The importance of water in cell biology

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The importance of water in cell biology
Martin Chaplin
London South Bank University
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The importance of water in cell biology
Outline of talk

• Hydrogen bonding versus non-bonded interactions
• (conflict between enthalpy and entropy)
• Protein hydration
• Carboxylic acid clusters and the cytoskeleton
• Intracellular water

3
Water Structure
Hydrogen atoms are not fixed
Highly variable dipole moment and dielectric
constant
4
Water Structure
No distinct lone pairs of electrons
Hydrogen atoms are not fixed

-
Highly variable dipole moment and dielectric
constant
Compact shape
5
Water Structure
No distinct lone pairs of electrons
Hydrogen atoms are not fixed

-
Highly variable dipole moment and dielectric
constant
Compact shape

Compact tetrahedral hydrogen bonding

-
-
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Water Structure
No distinct lone pairs of electrons
Hydrogen atoms are not fixed

-
Highly variable dipole moment and dielectric
constant
Compact tetrahedral hydrogen bonding
Larger and non-spherical van der Waals shape
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What is waters hydrogen bond?
Average values. In reality, there is much
vibration and variation cf. van der Waals minimum
energy position 3.0 - 3.6 Å
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Water Equilibrium Structure
H-bond 23 kJ/mol O-H covalent bond 492
kJ/mol O-H 0.97 Å O-HO 1.88 Å Commonly
found tetrahedral arrangement of water molecules.
Hydrogen bonds O-HO are not necessarily
straight. Forms networks due to the substantial
cooperativity in bond strengthening due to
electron overlap within molecular orbitals.
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Water Equilibrium Structure
Ubiquitous water tetrahedra
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Water Equilibrium Structure
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Water Equilibrium Structure
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Water Equilibrium Structure
The most successful explanation for the special
properties of water are found in the Mixture
models Dense clusters of water
Lower density clusters of water
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Water Equilibrium Structure
maximising hydrogen bonds lower density more
viscous
maximizing van der Waals interactions more
reactive
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Water Equilibrium Structure
maximising hydrogen bonds more viscous
maximizing van der Waals interactions more
reactive
DH is -ve stronger bonds DS is -ve more
ordered DG is 0 finely balanced DV is ve
lower density
Conflict between non-bonded interactions and
hydrogen bonding Different conditions and/or
solutes shifts the equilibrium
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Water Equilibrium Structure
a
b
Broad and shallow minimum Higher enthalpy but
greater entropy
Deep but localised minimum More negative
enthalpy but smaller entropy
Stronger hydrogen bonding a ? b Weaker
hydrogen bonding b ? a
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Icosahedral dynamic equilibria
Small clusters form larger clusters at lower
temperatures.
M. F. Chaplin, A proposal for the structuring of
water, Biophys. Chem. 83 (2000) 211-221.
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Water Equilibrium Structure
DH is ve DS is ve DV is -ve
expanded structure lower density water lower
diffusion more viscous
collapsed structure higher density water greater
partner switching more reactive
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Water Equilibrium shifts
collapsed structure higher density more reactive
expanded structure lower density more viscous
Cs gt Rb gt K
Ca2 gt Li gt Na
ClO4- gt H2PO4- gt Cl-
SO42-, HPO42-
Leu, Ileu, Lys, Arg
Asp, Glu
Stabilise central dodecahedron Weakly hydrated
ions with diffuse charge density.
Strongly hydrated with high charge density.
Ionic kosmotropes
Ionic chaotropes
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Protein hydration

• Every amino acid hydrates differently
• Hydrophobic amino acids, e.g. leucine
• low-density clathrate water surrounds
• Basic amino acids, e.g. lysine
• low-density clathrate water surrounds, some
  puckering
• Hydrophilic amino acid, e.g. threonine
• similar to bulk water
• Acidic amino acids, e.g. aspartic acid
• high density water surrounds with broken
  hydrogen bonding

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Protein hydration
Larger volume than average, lower density water
Smaller volume than average, higher density water
H. Zhao, Biophys. Chem. 122 (2006) 157183.
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Protein hydration
Conflicting effects mixed environments around
proteins. Weak H-bonding allows greater
flexibility. Strong H-bonding gives greater
stability and solubility. Ordered structure in
first shell around the protein, both hydrophobic
clathrate-like and H-bonded each helps the other
to optimise waters H-bonding network. Clathrate
formation over hydrophobic areas maximises
non-bonded interactions without loss of
H-bonds. Carboxylate groups usually only fit a
collapsed water structure creating a reactive
fluid zone. Diffusion of surface water is only
10 of bulk water, and similar to supercooled
water Protein rotation creates a surrounding
zone of broken hydrogen bonds.
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Rotational and translational diffusion
Translational diffusion involves breaking
water-water links at a distance from surface
Rotational diffusion involves breaking close
water-water and protein-water links.
Interfacial region around a protein (perturbed
water) comparable to protein volume
The surface area for translation and rotation is
the same but the velocity differential is
constant for all r for translational but varies
with r2 with rotation. At the breaking surface,
half the H-bonds are broken.
More hydration slows down rotation far more than
effect on translation
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Anchored proteins
Static anchoring creates static surface water
Static anchoring required to exert forces
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The Cytoskeleton
Actin, tubulin and intermediate filaments form
the cytoskeleton in eukaryotic cells. Together
they control mitosis, the shape of the cells and
organise the cytoplasm and nucleus. Tubulin
forms fat hollow and stiff microtubules making
tracks for the movement of organelles. Actin
forms thin flexible microfilaments. Intermediate
filaments form flexible and elastic
links. Actins all have acidic N-termini, Tubulins
have acidic C-termini and Intermediate
filaments have acidic central regions. The
surface area of these filament systems exceeds
that of all internal membranes 10-fold or more.
They are highly conserved but their 3-D
structures are known only in part. Fibres are
known through electron crystallography and
guesswork.
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Actin
Acidic N-terminus
ADP/ATP binding site
Actins are highly conserved with 375 Amino
acids. They form 10 of total intracellular
protein
1HLU Bovine b-actin profilin complex
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Actin Filament
Acidic N-terminus
Actin filaments in the cytoskeleton are highly
dynamic. Hydrolysis of ATP accompanies
polymerization of ATP-containing monomers but
destabilises the actin filaments. Water molecules
shield the binding surfaces
F-actin from Holmes KC and Eschenburg S
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Actin N-terminus
All known structures from UniProt Knowledgebase
The a-actins have four terminal acidic groups
whereas the b- and g-actins have three. The acid
groups are conserved as either aspartate or
glutamate.
The amino terminal residue in many eukaryotic
proteins is N-acetylated.
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Selected Actins
From mammals, birds, amphibia, crustacea and
fungus
a b / g
a b / g
a b / g
a b / g
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Tubulin
acidic C-terminus
Tubulin has two similar subunits of 450 amino
acids. It has a GTP binding domain near the
N-terminal, a beta-sheet core and alpha helices.
Two antiparallel helices lead to the highly
acidic external C-termini. There is a head to
tail arrangement of dimers with the beta-subunit
GTP at the open end. Only this GTP is hydrolysed
following polymerization.
1Z2B Bovine a/b tubulin colchicine-vinblastine
complex, introduces a curve not seen in native
tubulin
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Tubulin microtubules
b
b
a
a
25 nm
b
a
Acidic C-terminus
Different numbers of subunits may coil round. The
acidic negatively charged C-termini project into
the external solution.
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TubulinC-terminus
All known structures from the UniProt
Knowledgebase. The b-tubulins are mostly on the
left.
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Selected Tubulins
From mammals, insects, plants, alga, fungi and
protozoa
a b a b a b a b
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Intermediate filaments
Fibrous elastic proteins formed mainly from
coiled coils (ropes) of multi-stranded acidic
a-helices, 11 nm diameter. The central
rod domains contain 310 amino acids. Some
acidic groups form salt links across to other
strands but excess acidic groups are present on
the surface of the rod-like structure with
excess basic groups at the end. Between
a-helices are glutamate rich acidic bulges
(single p-helices) forming flexible linker
regions. As strands gather these group together.
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Intermediate filaments
From mammals, birds, amphibia, snakes and
fish, from cytokeratins, vimentins, desmins and
neurofilaments
Acidic p-loops
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Acid oligopeptide clusters
Acetyl-GLU-GLU-ASP-
Acid groups tend to cluster together to
share cations and to minimise water disruption
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Carboxylate effects
1 0 -1
-0.745
-0.774
2.23 Å
pKa2.86
kosmotrope
chaotrope
pKa4.74
Causes density increase, if hydrogen bonded
cf. sulfate (kosmotrope) 0.87, perchlorate
(chaotrope) 0.71
Aspartic and glutamic acids are usually
kosmotropic
Different carboxylates have different pKas
Na RCO2- pairs are always solvent separated (Na
holds on to water strongly) K RCO2- pairs move
from solvent separated to ion pair as pKa reduces
6-31G basis set used
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Carboxylate effects
1 0 -1
-0.745
-0.774
How can the pKa be shifted?
pKa2.86
kosmotrope
chaotrope
pKa4.74
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Carboxylate dipoles and pKas of the acids
2,2 dimethylpropanoate
1.9
1.8
1.7
Carboxylate group dipole, D
1.6
1.5
trifluoroacetate
1.4
0
1
2
3
4
5
6
pKa
Dipoles were calculated using ab initio molecular
dynamics with the 6-31G basis set, pKas from
Dean, Lange's Handbook of Chemistry (1999).
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Carboxylate effects
1 0 -1
-0.745
-0.774
How can the pKa be shifted?
pKa2.86
kosmotrope
chaotrope
pKa4.74
By small changes in charge
H-bonding to carboxylate increases O negative
charge and pKa Clathrate structuring around
carboxylate reduces O negative charge and pKa
Overlapping negative field from nearby groups
enhances counter ion association
Ion pair association discourages hydrogen bonding
but encourages clathrate formation
6-31G basis set
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Carboxylate effects
High pKa, H-bonding to water
Low pKa clathrate
Ions dissociate on rotation
Low density water
High density water
Static, fewer H2O collisions pulling it apart
Ion binding
Signals effect
Destroys signal
Solvent separated Na, increasing charge
Clathrate occupied ion pair K, reducing charge
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Na/K comparison
Attraction Na - water gt water water gt K -
water


Selective accumulation of K over Na where there
is low density water
Data from Collins KD, Biophys. J. 72 (1997) 65,
Millero FJ In Water and aqueous systems
(1972), and Khan A Chem. Phys. Lett. 388 (2004)
342
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Phosphates
Intracellular concentration 100 mequiv l-1
H2PO4- HPO42- pKa 7.21
6.67
Chaotrope zero
ionic strength Kosmotrope
gt100 mM ionic
strength As the ionic strength reduces the
chaotropic H2PO4- concentration increases.
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Transformation of water structuring
ATP
Free Protein Polymerized Protein
ADPPi
Static Greater H-Bonding Low density water
Rotation High density water Broken H-bonds
e.g. Rotating free G-actin
F-actin
filament
If there is more order in the protein fibre, then
there is more order in water
Protein fibres trap water, which has decreased
entropy. In order to attempt to keep the water
activity constant, therefore, the water has to
form bonds with a more negative enthalpy. This
results in stronger bonds, causing greater
structuring and lower density.
Enclosure of water involving capillary action.
This forms stretched confined water is much
more highly structured than the bulk water.
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Transformation of water structuring
water/water
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperative reinforcement
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Cooperativity and information transfer
Intracellular water favors K over Na
Static charge-dense intracellular macromolecular
structures prefer ion pair over freely soluble K
Ion paired K prefers local clathrate water
Clathrate water prefers local low density water
structuring
Low density water structuring can reinforce
neighboring site water structuring
Ca2 Na destroy low density water structuring
cooperatively
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Some surface water is well ordered
Water molecules connecting the haem groups and
protein residues of the two identical subunits of
Scapharca inaequivalvis haemoglobin. Note the
symmetry of the two pentameric rings. On binding
oxygen, the water molecules transfer information
between the subunits before the water cluster is
disrupted .
Royer et al. Proc. Natl Acad. Sci. USA 93, 14526
(1996).
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Some water is required for structure
A single water molecule in the ligand-binding
site of concanavalin A functions as a link
between Asp14, Asn16 and Arg228 of the protein
and the 2'-OH hydroxyl group of the trimannoside
ligand.
Li Lazaridis, J. Phys. Chem. B 109, 662 (2005).
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Some water is required for proton transfer
Water molecules in bacteriorhodopsin
photoisomerization of all-trans-retinal (pKa 13)
to 13-cis-retinal (pKa 8.45), drives a proton
from its Lys216-Schiff base to Asp85 releasing
the pentagonal hydrogen-bonded ring, flipping the
Arg82 towards the (arrowed) protonated water
molecule, releasing a proton through a water
wire) to the extracellular space. The Schiff base
is reprotonated from the cytoplasm through
another associated water wire.
Garczarek Gerwert, Nature 439, 109 (2005).
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Some water is required for electron transfer
Rapid electron transfer between two molecules of
bovine liver cytochrome b5. The electrostatic
interactions of the water molecules provide a
large donor-to-acceptor coupling that produces a
smooth distance dependency for the
electron-transfer rate. Only the water cluster
and the cytochromes are shown, and the protein
residues are hidden.
Lin et al. Science 310, 1311 (2005).
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Potential-energy funnels for the folding of
proteins
a The folding-energy landscape in the presence
of low hydration highlights the numerous barriers
to the preferred minimum-energy structure on the
folding pathway. There are many local minima that
might trap the protein in an inactive
three-dimensional molecular conformation. b
When a protein is sufficiently hydrated, a
smoothed potential-energy landscape is evident.
This allows proteins to attain their active
minimum-energy conformation in a straightforward
and rapid manner.
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There may be a spine of hydration running down
the bottom of the B-DNA minor groove particularly
where there is the AT duplex known to favor
B-DNA. Thus AT duplex sequences favor water
binding in the minor groove and also protein
binding there driven by the large entropy release
on this low entropy water's release. Protein
sliding along the DNA is assisted by uniform
complementary electrostatic interactions between
the positive protein and negative DNA, whereby
the protein follows the helical pathway of the
groove rather than jumping between the major
groove and the more negative minor groove
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More Water
_at_
www.lsbu.ac.uk/water
Any questions?
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Water Evidence, X-ray Diffraction
Standard deviation of all 50 peaks, troughs and
inflections is lt 0.8.
Narten, Danford Levy, Faraday Discuss. 43
(1967) 97-107.
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The importance of water in cell
biology Biological surfaces in contact with
water affect the arrangement of the first shell
of the surrounding water by means of polar,
dispersion and directed hydrogen-bonding effects.
The preferred orientations of this first shell
water are also affected by the favoured
orientations of neighbouring first shell water
molecules as well as the second and more distant
aqueous shells. If the surface is flexible, it
enables greater freedom of movement within the
surface water molecules and the surface will
respond to changes in this hydration layer.
However, if the surface is more fixed, the
adjacent water is more static and necessarily
more extensively structured. The surface
structuring of water and its consequential
structuring of the biological molecules are
affected by, and will affect, the thermodynamics
and kinetics for the binding of other molecules
and ions to the surface. In this presentation,
the organization of water at the surfaces of
biologically important molecules, including
proteins, DNA and the cytoskeleton, are described
and general conclusions drawn.
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Amongst the other evidence for icosahedral
clustering (IC)
Explains waters anomalies. Viscosity of
supercooled water indicates IC pentagonal
spines. Vibrational spectra supports IC large
and small clusters . Flicker noise spectrometry
shows IC clusters. The diameter of IC cluster is
minimum stable water droplet. Known clathrates
in water, e.g. magic number ions, NH4. The
cavity-cavity distance in IC is 5.4 Å, c.f.
supercooled water at 5.5 Å The icosahedral
nanodrop of water in a polyoxomolybdate.
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Water Equilibrium shifts
collapsed structure easily frozen more reactive
expanded structure lower density more viscous
Cs gt Rb gt K
Ca2 gt Li gt Na
ClO4- gt H2PO4- gt Cl-
SO42-, HPO42-
Lys, Arg, His
Asp, Glu
Hydrophobic solutes
Pressure , Temperature
Static surfaces with low charge density
Stirring, Microwaves etc.
Clathrate producers
Clathrate destroyers
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