more strongly held around the phosphate groups, due to their charged if rather diffuse electron distribution, but more ordered and more persistent around the bases with their more directional hydrogen-bonding ability. Water molecules are held rela- tively strongly, with residence times for the first hy- dration shell being about 0.5-1 ns. Because of the regular repeating structure of DNA, hydrating water is held in a cooperative manner along the double helix in both the major and minor grooves (Fig. 3). The cooperative nature of this hydration aids both the zipping (annealing) and unzipping (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 of hydration than DNA due to its extra oxygen atoms (i.e. ribose 02 ' ) and unpaired base sites. In DNA, the bases are involved in hydrogen-bonded pairings. However, even these groups, except for the hydrogen-bonded ring nitrogen atoms (pyrimidine N3 and purine N1) are ca- pa ble of one further hydrogen-bonding link to water within 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 where there 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) in sequential opposite strands (not base paired). This water is fully hydrogen bonded by accepting two further hydro-

of hydration in the major and minor grooves.

gen bonds from secondary hydration water. This hy- dration may occur regularly down the minor groove connecting strands but any cooperative effect is through the secondary hydration. These primary hydration water molecules 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 base pairing produces the narrower minor groove and more pronounced spine of hydration, whereas the G=C base pairing produces a wider minor groove with more exten- sive hydration, due in part to the 50% greater hydration sites. Such solvent interactions are key to the hydration environment, 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 hydrophobic surface, is stabilized at high water activity whereas A- DNA, with its shared inter-phosphate water bridges, is more stable at low water activity.

7. Conclusions

Water should never be assumed to be just an inert diluent in biochemical processes. It plays an intimate part in determining the structure and reactions of macro- molecules and its own structuring can inform other pro- cesses over significant distances. Water should be given greater prominence in both research and teaching. We should always be alert to the central role that water plays in the rich diversity of biological processes

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

M.F. Chaplin Biorhemistly and Molecular Biology Education 29 (ZOOI) 54-59 59

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

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