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Water: its importance to life

Article  in  Biochemistry and Molecular Biology Education · March 2001

DOI: 10.1111/j.1539-3429.2001.tb00070.x

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 BIOCHEMISTRY
and
MOLECULAR BIOLOGY
EDUCATION
ELSEVIER Biochemistry and Molecular Biology Education 29 (2001) 54-59
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. (02001 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. Water has the molecular formula H 2 0 but the
E-nioil address: chaplimf@sbu.ac.uk (M.F. Chaplin). 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
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 - - - Ohydrogen 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
55
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
Waals contacts to the surface whilst retaining maximal
hydrogen bonding.
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
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
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
57
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'
..
H-0
/
I
,
/
Minor groove
HHO\
H
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 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 0 2 ' ) 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-
\oHH
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
polysaccharides, Chime interactive figures and many rel-
evant references is given at http://www.sbu.ac.uk/water/.
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