Intracellular Water

The effect of intracellular protein on water structuring

The degree of density lowering inside cells is determined by the solutes, their concentration and the conformations and state of motion of the proteins; mobile proteins creating more disorder in the clustering compared with more static proteins. Many intracellular proteins are globular, so retaining rotational entropy notwithstanding the crowded environment. Water has conflicting effects in the mixed environments around proteins. Weak hydrogen-bonding between the protein and water molecules allows greater flexibility. Strong hydrogen-bonding endows the protein with greater stability and solubility. There is an ordered structure in the first shell around the protein, with both hydrophobic clathrate-like and hydrogen-bonded water molecules each helping the other to optimize water's hydrogen-bonding network. Changes in the proteins' conformations may result in water influx into the cell or efflux from the cell [949] as well as density changes. Protein carboxylate groups are generally surrounded by strongly hydrogen-bonded water whereas the water surrounding basic chaotropic groups (arginine, histidine, and lysine) tends towards a clathrate structuring. Clathrate formation over hydrophobic areas maximizes non-bonded interactions without loss of hydrogen bonds whereas carboxylate groups usually only fit a collapsed water structure creating a reactive fluid zone. The diffusional movement of the proteins will cause changes in the water structuring outside the first hydration shell.

At the breaking surface, half the hydrogen bonds are ruptured, therefore creating a zone of higher density water (as explained elsewhere). Protein rotation thus creates a relatively close and surrounding high-density water zone with many broken hydrogen bonds, and as shown by some NMR studies. The volume of this interfacial region of perturbed water around the proteins is comparable to the volumes of the proteins. [Back to Top ]

Such hydrogen-bonding induces a more negative charge on the carboxyl oxygen atoms, increasing the group's dipole and this leads to an increase in the carboxylate pK_a, as is shown below.

 

If the charges on the carboxylate oxygen groups are reduced, by neighboring electron-withdrawing substituent groups or imposed negative electric field, the pK_a is reduced. Note that the charge on the oxygen atoms lies between that on the ionic kosmotrope sulfate (–0.87; with hydrogen bonds to water) and the ionic chaotrope perchlorate (–0.71; preferring to be surrounded by clathrate water). Thus, only a 5% reduction in the negative charge on the carboxylate oxygen atoms is necessary to switch from kosmotropic to chaotropic behavior. It is found that Na⁺ ions prefer binding to the weaker carboxylate groups (pK_a > 4.5) whereas K⁺ ions prefer the stronger acids (pK_a < 3.5) [634].

 

It should be noted that, in the absence of an aqueous solution, Na⁺ ions bind with a covalent character to the glutamate carboxylate groups whereas K⁺ ions do not [3070]. [Back to Top ]

The importance of protein mobility

Actin is a highly conserved and widespread eukaryotic protein (42-43 kDa) ^e responsible for many functions in cells. Non-muscle cells contain 5-10% (of all protein) actin whereas muscle cells contain about 20%. All actin molecules contain a conserved acidic N-terminus with several neighboring aspartic or glutamic acids (emphasized right) and a post-translation acetylated terminal amino group (for example, N-Ace-Asp-Glu-Asp-Glu in rabbit muscle α-actin). When actin forms a filament structure this highly charged grouping is placed on the outer exposed edge of the helix, where it may be additionally used as a binding site for other proteins, such as myosin. The positionings of the acidic N-terminal antennae are shown in red in the cartoon below. Tubulin and the intermediate filaments are further structural proteins that form immobile structures with ordered hydration within cells, possessing conserved acidic groupings that serve similar functions. ^e

 

Cartoon of the semi-flexible actin filament, showing the positioning of the acidic N-termini (shown red)

Na⁺ and K⁺ ions behave differently when close to the carboxylate groups; K⁺ ions have a preference for forming ion-pairs whereas Na⁺ ions form solvent separated pairings [637, 2035]. This is due to the Na⁺ ions holding on to their water strongly with the inner shell polarized water molecules being ideally situated for forming strong hydrogen bonds to the carboxylate oxygen atoms. The K⁺ ions prefer an inner shell clathrate water structuring that is reinforced by its direct ion-pairing to the carboxylate group. Also, Na⁺ ions cannot take part in clathrate structuring whereas K⁺ prefers this environment [641]. As the distance between the neighboring amino acid carboxylate groups (about 7.3 Å) is about the same as the diameter of clathrate water clusters (about 7.6 Å), extensive ion-pairing of the adjacent carboxylate groups may be accommodated by neighboring clathrate clustering. K⁺ RCO₂^- pairs move from solvent separated to ion-pair with the reduction in the pK_a. K⁺ ions prefer strong acids (that is, low pK_a) whereas Na⁺ prefers weak acids (that is, higher pK_a). Direct ion-pair association discourages aqueous hydrogen-bonding to the carboxylate oxygen atoms but encourages clathrate formation surrounding the ion-paired carboxylate group.

 

 

When ion-paired the hydrogen-bonding to the carboxylate oxygen atoms is prevented, and a surrounding clathrate structuring is preferred. The clathrate clustering can signal their state to other neighboring carboxylate groups (see later).

When the static protein filaments convert to freely diffusing proteins, the ions are swept away due to the multiple contacts with surrounding water molecules and their inability to keep up with the rotating protein at increasing radial distances. This results in the conversion of any localized clathrate water structuring to high-density water involving hydrogen-bonded carboxylate groups. [Back to Top ]

Cooperative changes creating low-density water when actin is polymerized

 

Further support for this process is given by F-actin becoming more static in the presence of about 100 mM K⁺ [642].

Formation of K⁺-carboxylate ion-pairs leads to the formation of a surrounding clathrate water structuring that further leads to icosahedral water structuring (so ensuring maximal hydrogen-bond formation) and informing neighboring carboxylate groups. This signaling cooperatively reinforces the tetrahedrality of the water structuring found between these groups. The mechanism is indicated on the right (press the Forward button). Note that the clathrate arrangement allows cluster mobility (like a ball-and-socket joint), enabling the hydrogen bonding to search out cooperative partners. The expected greater rigidity of such structures with lower temperatures has been proven [1466].

 

The cooperative nature of this signaling explains the signal amplification seen in the conduction of impulses along the microtubules and actin [1434], ^e and perhaps linking to other parts of the cytoskeleton ^e [1307]. The importance of this cannot be overstated as microtubules are implicated in information processing and memory [1307]. The structuring of water may, therefore, be critical for the conscious mind [1337].

 

Although the clustering (above right) involves a significant drop in entropy (increase in coherence) , this is compensated by a more-negative enthalpy due to the stronger bonding of the fully-tetrahedral hydrogen-bonded structure. The phase transition between ordered and disordered water has been regarded as a mechanism to release energy for biological work rather than the membrane (pump) theory [1960]. It is consistent with Ling's association-induction polarized multilayer model [634], ^c as can be seen from the net dipoles emanating from the clathrate arrangement, but offers a more realistic explanation. The initial icosahedral size (3 nm diameter) also equals the water domain size proposed by Watterson [546]. Extensive (1.4 nm) long-lived (> 300 ps) aqueous dipole arrangements between biomolecules have been found in molecular dynamics studies [329]. Further support for this model is given by several studies concerning different types of intracellular water, such as "normal" bulk and "abnormal" osmotically inactive interfacial water [639]. Also, the water in the extreme halophile Haloarcula marismortui, which contains unusually large amounts of bound intracellular K⁺ ions, shows particularly slow translational diffusion [1253]. 

 

Idealized tetrahedral clustering caused by neighboring K⁺ carboxylate ion pairs

 

These orthogonal diagrams show the maximum limit of fully tetrahedral water clustering due to the cooperative clustering (see above) from two K⁺-carboxylate ion-pairs. This fully tetrahedral structuring, which resembles a mixture of hexagonal and cubic ice structures but with 5-fold symmetry (such that it cannot be easily frozen), is idealized in the cartoons. An interactive structure is given (Jmol).

 

 

In the presence of raised levels of Na⁺ 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⁺ or Ca²⁺ ion-pairings destroy the low-density clathrate structures and initiate a cooperative conversion of the associated water towards a denser structuring.

 

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

• Intracellular water favors 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 over freely soluble K⁺ ions.
• Ion paired K⁺-carboxylate groupings prefers a local clathrate water structuring.
• Clathrate water prefers local low-density water structuring.
• Low-density water structuring can reinforce the low-density character of neighboring site water structuring.
• Na⁺ and Ca²⁺ ions can destroy the low-density structuring in a cooperative manner. [Back to Top ]

 

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Footnotes

^a  Philosophical, historical, and scientific views of water in living systems have been reviewed [1011] [Back]

 

^b   The cytoplasm of cells is described in [892]. Nuclear magnetic resonance signal widths are much broader inside cells, showing that intracellular water is far more structured than extracellular or pure water; for example, see [884]. For a review of the relationship between the intracellular water and the cytoskeleton see [880]). It is noteworthy that the amount of 'free' versus 'bound' water [969], the concentration of K⁺ ions, and the cytoskeleton are all intimately linked together by the differences between normal and cancer cells [1333]. [Back]

 

^c   The polarized multilayer theory relies on the hypothesis that static proteins in the cell change their secondary structure from compact to extended forms, so that the newly formed extensive lengths of polypeptide 'polarize' the water using their amide (N-H and C=O) backbone. Although there is support for the existence of polarized multilayers of water [634], there is no experimental support for this explanation for their presence. Indeed, most experimental data is contra-indicative, resulting in only minority support from scientists in this area. [Back]

 

^d   The chaotropic/kosmotropic nature of these ions has been confirmed by FTIR [1072]. [Back]

 

 

 

 

 

Eukaryotic cells are dynamic structures, able to move, and divide. Such activities are achieved through the use of cytoskeletal filaments (actin, tubulins and intermediate filaments [3051], see cartoon above) and molecular motors (such as myosin) within the cell. The filaments lengthen, shorten and cross-link, regulated by other proteins. The actin filament is a helical ribbon formed of two parallel helical strands that each comprise a linear strand of small G-actin proteins (42 kDa) molecules with uniform orientation along the ribbon axis. The ribbon has a width of ∼8 nm, a thickness of ∼5 nm, and a helical repeat of 36 nm. An analysis of the known actin structures from the UniProt Knowledgebase showed the 32 complete α-actin structures recorded from fish, birds, snakes, and mammals all to have the conserved acidic N-terminal structure Acetyl- D/E- D/E- D/E- D/E- T/S/V- T- A- L- V- C- D- N- G- S- G- L- V- K- A- G- F- A- G- D- D- A- P- R- A- V- F- P- S- I- V- G- R- P- R- H- Q- G- V- M- V- G- M- G and the 70 complete β- and γ-actin structures recorded, from a similar range of species, to all have the conserved and closely related N-terminal structures of Acetyl- D/E- D/E- D/E- I/V/T- A/T- A/S- L- V- V/I/C- D/A- N- G- S- G- M/L- C- K- A- G- F- A/G- G/R- D- D/G- A- P- R- A- V/A- F- P- S- I- V- G- R- P- R- H- Q- G- V- M- V- G/R- M- G with even the variations limited to one or a very few molecules.

 

Eukaryotic tubulin is another structural protein that forms immobile structures with ordered hydration within cells [689]. It forms fat hollow and stiff microtubules (≈ 25 nm in diameter, containing low-density water) making tracks through the intracellular space for the movement of organelles. These tubular structures are built up from pairs of similar subunits (α and β), each of about 450 amino acids [1140]. Tubulins have a GTP binding domain near their N-terminal and two separate β-sheets each surrounded by α-helices. The microtubules are formed from head to tail arrangement of α/ β dimers with the beta-subunit GTP at the open end. Only this GTP is hydrolyzed following polymerization. Outside the molecular core, two exterior antiparallel α-helices lead to highly acidic C-termini, which are thus situated on the outside of the microtubule and away from the tubulin surface [1306]. The α-tubulin structure is highly conserved with C-terminal consensus sequence - A- R- E- D- M- A- A- L- E- K- D- Y- E- E- V- G- V- D- S- V- followed by an extremely variable but highly acidic C- terminus exemplified by the human α- tubulin (type ‘ubiquitous’) structure - E- G- E- G- E- E- E- G- E- E- COOH. The β- tubulin structure is also highly conserved with C- terminal consensus sequence - A- E- S- N- M- N- D- L- V- S- E- Y- Q- Q- Y- Q- D- A- T- A- followed by an extremely variable highly acidic C- terminus exemplified by the human β- tubulin (type ‘4Q’) structure - E- E- E- E- D- E- E- Y- A- E- E- E- V- COOH. As with the actins, these examples indicate that very strongly conserved structures exist for tubulins except for at an end (here the C-terminus) where glutamic acid and aspartic acid occur apparently interchangeably and in variable but always very acidic sub-structures. An improper functioning of tubulin has been linked with intracellular water clustering in the origin of cancer [1901].

 

Intermediate filaments are fibrous, flexible and elastic proteins formed mainly from coiled coils (‘ropes’, ≈ 11 nm diameter) of multi-stranded acidic α-helices [1141]. There are some families of associated proteins such as the nuclear lamins, sarcomere desmins, neuron neurofilaments and epithelial cytokeratins. In addition, the vimentins (see right) generally support the organelles and maintain cellular integrity. All these proteins possess α-helical central ‘rod’ domains (≈ 48 nm long) that contain about 310 amino acids plus more variable end domains, while some acidic groups form salt links with basic groups in other strands to build up and lengthen the resulting ‘elastic rope’ structure, there exists an excess of acidic groups on the surface of the filaments. Within the α-helices are acidic bulges (formed from single π-helical loops) containing clusters of three glutamic acids within four residues and creating flexible ‘linker’ regions. As different strands come together to form the fibrous ‘ropes’ these acidic clusters are grouped together with contributing acidic clusters from about 20 helices on the surface of the filaments. [Back] 

 

^f  Time-resolved infrared pump-prime spectroscopy of acetate in D₂O suggests the bridge structure (right) as the most stable (single-hydrated) structure [1804], but other water molecules are also involved.

 

The optimal configurations, energy parameters, and normal vibrational frequencies of the hydrates of the acetic acid and acetate molecular hydrates have been calculated using density functional theory. The preferred structures CH₃COOH⋅(H₂O)₂, CH₃COOH⋅(H₂O)₈, CH₃CO₂^-⋅(H₂O)₂, CH₃CO₂^-⋅(H₂O)₂, CH₃CO₂^-⋅(H₂O)₆, and CH₃CO₂^-⋅(H₂O)₁₆ [3151]. [Back]

 

^g   Ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy have shown somewhat higheramounts of bulk-like water in Escherichia coli and Saccharomyces cerevisiae [3494]. [Back]

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