Water is a major contributor to a protein's 3-D structure and the protein controls the structuring of its surrounding water.
The contribution of water to protein
Water in protein recognition and binding
Water in protein function
Protein folding and denaturation
The contribution of water to protein
Protein hydration is very important for their three-dimensional structure
and activity [472, 1093, 1345, 2005].
Indeed, proteins lack activity in the absence of hydrating water. The aqueous structuring around proteins is affected out to at least 1 - 1.5 nm from its surface or 2 - 3 nm between neighboring proteins, as shown by terahertz spectroscopy [1368, 2102],e with even small proteins (e.g. bovine serum albumin, 66,463 Da) affecting the whole of its unstirred (Nernst) layer of about 20,000 neighboring water molecules . Additionally, the presence of glycans attached to (glyco)proteins impose a long-range order on the water structure out to several nanometers, dependent on the orientation of the glycan . Some water molecules interact with the surface, reorienting both themselves and the surface groups whereas other water molecules link these to the bulk in an ordered manner whilst remaining in dynamically active . In solution
proteins possess a conformational flexibility, which encompasses
a wide range of hydration states, not seen in the crystala or in non-aqueous environments. Equilibrium between these
states will depend on the activity of the water within its microenvironment; that is, the
freedom that the water has to hydrate the protein .
Thus, protein conformations demanding greater hydration are
favored by more (re-)active water (for example, high density
water containing many weak bent and/or broken hydrogen bonds)
and 'drier' conformations are relatively favored by lower
activity water (for example, low-density water containing
many strong intra-molecular aqueous hydrogen bonds). Surface
water molecules are held to each other most strongly by the positively-charged
basic amino acids. The exchange of surface water (and hence
the perseverance of the local clustering and the overall system
flexibility) is controlled by the exposure of the groups to
the bulk solvent (that is, greater exposure correlates
to greater flexibility and freer protein chain movement) . Hydration also affects the reactions and interactions of coenzymes and cofactors; thus, the various redox potentials (and hence whether they oxidize or reduce) of some iron-sulfur proteins are accounted for by differential hydration rather than direct protein binding effects .
The folding of proteins depends on the same factors as control
the junction zone formation in some polysaccharides; that is, the incompatibility between the low-density
water (LDW) and
the hydrophobic surface that drives such groups to form the
hydrophobic core.b This drive for hydrophobic groups to mostly cluster away from the protein surface (in water soluble proteins) is controlled by the charged and polar group interactions with each other and water. Interestingly, in pure water and in the absence of screening dissolved ions, some buffer-insoluble proteins are quite soluble due to the weak (here unshielded) interactions between the protein's intrinsic charges . Non-ionic kosmotropes,
which stabilize low-density water, consequentially stabilize the structure of proteins. In addition,
water acts as a lubricant ,
so easing the necessary peptide amide-carbonyl hydrogen bonding
changes. The biological activity of proteins appears to depend
on the formation of a 2-D hydrogen-bonded network spanning
most of the protein surface and connecting all the surface
hydrogen-bonded water clusters .
Such a water network is able to transmit information around
the protein and control the protein's dynamics, such as its
domain motions . Much life, however, functions optimally at about 37 °C where this spanning network is about to break down on heating, perhaps to help compensate for the biosystem's entropic changes .
Intramolecular peptide (amide) hydrogen bonding makes a major
contribution to protein structure and stability. Equilibrium H/D fractionation may be used to quantitatively assess hydrogen bond strengths and has shown them to be marginally stronger on average in α-helices rather than β-sheets by ~0.8 kJ mol-1 . However such hydrogen bonds are only
effective in the absence of accessible competing water. Even
just the presence of close-by water molecules causes peptide hydrogen bonds to lengthen ,
so loosening the structure. Water mediated hydrogen bonding between peptide links has been found to be particularly important in the structure of collagen where only a third of the peptide links directly hydrogen bond to other peptide links  and the stability of the triple helix is sequence dependent . Water molecules can bridge between
the carbonyl oxygen atoms and amide protons of different peptide
links to catalyze the formation, and its reversal, of peptide
hydrogen bonding as well as forming long-lived linkages stabilizing
protein-ligand and protein-protein interfaces (for example, ). The
internal molecular motions in proteins, necessary for biological
activity, are very dependent on the degree of plasticizing,
which is determined by the level of hydration .
Thus internal water enables the folding of proteins and is
only expelled from the hydrophobic central core when finally
squeezed out by cooperative protein chain interactions .
Many water molecules (similar in amount to individual amino
acids) remain behind buried in the core of the proteins, so
forming structurally-important hydrogen bonded linkages. The
position of the ESCS equilibrium around enzymes has been shown to be important
for their activity with the enzyme balanced between flexibility
(CS environment) and rigidity
(ES environment). Addition of non-ionic chaotropes only, or kosmotropes only, both
may inhibit the activity of enzymes (by shifting the equilibrium
to the right or left respectively) whereas an intermediate
mixture of kosmotropes and chaotropes restores optimum activity .
Also, the ease of enzyme-substrate contact may be controlled
by the level of water structuring in the protein environs
. The crystal structures of proteins suggest that the commonest water polygons surrounding proteins are (H2O)3 trimers (typical of CS environments) and (H2O)5 pentamers (typical of ES environments) with lower amounts of tetramers, hexamers and heptamers
The first hydration shell around proteins (~0.3 g/g) is ordered;
with high proton transfer rates and well resolved time-averaged
hydration sites; surface water showing coherent hydrogen-bond patterns with large net dipole fields .
The hydrogen bonds holding these water molecules to the protein are stronger with longer lifetimes than bulk water  and this water is unavailable to colligative effects. As hydration sites may be positioned close together and therefore
mutually exclusive, it has been argued that the solvation
is better described as a water distribution density function
rather than by specific water occupied sites. For example,
no more than 164 of the 294 high-density hydration sites around
myoglobin are occupied at any moment and there is no correlation
between the maximum site density, occupancy and residence
The first hydration shell is also 10-20% denser than the bulk
water and probably responsible for keeping the molecules sufficiently
separated so that they remain in solution 
(that is, solutions are kinetically stable but often
thermodynamically unstable).c Although a significant amount of this density increase has
been shown to be due to simple statistical factors dependent
upon the way that the surface is defined in depressions ,
much is due to a protein's structure with the excess of polar
hydration sites (tending to increase surface density)d easily counteracting the remaining non-polar surface groups
(tending to produce low density surface water). This water
is required for the protein to show its biological function
as, without it, the necessary fast conformational fluctuations
cannot occur. Thus proteins have no activity (and enter a glassy state, at about 220 K) when the surrounding water becomes predominantly low-density .
Using X-ray analysis, the hydration shell shows
a wide range of non-random hydrogen-bonding environments and
energies. Proteins are formed from a mixture of polar and
non-polar groups. Water is most well-ordered round the polar
groups where residence times are longer, but where they will interfere with water's natural hydrogen bonding, than around non-polar
groups where aqueous clathrate structuring may form. Interestingly, the clathrate pentagonal structuring has been found to extensively surround the helices in the four-helix bundle winter flounder antifreeze protein, so retarding the formation of harmful ice crystals in fish living in near-freezing water . Both types of group create order in the water molecules
surrounding them but their ability to do this and the types
of ordering produced are very different. Polar groups are
most capable of creating ordered hydration through hydrogen
bonding and ionic interactions (a excellent guide to amino
acid hydrogen bonding is given elsewhere). This is most
energetically favorable where there is no pre-existing order
in the water that requires destruction. The ordering created in the water surrounding proteins extends the proteins' electrostatic surfaces well away from their physical (that is, amino acid) surfaces giving them far greater electrostatic visibility to visiting ligands . This non-specific electrostatic effect of the water effect is additional to any specific directed hydrogen bonding that may extend away into the bulk from the surface
The line is the best straight line from these amino acid data but is expected to lie to the low density side of aqueous structuring.
The graph opposite shows the molar volumes of the amino acids  (using the one-letter code). Leucine (L) has the largest molar volume relative to that expected from its molecular weight and forms low density clathrate water structures if exposed to the solution. It is followed by isoleucine (I), valine (V), phenylalanine (F), lysine (K) and arginine (R). Aspartic acid (D) has the smallest molar volume relative to that expected from its molecular weight and forms higher density water structuring. It is followed by asparagine (N), glutamic acid (E), serine (S), cysteine (C), glutamine (Q), histidine (H), threonine (T) and glycine (G). The hydration properties of alanine (A), proline (P), methionine (M), tyrosine (Y) and tryptophan (W) are slightly kosmotropic .
The water is slow
to exchange, showing the dynamic behavior of bulk water 25 °C
colder . Low-density
water (such as ES)
is promoted [148, 276]
surrounding this dense hydration and polyelectrolyte double
layer (as described in the 'Polysaccharide
hydration' section). Non-polar groups promote clathrate
structures  (such
surrounded by denser water. It is no surprise, therefore that
the degree of hydrophobic hydration is correlated with the
hydration of the polar groups. Clathrate shells contain loosely
held water with greater rotational freedom than in the bulk
. However, under
favorable conditions clathrate hydrophobic hydration may exert
pressure on non-polar C-H bonds pushing them in, so contracting
their bond length and increasing their vibrational frequency.
This blue-shifting (that is, the vibration frequency increases and intensity reduces) 'push-ball' hydration 
should not necessarily be thought of as 'typical' hydrogen bonding even
if the CH···OH2 distances
are suitably close (see ). They can be considered as part of a continuum
of hydrogen bonding behavior, however, where sometimes the OH2 behaves as a much more weakly interacting base than usual
and the C-H behaves with reversed dipolar behavior compared
with the more usual O-H hydrogen-bonding partners .
There are significant differences in the directional rates
of water diffusion perpendicular and parallel to the protein
surface that are maximal at about 6 Å but still determinable
at 15 Å from the surface .
It is clear that evolutionary processes have made use of the
organization in this water surrounding proteins to create
preferred diffusive routes and orientation for metabolites
and favored conformational changes and interactions. Such
diffusive paths lead to binding sites with their own helpful
hydration. It has been suggested that pressure waves formed
from flickering water clusters (for example, as described elsewhere) may link protein molecular
vibrations, so carrying information through the intracellular
and powering product movement between enzymes in biochemical
Water in protein recognition and binding
Water molecules form an integral part of most protein-protein , protein-DNA  and protein-ligand  interactions, aiding the mutual recognition and both the binding thermodynamics and binding kinetics . Water's small size, polarity and conformational flexibility, together with the strength and directionality of the interactions, ensures good fits whilst retaining flexibility and ease of reversibility. The driving force for binding depends not only on the interaction of the biological molecules with each other but the energetic cost for the necessary removal of hydration water and the energetic gain for the subsequent molecular rearrangement of the hydration water molecules [1793, 1805]. The use of water may also be useful in broadening the specificity of such links; for example, the peptide-binding protein OppA uses several flexibly adaptive water molecules to hydrogen bond and shield charges when binding to lysine-X-lysine tripeptides, where X is any one of the twenty common amino acids .
In some cases, structured and structuring water molecules have also been found to be very important for inhibitor recognition . [Back to Top ]
Water in protein function
The energetic optimization of mutual
hydrogen bonded networks between protein, water and
ligand is an intrinsic part of the molecular recognition
process in enzymes, binding proteins and biological
macromolecules generally .
Note that water bound and oriented in empty ligand
binding sites will reduce the entropy of activation
when replaced by the ligand .
Water molecules are arranged/arrange themselves around 'active' sites such that they facilitate the reactions. This arrangement often leads to the localized water molecules being activated for reaction either directly or catalytically. Also, it may lead to small empty spaces (dehydrons) and basic reactivity of surface water .
Figure 1. The water
network links secondary structures within the protein
and so determines not only the fine detail of the
protein's structure but also how particular molecular
vibrations may be preferred. The above chain of ten
water molecules, linking the end of one α-helix
(helix 9, 211-227) to the middle of another (helix
11, 272-285) is found from the X-ray diffraction data
of glucoamylase-471, a natural proteolytic fragment
of Aspergillus awamori glucoamylase .
Figure 2. The above centrally-placed water molecule makes strong
hydrogen bonds to residues in three separated parts
of the ribonuclease molecule holding them together.
This water molecule and its binding site are conserved
across the entire family of microbial ribonucleases
Water molecules have also proved integral
to the structure
and biological function of a dimeric hemoglobin . The
internal water molecules in proteins have been surveyed
Both proton-transfer processes [907, 160b] and electron-transfer
may be facilitated by ordered water molecules connecting proton
donor to acceptor sites or electron donor to electron acceptor
sites respectively. In both cases, the transfer is faster
where the linking water molecules possess stronger hydrogen
Further, a network of hydrogen bonded water molecules plays a catalytic role in water oxidation in photosystem II .
Local water structures and proton-conducting hydrogen-bonded water wires can form rapidly (~10 ns or so) in response to control by relatively small-scale rearrangements of the protein matrix .
[Back to Top ]
aTo avoid such activity loss,
proteins generally avoid crystal formation, perhaps by evolutionary
design involving surface kosmotropic lysine residues that minimize self-aggregation .
b A historical review of the 'hydrophobic effect' in proteins is available .
comparison of the hydrophobicity of the amino acids is
given in the table opposite. Further discussion of relative
hydrophobicities is given
elsewhere as is a classification of hydrophobicity
It should be noted that such hydrophobic interactions
are particularly important in stabilizing interdomain
and quaternary interactions. [Back]
c The effect of strongly-bound
surface water molecules on preventing protein-protein interactions
is described in .
d Alternatively to this molecular
group approach, some of this increased density may be attributed
to electrostriction pressure (that is, local pressure
increase due to the localized electric field) .
e Other techniques, such as NMR, are incapable of showing this.