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The importance of water in cell biology. Martin Chaplin. London South Bank University. The importance of water in cell biology. Outline of talk. Hydrogen bonding versus non-bonded interactions (conflict between enthalpy and entropy) Protein hydration

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the importance of water in cell biology
The importance of water in cell biology

MartinChaplin

London South Bank University

the importance of water in cell biology2
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
slide3

Water: Structure

Hydrogen atoms are

not fixed

Highly variable dipole

moment and dielectric constant

slide4

Water: Structure

No distinct lone pairs

of electrons

Hydrogen atoms are

not fixed

+

-

Highly variable dipole

moment and dielectric constant

Compact shape

slide5

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

+

-

-

slide6

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

slide7

What is water’s hydrogen bond?

Average values.

In reality, there is much vibration and variation

cf. van der Waals minimum energy position 3.0 - 3.6 Å

water equilibrium structure
Water: Equilibrium Structure

H-bond ~23 kJ/mol; O-H covalent bond ~492 kJ/mol

O-H 0.97 Å O-H···O 1.88 Å

Commonly found tetrahedral arrangement of water molecules. Hydrogen bonds O-H····O are not necessarily straight. Forms networks due to the substantial cooperativity in bond strengthening due to electron overlap within molecular orbitals.

water equilibrium structure9
Water: Equilibrium Structure

Ubiquitous water

tetrahedra

water equilibrium structure12
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

water equilibrium structure13

collapsed structure

expanded structure

Water: Equilibrium Structure

maximizing van der Waals interactions

more reactive

maximising hydrogen bonds

lower density

more viscous

water equilibrium structure14

collapsed structure

expanded structure

Water: Equilibrium Structure

maximizing van der Waals interactions

more reactive

maximising hydrogen bonds

more viscous

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

slide15

Water: Equilibrium Structure

collapsed structure

expanded 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

slide16

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.

water equilibrium structure17
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

water equilibrium shifts
Water: Equilibrium shifts

expanded structure

lower density

more viscous

collapsed structure

higher density

more reactive

Cs+ > Rb+ > K+

Ca2+ > Li+ > Na+

ClO4- > H2PO4- > Cl-

SO42-, HPO42-

Leu, Ileu, Lys, Arg

Asp, Glu

Stabilise central dodecahedron

Weakly hydrated ions with diffuse charge density.

Stronglyhydratedwith high charge density.

Ionic kosmotropes

Ionic chaotropes

protein hydration
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

slide20

Protein hydration

Larger volume than average,

lower density water

Smaller volume than average,

higher density water

H. Zhao, Biophys. Chem. 122 (2006) 157–183.

protein hydration21
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 water’s 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.

rotational and translational diffusion
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

anchored proteins
Anchored proteins

Static anchoring creates static surface water

Static anchoring required to exert forces

the cytoskeleton
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.

actin
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

actin filament
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

actin n terminus
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.

selected actins
Selected Actins

From mammals, birds, amphibia, crustacea and fungus

a

b

/

g

a

b

/

g

a

b

/

g

a

b

/

g

tubulin
Tubulin

b

a

b

a

GDP

GTP

GDP

GTP

+ 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

tubulin microtubules
Tubulin microtubules

GDP

GTP

GDP

GTP

b

a

b

a

~25 nm

a

b

Acidic C-terminus

Different numbers of subunits may coil round. The acidic negatively charged C-termini project into the external solution.

tubulin c terminus
TubulinC-terminus

All known structures from the UniProt Knowledgebase.

The b-tubulins are

mostly on the left.

selected tubulins
Selected Tubulins

From mammals, insects, plants, alga, fungi and protozoa

a

b

a

b

a

b

a

b

intermediate filaments
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.

intermediate filaments34

From mammals, birds, amphibia, snakes and fish,

from cytokeratins, vimentins, desmins and neurofilaments

Intermediate filaments

Acidic p-loops

acid oligopeptide clusters
Acid oligopeptide clusters

Acetyl-GLU-GLU-ASP-

Acid groups tend to cluster together to share

cations and to minimise water disruption

carboxylate effects

O

C

O

Carboxylate effects

+1

0

-1

-0.745

-0.774

2.23 Å

chaotrope

pKa=2.86

kosmotrope

pKa=4.74

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

carboxylate effects37
Carboxylate effects

+1

0

-1

-0.745

-0.774

How can the pKa be shifted?

chaotrope

pKa=2.86

kosmotrope

pKa=4.74

carboxylate dipoles and pka s of the acids
Carboxylate dipoles and pKa’s 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).

carboxylate effects39
Carboxylate effects

+1

0

-1

-0.745

-0.774

How can the pKa be shifted?

chaotrope

pKa=2.86

kosmotrope

pKa=4.74

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

carboxylate effects40
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

Destroys signal

Signals effect

Solvent separated Na+, increasing charge

Clathrate occupied ion pair K+, reducing charge

na k comparison

-

-

-

-

+

+

+

+

Na+ partitions away from low density water

K+ prefers low density water

Na+ does not shows magic number Na+(H2O)20

K+ shows magic number K+(H2O)20

Na+/K+ comparison

Attraction

Na+ - water > water – water > 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

phosphates
Phosphates

Intracellular concentration ~100 mequiv l-1

H2PO4- HPO42- pKa 7.21 6.67

Chaotrope zero ionic strength

Kosmotrope >100 mM ionic strength

As the ionic strength reduces the chaotropic

H2PO4- concentration increases.

transformation of water structuring
Transformation of water structuring

ATP

Free Protein Polymerized Protein

ADP+Pi

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.

slide56

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

slide57

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).

slide58

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).

slide59

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).

slide60

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).

slide61

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.

slide62

There may be a spine of hydration running down the bottom of the B-DNA minor groove particularly where there is the A=T duplex known to favor B-DNA. Thus A=T 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

more water
More Water

@

www.lsbu.ac.uk/water

Any questions?

slide65

Water: Evidence, X-ray Diffraction

Standard deviation of all 50 peaks, troughs and inflections is < 0.8%.

Narten, Danford & Levy, Faraday Discuss.43 (1967) 97-107.

slide66

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.

slide67

Amongst the other evidence for icosahedral clustering (IC)

Explains water’s 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.

water equilibrium shifts69
Water: Equilibrium shifts

expanded structure

lower density

more viscous

collapsed structure

easily frozen

more reactive

Cs+ > Rb+ > K+

Ca2+ > Li+ > Na+

ClO4- > H2PO4- > Cl-

SO42-, HPO42-

Lys, Arg, His

Asp, Glu

Hydrophobic solutes

Pressure , Temperature

Static surfaces with

low charge density

Stirring, Microwaves etc.

Clathrate producersClathrate destroyers