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One leaf of a hydrated membrane bilayer made of

1,2-dipaltmitoyl-sn-glycero-3-phosphatidylcholine

 

One leaf of a hydrated membrane bilayer made of DPPC

Biomembrane Hydration

Biological membranes primarily consist of proteins, lipids, and water.


V Protein hydration

V Phospholipid hydration

V Cholesterol and membrane hydration

V Triacylglyceride hydration

 

Life requires water

Lipid membranes require water to form and act

Phospholipid hydration

The insolubility of phospholipids in water drives lipid self-assembly into membranes, allowing minimum exposure of the hydrophobic parts with water whilst solvating the hydrophilic phospho- 'head' groups. The formation of 'flat' membranes, as opposed to tightly curved micelles comes about due to the rectangular lipid structure and the inclusion of proteins. The strongly-held hydrophilic coating forms a barrier to the facile fusing of membranes. Some phospholipids are preferably found on particular sides of the membrane; for example, phosphatidylserine is always found on the inside of plasma membranes [3006]. This is due to the hydration compatibility of the local head groups.

 

Early descriptions of membranes revolved around the 'fluid mosaic model' [2592a] (see figure below), where the 'backbone' of the membrane is the phospholipid bilayer. Over the years further studies demonstrated the need for a more complex model description (as in [2592b]). This newer model, however, is still misleading as it does not include the bound and included water, that is absolutely required for the formation, structuring, dynamics, stability and varied biological functions of biomembranes. It is now recognized that water forms an essential, integral and major constituent in the highly complex structure of biologically active membranes [2767]. This water has very different properties than bulk water.

 

Contemporory picture of the Fluid Mosaic Model, from

 

Contemporory picture of the Fluid Mosaic Model, taken from https://en.wikibooks.org/wiki/A-level_Biology/Biology_Foundation/cell_membranes_and_transport

Membranes divide aqueous environments and, amongst other activities, control the passage of materials between them, with water permeation critical to osmotic equilibrium. They are built from a bilayer of phospholipids that each consist of a hydrophilic head group within the aqueous environment on both sides and hydrophobic fatty acid 'tails' within the membrane interior. In the model above, sections through the membrane are shown. These hydrophobic areas are not allowed to contact water, so such membranes have no edges, forming continuous surfaces with an outside and an inside. The phospholipids may diffuse laterally, giving rise to the 'fluidity' of the membrane (with the membrane behaving like a 2-dimensional liquid) but only extremely rarely change sides of the membrane ('flip-flop' ). Although often ignored, the biological functions, structuring, and movement of the membrane lipids and proteins depend substantially on their hydration [2597] and the contacting aqueous phases; for example, water molecules hydrogen bond between the carbonyls of neighboring dipalmitoyl phosphatidylcholine groups to spread the head groups and enable them the room to twist such that their dipole moments are in a nearly anti-parallel arrangement [2597]. Membrane proteins often have strongly hydrogen-bound helices for their main structural support, with the helices usually either lying on the surface and parallel to the membrane surface or perpendicular to the surface and crossing the membrane. Due to their hydrophobic environment, these backbone hydrogen-bond strengths may be as much as 25 kJ ˣ mol-1 greater than the water-water hydrogen bonds [3044]. Where they are weaker, they indicate a bending or cleavage site in the protein's function or activity [3044].

 

Cell membranes are heterogeneous being self-assembled from hundreds of different lipids plus many different membrane proteins and other materials, such as cholesterol. The lipids are able to move, with their associated water, within their two-dimensional surface. Their surfaces are highly corrugated (not planar as often displayed in the fluid mosaic model, see above, that would result in slow perpendicular motion relative to lateral diffusion) resulting in the very reduced lateral diffusion of the water, and other, molecules along the surfaces [2589], as such lateral motion also involves perpendicular motion pushing other molecules out of the way. The ratio of the lateral to the perpendicular motion will depend on the membrane topographical and chemical structure at issue. Adequate hydration is needed for the structure, dynamics, and function of the membrane proteins, not only at their external aqueous interfaces but also within the membrane [2591], and with hydration also needed for the fluidity (as a plasticizer) of the phospholipid biomembrane itself. Hydration also mediates adhesion and fusion between different membranes and between membranes and proteins plus its importance in membrane protein structure and activity. The aqueous interface next to the membranes forms a functional unit linking the membrane to the water-soluble proteins of the cytosol, facilitating the rapid exchange of structural, dynamic and physiological information [1553].

 

The complexity of membrane structure, however, does not mean that they are randomly organized as different areas of a membrane will have different constituents and the different phospholipids are tightly controlled (although this is currently poorly understood) [2765]. This diversity makes the study of membrane hydration difficult with most 'model,' studies involving membranes constructed from a single phospholipid type. The results of such studies (some given below) can only be indicative of the biological reality. Also, it should be remembered that gasses such as oxygen, hydrogen, and nitrogen are much more soluble in the oily part of biomembranes than in water and may have an (unexpected) effect on their properties.

 

Palmityl oleyl phosphatidylethanolamine

 

Palmityl oleyl phosphatidylethanolamine
Opposite shows the charge distribution in a typical phospholipid molecule, palmitoyl oleoyl phosphatidyl-ethanolamine. a The ionic groups generate strong electric fields in the plane of the membrane and will interact with neighboring head groups and imposed electric fields. In the absence of proteins, most lipid membranes are expected to be negatively charged (i.e. negative zeta potential) due to the steric arrangement with more closely held hydrogen phosphate anions, together with counter-ion attraction that allows anions closer than cations. However, they will allow proton transfer in the plane of the surface by hopping between anion groups. They have the capability of being directed by differences in the head groups within different raft (dynamic domains > 40 nm in size) areas. Asymmetry in the number of lipids per side (inner and outer leaflet) of a membrane may be caused by hydrogen-bond interactions and hydration between the head groups [3006]. Thus, they affect these raft area [2634].

 

Examples of phospholipids,

R1 and R2 are fatty acid chains

 

examples of phospholipids, R1 and R2 are fatty acid chains

The different sides of biomembranes are quite different from each other with different phospholipid and protein compositions [3006]. As most biomembranes present curved surfaces, they are naturally asymmetric with the inner leaflet being more tightly curved, than the outer leaflet, and with a lesser surface area. This asymmetry may be achieved by a different number of lipids in the outer and inner leaflet, a difference in transmembrane head-group hydration and/or a different head group orientation for the interacting phosphate groups [2962]. Both sides involve highly hydrophilic head groups of phospholipids, with some examples given left. The hydrophobic tail groups (R1 and R2 left) are also varied and often consist of one saturated and one kinked unsaturated group in each phospholipid (see palmitoyl oleoyl phosphatidyl ethanolamine above). Due to their different positioning in the glycerol backbone, the hydrophobic chains are axially displaced by about two methylene (CH2) units towards the middle of the bilayer. It follows that molecules that interfere with the surface hydration also change the local membrane curvature with possible further effects on the biomembrane enzymology and transport properties [3006].

 

The head-groups extend to different extents away from the bilayer according to their structures, charges and neighboring steric and electrostatic interactions. They strongly bind many water molecules in subtly different manners and their interfaces are somewhat rugged and heterogeneous with highly variable diffusibility with some water being trapped and almost static [2585]. The number of water molecules associated with each head group varies with the head group, its neighbors and the method used in the determination. In particular, the negatively charged phospholipids such as phosphatidyl-serine and phosphatidyl-inositol, although generally in a minority, appear to have very important roles in stiffening the membranes and attracting and binding the proteins to the membrane surface; processes modulated by cations [2754].

Density of material through a DPPC membrane.; from [2586]

 

Density of material through a 1,2-dipaltmityl-sn-glycero-3-phosphocholine membrane.; from [2586]

 

Shown on the right is the density profile of material across a 1,2-dipaltmitoyl-sn-glycero-3-phosphatidylcholine membrane [2586] (DPPC), used as an example. The molecular structure beneath the graphs is indicative only, with the actual 3-dimensional structure folding up in different ways causing about a 10% shortening in length and giving rise to corrugated (i.e. not planar) surfaces. It shows how the water density (blue) mixes with the phosphate (violet) and carboxylate (red) in the head-groups but reduces up to the methylene chain of the fatty acid (gray) with effectively none within the interior. Both the water diffusion rate [2789] and the water activity reduces (qualitatively rather than quantitatively) in line with the water density line (blue). The reduction in the water motions together with the reduction in the entropy of the water near the phospholipid bilayers is extended by the presence of a peripheral membrane-bound protein. These effects range up to about a nanometer above that protein so aiding molecular recognition, binding, and protein−protein interactions [2789]. Similar to that occurring in proteins, a dynamical transition in the bilayer interior is found at about 188 K using 2H electron spin echo envelope modulation (ESEEM) NMR-like spectra. This is due to the onset of isotropic water movement. Another transition at about 100 K is ascribed to the restricted reorientational motions of water together with lipid chain flexibility around defective free-volume holes [2923].

 

The water activity also varies parallel to the membrane, across its surface, due to differences between the phospholipids and the presence of membrane proteins. This causes differences in the lipid packing and the flow of water.

 

The head-groups are marked by higher absolute density, due to the presence of greater oxygen and phosphorous density plus more densely held water. The middle of the membrane shows slightly lower absolute density due to the chain ends with associated vacant space caused by their poor interactions. The surface area taken up is about nm2 that increases by about 1% °C-1. About 10-12 H2O molecules may be included within the head group volume, with half of these associated with the phosphate and any remaining affected water molecules lying above this surface. Using molecular dynamics, these affected water molecules are shown to stretch at least two further molecular diameters (~10-12 H2O) before there is a little discernible effect on their diffusivity [2585]. Using terahertz spectroscopy three types of water have been found amongst the lipid head groups; (1) bulk-like, (2) frozen held firm by two or more hydrogen bonds, and (3) molecules with only one (or perhaps no) hydrogen bond allowing rapid rotational diffusion in more hydrophobic areas [2587].

 

Cartoon showing the gel-fluid transition in phospholipid bilayers

 

cartoon showing the gel fluid transition in phospholipid bilayers
Interactions with water can be estimated from known interactions of other related molecules in solution. The negatively charged phosphate groups (shown red, right) within the head groups are expected to hydrogen bond two water molecules strongly through each of their strongly charged singly bonded oxygen atoms and one water molecule less strongly through each of their doubly single-bonded (ether) oxygen atoms (i.e. 6 H2O per lipid); most of these water molecules are likely to be so strongly bound as to be unfreezable.

 

The deeply-located carboxylate ester groups within the head groups (shown orange, above right) are expected to hydrogen bond weakly to two water molecules through their doubly bonded (keto) oxygen atoms and one water more weakly through their doubly single-bonded (ether) oxygen atoms.

 

 

Melting temperature with water content, from [2605]

 

Melting temperature with water content, from [2605]
Some of these inner hydrogen bonds may, however, be disallowed for steric reasons, particularly as one of the two ester groups is buried deeper into the membrane (i.e. 3- 6 H2O per lipid). These water molecules may (mostly) link to oxygen atoms of neighboring ester or phosphate groups. Water hydrogen-bonded to the phosphate groups also (mostly) hydrogen bonds to other phosphate groups or further water molecules [2766]. Phosphate groups may also form ion-pairs or water-separated ion-pairs to cations.

 

Hydrogen bonding to the remaining head group depends on its structure, but for the choline group in the example, they are likely to form (freezable) partial clathrate shells (~10-12 H2O per lipid) with no direct hydrogen bonds. Other H2O may be trapped dependent on the neighboring structures. Overall there are about 28 H2O molecules directly affected (using terahertz spectroscopy) per lipid molecule out to at least one nm from the surface (~41 % w/w, xw ~ 0.97) [2590]. Other molecules further out to about 3 nm will be orientated by the inner water [2963]. The higher osmotic pressure generated by these inner water molecules (>70 MPa, lower water activity <0.5, [1553]) is partially compensated by the surface pressure exerted by and between the head groups, but will extend somewhat into the neighboring aqueous phase to repel approaching similar membranes (as shown by supported lipid membranes). This osmotic force is also known as the hydration force.

 

If excess water is attracted towards and into the surface then the swelling may result in membrane leakage. If less water is available, the membrane is dehydrated with reduced overall thickness, a reduced area per lipid, reduced permeability and reduced fluidity [2605] making the membrane sub-optimal or even totally inactive. This is particularly noticeable at hydration levels less than 12 H2O per lipid (see figure above left). The membranes represent one of the major sites of irreversible damage by dehydration, including that caused by ice formation removing the liquid water. However, the lipid structure may be maintained in the dehydrated state if it is dried (or frozen) in the presence of trehalose that binds to the carbonyl groups replacing about half the water molecules per lipid molecule. In this way, trehalose encourages the fluidizing of the dry bilayers by preventing the head groups directly interacting to form static condensed structures. Although trehalose prevents damage to the membrane it is insufficient for the active working of the membrane that requires the water molecules' dynamic movement.

 

Small amounts of water can enter the membrane, depending on the membrane structure, due to their small size and their few or zero retaining hydrogen bonds when lying close to the interface. They start by hydrogen bonding to the ester carbonyl groups, then moving between nano pools created from kinks in the lipid acyl chains. The surface packing may aid this process. This allows the possibility of the slow movement of water across membranes dependent on the osmotic pressure (water activity) difference between the solutions on opposite sides. This movement may be assisted by the lateral flipping of membrane lipids. The rate of flow may be different in different directions even if under the same pressure. The water's reduced entropy in the restricted water pools can be compensated by the disorder entropy they allow in the acyl chains [2588]. If, however, the membranes are dehydrated, containing less than the required (~20 H2O) water, the bilayer will stiffen, c disallowing such transport. Other molecules can cross the membrane by passive diffusion or via specific transporting membrane proteins; either active or passive [2772]. Passive diffusion depends mostly on the partitioning coefficient of the (hydrophobic) material into the membrane and the concentration difference across the membrane.

 

Aqueous ions affect the surface pressure of DPPC monolayers and are expected to equally affect biomembranes. The presence of the ions close to the interface was found to increase the surface pressure at a fixed area per molecule, with this increase dependent on the ion in order of the Hofmeister series. Thus SCN- gives a far greater increase in pressure than Cl-. It is thought that these anions partition into and interact with the head-group volumes but not the hydrophobic tail volumes [2657].

 

The curvature of membranes depends on the size and aqueous interactions of the surface polar groups, the volume to length ratio of the hydrophobic tails and the differences between the two sides of the membranes [3006].

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Cholesterol

 

Cholesterol

Cholesterol and membrane hydration

Cholesterol b is found in the membranes of almost all Eukaryotes, where it is required for life, but is absent in Prokaryotes. The amount of cholesterol present depends on the membrane but may vary up to half the molecules present. It is used to make the membrane stronger (condensing its structure), thicker, less fluid and less permeable to both hydrophilic and hydrophobic molecules. Cholesterol seems to have the perfect structure for this purpose both in its small head group and the length of its tail [2610]. Although cholesterol generally stiffens membranes, it has far less effect on some structures, such as dioleoylphosphatidyl-choline (DOPC) bilayers [2683]. Cholesterol also eases transitions in the phase structure of the membranes (packing transitions). This is particularly relevant to the interference of anesthetics with the transmission of the nervous impulses; the changes in phases interfering with the opened and closed ion channels [3006].

 

The transition in phospholipid bilayers on addition of cholesterol

 

cartoon showing the transition in phospholipid bilayers on addition of cholesterol
The polar alcohol group sits on the surface at the level of the carboxylic ester groups. It pushes the phospholipid head groups apart so enhancing their (head group) mobility and accelerating the translational diffusion of local water along the membrane surface. Within the bilayer, cholesterol strengthens the hydrocarbon hydrophobic connections so dramatically slowing and reducing penetration by water [2606], hydrophilic or hydrophobic molecules [2607].

 

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Triacylglyceride hydration

Tripalmitoyl glyceride

tripalmitoyl glyceride

Biological fats and oils consist of mixtures of triacylglycerides (triglycerides, see right); esters derived from glycerol and three fatty acids. As adipose tissue, they act as a store of energy and (metabolic) water (for example, in camel humps and bushman's bottoms).

 

Water has low (< 0.05% v/v) solubility in triglycerides due to their hydrophobicity but can hydrogen bond to the ester oxygen atoms (shown red right) of single triglyceride molecules or hydrogen bond between neighboring molecules [2764]. Thus, they may have considerable influence on the triglyceride fluidity and packing, as with membranes (above). Also, the water may promote hydrolysis and auto-

oxidation reactions. Little has been published of the molecular mechanisms in this area, however.

 


Footnotes

a   Lipid publications rarely use the IUPAC nomenclature where, for instance, palmitic acid is hexadecanoic acid. [Back]

 

b   Cholesterol is a natural steroid that we need to biosynthesize to live. We also get a lesser amount from animal sources in our diet. For many years, however, excessive cholesterol in our diet has been linked to narrowing of the arteries (arteriosclerosis), heart attacks and strokes, with statins being prescribed to counteract its effects. Recently a (controversial) study has shown that high levels of low-density lipoprotein cholesterol (LDL-C) in the blood is inversely associated with mortality in most people over 60 years [2609], a fact that appears inconsistent with the long-standing cholesterol hypothesis. [Back]

 

c   Stiffness (flexibility) of membranes can be described using the bending modulus KC. For an ideal bilayer, the intrinsic curvature is zero, with the energy required to deform the membrane from its intrinsic curvature to some other curvature Ebend (r) given by

Ebend (r) = KCCx,y 2/2 = (KC/2)(d2z/dxsquared + d2z/dysquared )squared

where Cx,y is twice the mean curvature at location (x,y) in the plane of the membrane that has fluctuations in the z-direction (see [2683] for more details).

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This page was established in 2016 and last updated by Martin Chaplin on 9 November, 2017


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