New Age of water

Entire biochemistry and cell biology textbooks will have to be rewritten on how water in the
cell and extracellular matrix is stage-managing the drama of life. This continues the exclusive
series started in SiS 23.
http://www.i-sis.org.uk/TIOCW.php

The Importance of Cell Water

Whats the Cell Really Like?
Whats the Bacterium Really Like?

The Importance of Cell Water

Prof. Martin Chaplin presents a new theory on the structure of water in the cell that switches
between low-density and high-density clusters
Although we understand much of what goes on inside cells at a molecular level, we don't
know how all the molecules can work together as a whole. Much useful biochemistry has
been discovered using dilute preparations from homogenised dead cells, but living cells are
very different, and contain more concentrated solutes and more organised proteins. Indeed,
test tube experiments may mislead us, and it should come as no surprise to find that living
cells possess characteristics that are very much more than the sum of their parts.
The study of the live cell is fraught with difficulty, as most procedures change it from its
native state. The key to understanding the cell comes from acknowledging the one constituent
that has often been ignored: water. The significance of water for the cell becomes clear when
we seek to solve big puzzles, such as 'How are potassium ions able to maintain a high
concentration inside cells whereas sodium ions are found mainly outside?' and 'How do cells
remain functional even when large holes are made in their surface membranes?'
There are at least four views as to how the water inside the cell affects its function:
1. The water mostly acts as an uncomplicated
environment for the cellular processes, which
are determined by the structure of the
macromolecules only. Although this view
seems the one most promoted in current
textbooks by default, it is rapidly losing
favour due to its inability to explain natural
processes.
2. The water forms polarised multi-layers over
extended protein surfaces, as proposed for
many years by Gilbert Ling [1]. There is

much experimental support for the

foundations of this theory but little
experimental support for the required
structural changes in the proteins or the
involvement of extended protein surfaces, as
proposed.
3. The water is involved in intracellular changes
between 'sol' and 'gel' states as more recently
promoted by Gerald Pollack [2]. This is an
interesting and useful idea but without a clear
molecular mechanism.
4. The water actively changes the density of its
hydrogen bonded structuring to enable
diverse intracellular processes, in a manner
compatible with the basic ideas of both
Gilbert Ling and Gerald Pollack.
The theory that I shall describe in this article (which I presented at the Gordon Research
Conference on Interfacial Water and Cell Biology in June 2004) belongs to the fourth, new
category. I propose that changes in the density and clustering of intracellular water are
modulated by the mobility of key proteins, which in turn are controlled by the energy status
and ionic content of the cell.

The nature of water

Water possesses many properties that seem strange, or anomalous [3]. Some of these, such as
its high melting and boiling points can be simply explained as due to water's hydrogen bonded
clustering. Over the last 10 years, a broad range of evidence has accumulated concerning a
two-state structuring within liquid water, which can explain many of the remaining anomalies
[4, 5]. This theory involves the presence in liquid water, of clusters with a lower density
comparable with that of ice. The water molecules in such clusters flicker between partners as
their hydrogen bonds are constantly making and breaking. Over a long time scale, they appear
as favoured arrangements. These low-density water clusters do not consist of ice-like crystals,
due to their lack of long-range order, but they do contain water molecules linked by hydrogen
bonds in an expanded, 4-coordinated tetrahedral arrangement. At the smallest scale, the water
may be thought of as an equilibrium between two water tetramers (see Fig. 1): structure A,
held closely by non-bonded interactions, forming a more dense structure, and structure B,
with molecules held further away and linked by hydrogen bonds to form a less dense structure
There is little difference in energy between the structures A and B, so the equilibrium is easily
affected by the presence of solutes and surfaces. An increase in temperature or pressure will
shift the equilibrium to the left.

Figure 1. Equilibrium between two water tetramers.

Although the natural structuring in water at ordinary temperatures tends towards the
'collapsed' structure A, the low density structure B can grow to form larger non-crystalline
clusters based on dodecahedral (12-sided) water cluster cores, similar to those found in the
crystalline 'clathrate hydrates'; as for example, the extensive icosahedral (H2O)280 aggregate
built up from tetrahedrally hydrogen-bonded water molecules surrounding a dodecahedron
made up of 20 water molecules, the basic clathrate cage (Fig. 2).

Figure 2. Extensive icosahedral (H2O)280 structure of water built up from tetrahedrally

hydrogen-bonded water molecules.

Intracellular water contains lower density water with more potassium ions
The differences in intracellular and extracellular environments of cells is primarily due to the
extensive surface area and high intracellular concentration of solutes that promote the lowdensity clustering of water and restricted diffusion inside cells. The extensive surface of
cellular membranes (e.g., each liver cell contain ~100 000 m2 membrane surface area)
favours the formation of low-density water inside cells, as the membrane lipids contain
hydrophilic head groups that encourage this organization of the associated interfacial water.
Other surfaces attract the water, so stretching the hydrogen-bonded water contained by the
confined spaces within the cells.
The difference in ionic concentrations is particularly evident in sodium (Na+; intracellular, 10
mM; extracellular, 150 mM) and potassium (K+; intracellular, 159 mM; extracellular, 4 mM).
Na+ ions create more broken hydrogen bonding and prefer a high aqueous density, whereas
K+ ions prefer a low-density aqueous environment, as proven by Philippa Wiggins [6]. The
differences in intracellular and extracellular distributions of potassium and sodium are due to
differences in the affinity of these ions for water. The interactions between water and Na+ are
stronger than those between water molecules, which are in turn stronger than those between
water and K+ ions, all due to the differences in surface charge density of the ions - that of the
smaller Na+ ion being nearly twice that of K+ ions. Ca2+, with an intracellular concentration
0.1 M and an extracellular concentration of 2.5 mM, has a surface charge density more than
twice that of Na+, and has even stronger destructive effects on low-density hydrogen-bonding
than Na+ ions.
Other studies confirm the preference of K+ ions for low-density water over Na+ ions. The ions
partition according to their preferred aqueous environment; in particular, the K+ ions are
preferred within the intracellular environment and naturally accumulate inside the cells at the
expense of Na+ ions. This process occurs simply as a result of the water structuring without
the help of putative ion-pumps in the cell membrane.
Besides, membrane ion-pumps cannot produce these large differences in ionic composition,
simply because the (ATP) energy required far exceeds the energy available to the cell. Also,
many studies, as for example, the extensive series carried out by Gilbert Ling, have shown
that cells do not need an intact membrane or active energy (ATP) production to maintain the
ionic concentration gradients.

The effect of intracellular protein on water structuring

The degree to which the density of cell water is lowered is determined by the solutes and the
state of motion of protein. Water has conflicting effects in the mixed environments around
proteins due to the variety of amino acids making up their surfaces. Weak interactions
between the protein and surface water molecules allow greater protein flexibility. Strong
interactions endow the protein with greater stability and solubility.
There is generally an ordered structure in the layer of water molecules immediately
surrounding the protein, with both hydrophobic clathrate-like and hydrogen bonded water
molecules each helping the other to optimize water's hydrogen bonding network. Protein
carboxylate groups are generally surrounded by strongly hydrogen-bonded water whereas the
water surrounding the basic groups arginine, histidine and lysine tends towards a more-open
clathrate structuring. The formation of partial clathrate cages over hydrophobic areas

maximizes non-bonded interactions between the water and the protein without loss of
hydrogen bonds between the water molecules whereas carboxylate groups usually only fit a
collapsed water structure (see below) creating a reactive fluid zone.
The rotation of the proteins will cause changes in the water structuring outside this closest
hydration shell. At the breaking surface, hydrogen bonds are ruptured, creating a zone of
higher density water. Protein rotation thus creates a surrounding high-density water zone with
many broken hydrogen bonds.

The importance of protein carboxylate groups

Protein has two acidic amino acids, aspartate and glutamate, with carboxylate (-CO2-) side
chains. Normally, aqueous hydrogen bonding to these carboxylate oxygen atoms both attracts
water molecules causing a localised high density water clustering and reduces the acidity of
the carboxylic acids. Otherwise, when the surrounding water molecules prefer to hydrogen
bond to themselves as with the formation of a clathrate cage, the acidity of the carboxylate
groups is increased. It is found that Na+ ions prefer binding to the weaker carboxylic acids
whereas K+ ions prefer the stronger acids [1].
Na+ and K+ ions also behave differently when close to the carboxylate groups; K+ ions have a
preference for forming ion pairs, where there is direct contact between the K+ and carboxylate
ions, whereas Na+ ions form solvent separated pairings where water molecules lie between the
Na+ and carboxylate ions, forming strengthened hydrogen bonds to the carboxylate groups
[7]. This is due to the Na+ ions holding on to their water more strongly. The K+ ions prefer to
be within a clathrate water cage and this preference both reinforces its direct ion pairing to the
carboxylate group and discourages aqueous hydrogen bonding to the associated carboxylate
groups.
The direct association of K+ ions with the aspartate and glutamate groups in proteins is the
central theme of Ling's fixed charge hypothesis where evidence for the molecular mechanism
for the association includes (1) the low intracellular electrical conductance, (2) the strongly
reduced mobility of intracellular K+ ions, (3) the one to one stoichiometric absorption of K+
ions to the carboxylate groups and (4) identification of the K+ ion absorption sites as the
aspartate and glutamate side chains of the intracellular proteins.

The importance of protein mobility

Actin is a highly conserved and widespread eukaryotic protein (42-43 kDa) responsible for
many functions in cells. Non-muscle cells contain actin in amounts 5-10% of all protein,
whereas muscle cells contain about 20%. Actin is converted between a freely rotating
monomer molecule (G-actin; about 4 - 6 nm diameter) and a static right-handed double helical
polymer protein filament (F-actin; up to several microns in length) by ATP; a process
involving the conversion of an -helix to a -turn in one of its structural domains. Each
molecule of the freely rotating G-actin can stir a large volume of water, whereas F-actin has a
much more ordered structure so creating more order in its surrounding water. The protein
fibres trap water, reducing its movement and compensated by greater hydrogen bonding.
Also, capillary action stretches the confined water, so ensuring that it is of lower density and
hence more highly structured than the bulk water.

All actin molecules contain a conserved negatively charged N-terminus, for example the Nacetyl-aspartyl-glutamyl-aspartyl-glutamyl sequence in rabbit muscle -actin. When G-actin
polymerises in the cell under the action of ATP to form F-actin, this highly carboxylated
antenna is placed on the exposed outer edge of the helix, where it may be additionally used as
a binding site for other proteins, such as myosin. Tubulin, another intracellular structural
protein that forms immobile structures within cells, possesses an even more extensive
negatively charged acidic C-terminal conserved antenna of about eight carboxylate groups
that serves similar functions.
F-actin's multiply negatively charged N-terminus attracts positively charged cations. Under
conditions when the carboxylic acids are weaker, both K+ and Na+ ions may form solvent
separated species. This competition results in a preference for Na+ ions and high-density
water. However, the natural rotation of the protein will tend to sweep such ions, and their
associated water, away. If the protein is prevented from rotating, Na+ ions tend to destroy any
low density structuring around carboxylate groups of the protein. However, the intracellular
Na+ ion concentration is generally far lower than that of K+ ions, which allows K+ ions to
compete successfully for these sites, forming ion pairs and encouraging clathrate formation.

Cooperative conversion of the water structuring

Binding of K+ ions by the carboxylate groups lowers the ionic strength of the intracellular
solution. As this ionic strength decreases, the acidity of phosphate groups decreases, resulting
in the conversion of the intracellular doubly charged HPO42- ions to the singly charged H2PO4ions, more favourable to low density water clustering. All intracellular phosphate entities will
behave similarly. The cooperative effects of the change between static filament formation and
freely diffusional protein are summarized in Fig. 3.

Figure 3. A summary of the cooperative effects when mobile proteins such as actin are
polymerised,
6

Formation of K+-carboxylate ion pairs leads to the formation of a surrounding clathrate water
structuring that further guides icosahedral water structuring (so ensuring maximal hydrogenbond formation) and informing neighbouring carboxylate groups. This signalling
cooperatively reinforces the tetrahedrality of the water structuring found between these
groups. The clathrate cage allows rotational mobility (like a ball-and-socket joint), enabling
the hydrogen bonding to search out cooperative partners (Fig. 4).

Figure 4. This diagram shows the clustering around two K+-carboxylate ion pairs (about 4
nm apart) as may be attached to part of two protein's structures. There are 7-8 shells of water
around each surface as is typically found between intracellular proteins. The K+ ions are
shown as violet and the water network is shown as linked (i.e. hydrogen bonded) oxygen
atoms (shown red) without showing their associated hydrogen atoms. The hydrogen bonding
initially forms clathrate cages around the ion pairs, followed by a more extensive icosahedral
arrangement. This is then followed by extension of the hydrogen bonding along 'rays'
connecting the neighbouring sites. Once these 'rays' link, the hydrogen bonding of each
reinforces the other in a cooperative manner, so strengthening the linkage and reinforcing the
overall low density aqueous environment. As the aqueous clathrate cage possesses a more
negative charge on its interior and a more positive charge on the outside, there is a marked
polarization in the water molecules that reinforces the hydrogen bonding interactions.
Although the clustering involves a major drop in aqueous mobility, the stronger 4-coordinated
bonding compensates this. This theory offers a molecular explanation for Ling's associationinduction polarized multilayer model (see "Strong medicine needed in cell biology", this
issue). The initial icosahedral size (3 nm diameter), surrounding each ion pair, also equals the
water domain size proposed by John Watterson. The tetrahedral structuring possesses fivefold symmetry, which prevents easy freezing in line with the pronounced supercooling found
for intracellular water.

Extension of the clathrate network and its associated low density water enables K+ ion
binding to all aspartic and glutamic acid groups, not just the key ones within the crucial Nterminal acidic centres. Thus, the sol-gel transition of Pollack (see "Biology of least action",
SiS 18) may be interpreted as due to the formation of low density water clustering (the gel
state) due to clathrate clustering around K+-carboxylate ion pairs.
In the presence of raised levels of Na+ and/or Ca2+ ions, as occasionally occurs during some
cell functions, these ions will replace some of the bound K+ ions. These newly formed solvent
separated Na+ and/or Ca2+ ion pairings destroy the low-density clathrate structures and initiate
a cooperative conversion of the associated water towards a denser structuring.

Conclusion
In conclusion, the aqueous information transfer within the cell involves the following:

Intracellular water favours K+ ions over Na+

ions.
Freely rotating proteins create zones of higher
density water, which tend towards a lower
density clustering if the rotation is prevented.
Static charge-dense intracellular
macromolecular structures prefer K+ ion pairs
to freely soluble K+ ions.
Ion paired K+-carboxylate groupings prefer
local clathrate water structuring.
Clathrate water prefers local low density
water structuring.
Low density water structuring can reinforce
the low-density character of neighbouring site
water structuring.
Na+ and Ca2+ ions can destroy the low density
structuring in a cooperative manner.

Article first published 13/10/04

References
1. Ling GN. Life at the cell and below-cell level.
The hidden history of a functional revolution
in Biology, Pacific Press, New York, 2001.
2. Pollack GH. Cells, gels and the engines of
life; a new unifying approach to cell function,
Ebner and Sons Publishers, Washington,
2001.
3. Detailed descriptions and references on my
website
http://www.lsbu.ac.uk/water/anmlies.html).

4. Chaplin MF, A proposal for the structuring of

water, Biophys. Chem. 2000, 83, 211-21.
5. Urquidi J, Singh S, Cho CH and Robinson
GW, Temperature and pressure effects on the
structure of liquid water, J. Mol. Struct. 1999,
485-486, 363-71.
6. Wiggins PM. Water in complex environments
such as living systems, Physica A 2002, 314,
485-91.
7. Collins KD. Charge density-dependent
strength of hydration and biological structure,
Biophys. J. 1997, 72, 65-76.
Martin Chaplin is Professor of Applied Science, London South Bank University, UK, with
special interests in the interactions between water and biological molecules.

What's the Cell Really Like?

It takes life-long commitment, profound knowledge and artistry to show the world what the
cell is really like. Dr. Mae-Wan Ho reports
In quest for the secret of life of cells, generations of biologists have dedicated their own lives
to finding ways of fixing and freezing tissues so that the structure of cells can be preserved as
close to their living state as possible.
But the living 'state' is not a static configuration of structures, but a dynamic process in which
structures are constantly changing, constantly being broken down and reformed. And no
matter how perfectly preserved, a fixed, frozen section of a cell, like a good photograph of a
person, can give no more than an instantaneous snapshot of its life-process.
While we have no difficulty in telling a good snapshot of a person from a bad one - especially
if we already know something about the life of the person - there are considerable problems in
sorting out actual structures from artefacts of preservation in the case of the cell, especially if
we have no idea what the cell is like in real life.
A great deal of aesthetics is involved, both in devising the methods for preservation and in
judging which picture best captures the living state. But it is by no means a purely arbitrary
aesthetic judgment. On the contrary, it is based on a deep understanding of the living cell and
the physics of preservation techniques.
One person who has combined those qualities to an impressive degree is Dr. Ludwig
Edelmann in the Saarland University, Homburg, Germany, who has produced some of the
most breathtakingly beautiful, 'true-to-life' portraits of cells that I have ever seen. The tenacity
and patience with which he pursues his goal is astonishing.
One schedule for preserving rat liver goes as follows: Small pieces of fresh liver were rapidly
'cryofixed' at low, sub-zero temperatures without any chemical fixatives, by placing them on a
cold metal mirror. These cryofixed samples were then transferred to a microscope table
cooled with liquid nitrogen and cut into thinner slices not thicker than 0.3mm, then transferred
into a small metal container (4mm diameter) for a prolonged period of freeze drying at a
greatly reduced pressure, so that the ice can sublimate away slowly without disturbing the fine
structures of the cells.
The temperature is increased very slowly, at the rate of 0.2C per hour from -90C to -30C,
followed by a rate of 1C per hour from -30 to -10C, which took about 13 days, and then
maintained at -10C for a further 10 h. In preparation for embedding, the temperature was
lowered to -20C and the specimens soaked with components of the resin for 6 hours before
warming to room temperature, and the specimens transferred into pure Spurr's resin for 2 to 4
h. Only then were the specimens transferred into embedding moulds containing fresh resin,
and allowed to polymerised for 1 day at 60C to give a small solid block out of which ultrathin
sections of 60-70nm could be cut with a special diamond knife and stained with uranyl acetate
and lead citrate for electron microscopy.
The schedule for rat liver, is not the same as for other tissues. In fact, each cell type or tissue
requires a special treatment to give its best results.

10

Some of the criteria of good results are obvious: high definition of structures, new structures
or increased resolution of known structures observed, no shrinkage or swelling, and no
breakage of structures. But other criteria are not so clear, and amount to an aesthetic
judgement as what is more life-like: a regime in which structures appear as if caught in the
midst of casual conversation and trafficking, with each minute entity engaged in its own
activity while 'watching' what its neighbours are up to (see Fig. 1). It is a regime of dynamic,
spontaneous order in which the structures appear minimally stressed and maximally
correlated. It makes you catch your breath in reverence of the beauty of life that has just been
unveiled.

Figure 1. Rat liver cell, magnified 82 000x

Edelman's holy grail for the most life-like picture of the cell goes back a long, long way.
Reading Erwin Schrdinger' book, What is Life? convinced him that the living cell is in a
state of low entropy, or high degree of dynamic order - an idea that is probably best
formulated, he tells me, in the "Association-induction hypothesis" (AIH) that Gilbert Ling
proposed in 1962 (see "Strong medicine for cell biology", SiS review). From Ling, he learned
that the living cell is primarily an assembly of water, proteins and associated potassium ions,
and that the states of water as well as proteins in the living cell are very different from those
of bulk water and isolated proteins. Dead cells or cells fixed by chemicals immediately
changes this low-entropy (highly ordered) state of water, proteins and potassium ions.
This has spurred him on to find the method that best preserves this living state, and it is slow
freeze-drying of cryofixed biological tissues instead of using chemical fixatives and solvents.

11

In the course of developing these techniques, Edelmann also confirmed a major prediction of
AIH, that cellular potassium is adsorbed at negatively charged sites of cellular proteins, and
not freely dissolved in cell water as was generally assumed. This assumption inevitably led to
the major dogma of contemporary cell biology that Gilbert Ling has thoroughly
deconstructed: that a sodium /potassium pump is responsible for pumping sodium ions (Na+)
out of the cell and potassium ions (K+) into the cell, thereby keeping intracellular K+
concentration high and Na+ concentration low.
The most spectacular visualization of potassium adsorption was achieved using a method
developed by Ling, which was to reversibly replace potassium ions of living muscle cells with
chemically similar heavy ions such as caesium or thallium before cyrofixation and freezedrying. Electron micrographs of thin sections of this muscle demonstrated directly the
localisation of the electron-dense heavy metal ions at the myosin protein bands as predicted
(see Fig. 2). Edelmann has demonstrated similar localised methods.

Figure 2. Muscle preloaded with Thallium (a) and containing Potassium (b).
These findings convinced Edelmann that proteins of living cells must have a different
structure compared to isolated proteins, which do not selectively adsorb potassium or other
similar ions.

12

In his search of the protein structure in living cells, Edelmann obtained images that have
never been seen before. The outer membrane of the cell as well as membranes inside the cell
appear in negative contrast, i.e., bright, as opposed to dark, as is usually seen, while proteins
of subcellular compartments appear very homogeneously distributed instead of being
heterogeneous or fibrous, suggesting that the latter may be artefacts. For comparison with
Figure 1, see an area of a liver cell with mitochondria obtained after freeze-substitution
involving dehydration at low temperature of a cryofixed specimen with acetone,
supplemented with the chemical fixative Osmium tetraoxide (Fig. 3).

Figure 3 . Rat liver cell freeze-substitution method, compare with Fig. 1.

Edelmann believes that dehydration with organic solvents, as opposed to freeze-drying, both
alters the conformation of proteins and removes associated water layers around proteins that
are essential for maintaining the original protein structure. He and the world have both been
richly rewarded by his sustained efforts.
Article first published 15/10/04

Source
Edelman L. Freeze-dried and resin-embedded biological material is well-suited for
ultrastructure research. J Microscopy 2002, 207, 5-26.

13

What's the Bacterium Really Like?

Far from being a bag of macromolecules dispersed at random inside a rather tough cell wall,
the bacterium is highly and spontaneously organised into nested functional compartments
through interactions between the macromolecules and cell water. Dr. Mae-Wan Ho reports

The sophisticated internal architecture of a bacterium

A bacterium is the simplest organism that exists, even though it is by far the oldest, with a
direct lineage going right back to the beginning of life on earth some 3.8 billion years ago.
Plants and animals are referred to as eukaryotes, meaning organisms whose cells have a 'true'
nucleus, while the bacteria, which have no nucleus, are referred to as prokaryotes. This is
indicative of their primitive status as "proto-cells", or forerunners to eukaryotes. But this
prejudice is unjustified, say Michael Hoppert and Frank Mayer of Gttingen University in
Germany. Frank Mayer, in particular, spent many years studying bacteria, and can vouch for
the sophisticated internal architecture that exists in a bacterial cell.
Much of the prejudice against bacteria stems from their small size and the tough cell wall,
which make them difficult to study. The much larger plant and animal cells show up many
organelles inside such as mitochondria (where food is oxidised to provide energy), lysosomes
and peroxisomes (where macromolecules are degraded back into building blocks), and many
membrane-bound compartments as well as a cytoskeleton of fibrous proteins that fill the
cytoplasm. But a typical electron micrograph of a bacterium on the same scale would reveal
an amorphous blob inside.
For a long time, people thought that the bacterium is little more than a rather tough envelope
filled with macromolecules scattered randomly throughout the cytoplasm, and that its
metabolism is extremely "helter-skelter and inefficient".
In fact, bacteria are stunningly efficient, as is clear from the speed with which they can
multiply - doubling every 20 minutes or so in the laboratory - and it makes much more sense
to suppose that, even without a membrane, the molecules required for a particular activity are
grouped together in what can be called "functional compartments".
The idea of functional compartments is not new, and has been proposed even for eukaryotic
cells since the first part of the last century (see The Rainbow and the Worm, the Physics of
Organisms). But the evidence for that has so far been indirect.
When bacteria are sufficiently magnified - about one million times - with a powerful enough
electron microscopy, an astonishing amount of sub-cellular organisation becomes evident; and
it is possible to see several well-defined compartments immediately.
Inside the outer cell wall layers, referred to as the 'capsule', an E. coli cell is further enclosed
by two membranes with a space in between - the periplasmic compartment - where nutrients
and wastes are captured and sorted, and where a cell-shape controlling network of polysugars
and peptides, the peptidoglycan, is located. At the centre of the cell is the densely packed
DNA strands of the bacterial genome, folded into a compact body, a nucleoid, forming a
loosely defined compartment devoted to storage and use of genetic information. In between

14

the nucleoid and the inner membrane is the cytoplasm, filled with ribosomes (organelles for
protein synthesis) and multi-enzyme or multi-protein complexes performing a variety of
functions. The most obvious multi-protein complex, connected to the inner membrane, is the
flagellar-motor that turns a long, helical flagellum to propel the bacterium through its aqueous
environment. Chaperonins and proteosomes are respectively responsible for folding new
proteins and disposing of used, obsolete ones. DNA polymerase complexes attached to the
DNA strands are responsible for replicating the genetic information. The pyruvate
dehydrogenase complex links three sequential reactions together, delivering the metabolites
from one reaction to another via a flexible arm of the protein.
But where is the cytoskeleton? Using antibody-staining techniques, Frank Mayer has found
evidence of abundant fibrous proteins that form a web-like structure just inside the inner
membrane, to which the ribosomes - organelles for synthesizing proteins - are attached.
Thus, there is no doubt that the bacterial cell is just as highly organised as cells of 'higher'
organisms.

Spontaneous order out of chaos due to interactions with cell water

But how are these functional compartments formed? Studies in the laboratory of Hoppert and
Mayer suggest that they form spontaneously as the result of the intrinsic properties of the
biological molecules themselves and the way they interact with water in the cytoplasm. Also,
the specific structure of water itself could influence the level of enzyme activity in particular
microenvironments.
For example, there are compartments called inclusion bodies in certain bacteria, which appear
to be storage granules consisting of starch or fats that are not soluble in water. Among the fats
often found in inclusion bodies are long chains of fatty acids called polyhydroxyalkanoates
(PHAs). Like most fats, they are hydrophobic (water-hating). However, the enzymes that
synthesize PHAs are soluble in water. So, while they are synthesized, PHAs are linked to the
enzymes and form a complex, part of which is water-soluble and part of which is not.
Eventually, the complex rounds up into a spherical compartment in which the water-soluble
enzymes form the outer shell, shielding the water insoluble PHAs inside. Water is expelled
from the interior, creating an inner compartment separate from the cytoplasm by the watersoluble enzyme molecules. As the PHA inclusion bodies mature, amphiphilic molecules
(molecules that love water at one end and hate it at the other) - specific proteins and
phospholipids - are added to the growing circumference of the boundary layer, while more
PHAs are added to the interior.

Reversed micelles offer clue to spontaneous organisation

Hoppert and Mayer studied enzyme activity in artificial 'reversed micelles'. A conventional
micelle is formed when water surrounds amphiphilic molecules that tend to form a sphere,
with their water-hating ends inside the sphere and their water-loving ends outside in the water.
A reversed micelle is just the opposite. The water-loving ends are inside the sphere interacting
with water, while the water-hating ends are outside in touch with a sea of organic solvent.
Depending on the size of the reversed micelle and the location of the water molecules within,
the water can adopt two different structures. Water close to the periphery of the micelle in
direct contact with the barrier molecules differs from that in the centre, and both are different

15

from bulk water (see Fig. 1). The low-density water forms a lot more intermolecular hydrogen
bonds than bulk water, and tends to resemble ice. It also has less charge, is less reactive and
more viscous than bulk water. High-density water molecules, by contrast, are less likely to
form hydrogen bonds with their neighbours than in bulk water, and are also less free to move
about.

Figure 1. A reversed micelle with enzyme trapped inside.

In the living cell, all compartments and macromolecular assemblies affect water structure,
according to Hoppert and Mayer, so there is a non-random variation in high and low density
water throughout the cell (see "The importance of cell water", this series), and in turn this
would affect the function of the proteins.
It is possible to measure the vibrational frequency of proteins dissolved in low-density water
using the reverse micelle system. This revealed that low-density water decreases the

16

vibrational frequency compared with proteins dissolved in bulk water. The vibrational
frequency affects enzyme activity. For example, lowering the vibrational frequency of an
enzyme may increase the temperature at which the enzyme achieves optimum rate of reaction.
Enzymes seem to like low-density water best, where they are presumably more free to move
around.
Hoppert and Mayer found that the enzyme activity also depends on the size of the reverse
micelles, and remarkably, all enzymes reach optimum activity at a particular size that is
approximately the spacing of the periplasmic space in the living cell, about 2 to 10
nanometres wide. Hoppert and Mayer introduced the term nanospaces to describe them. An
organisation into nanospaces is not only found in bacteria; it is also common in any other
living cell. This suggests that enzymes may be sitting in a microenvironment of structured
water that promotes optimum activity inside the cell. Thus, the layered structure of dense and
light water within the cell is part and parcel of the subcellular organisation that enables the
cell to function most efficiently.
Article first published 16/10/04

17