bacteria. The largely enhanced phase contrast realized

by using Zernike phase plates has been proven to be

beneficial in 3D-EM such as the single particle analysis

and cryo-tomography.

Cross-References

▶ 3D Electron Microscopy Based on Cryo-Electron

Tomography

▶Electron Crystallography

▶Electron Microscopy

▶ Protein Structure Comparison Methods

▶ Protein–Protein Interactions

▶ Single Particle Tracking

▶ Structural Genomics

▶X-Ray Diffraction and Crystallography of

Oligosaccharides and Polysaccharides

References

Boersch H. €Uber die kontraste von atomen in elektron-

mikroskop. Z Nat. 1947;2a:615–33.

Danev R, Nagayama K. Transmission electron microscopy with

Zernike phase plate. Ultramicroscopy. 2001;88:243–52.

Danev R, Nagayama K. Single particle analysis based on

Zernike phase contrast transmission electron microscopy.

J Struct Biol. 2008;161:211–18.

Danev R, Nagayama K. Phase plates for transmission electron

microscopy. Methods Enzymol. 2010a;481:343–69.

Danev R, Kanamaru S, Marko M, Nagayama K. Zernike phase

contrast cryo-electron tomography. J Struct Biol. 2010b;171:

174–81.

Danev R, Okawara H, Usuda N, Kametani K, Nagayama K.

A novel phase-contrast transmission electron microscopy

producing high-contrast topographic images of weak objects.

J Biol Phys. 2002;28:627–35.

Danev R, Gleaser RM, Nagayama K. Practical factors

affecting the performance of a thin-film phase plate for

transmission electron microscopy. Ultramicroscopy. 2009;

109:312–25.

Fukuda Y, Fukazawa Y, Danev R, Shigemoto R, Nagayama K.

Tuning of the Zernike phase plate for visualization of

detailed ultrastructure in complex biological specimens.

J Struct Biol. 2009;168:476–84.

Kaneko Y, Danev R, Nitta K, Nagayama K. In vivo subcellular

ultrastructures recognized with Hilbert-differential-contrast

transmission electron microscopy. J Electron Microsc.

2005;54:79–84.

Nagayama K. Phase contrast enhancement with phase plates in

electron microscopy. Adv Imag Electron Phys. 2005;138:

69–146.

Nagayama K. Anti-charging phase plates. JPN-Patent, Tokugan

2012-039409. 2012.

Rochat RH, Liu X, Murata K, Nagayama K, Rixon F, Chiu W.

Seeing the genome packaging apparatus in herpes simplex

virus type I (HSV-1) B-capsids. J Virol. 2011;85:1871–4.

Phase Plate

▶ Phase Contrast Electron Microscopy

Phase Transitions and Phase Behaviorof Lipids

Rumiana Koynova and Boris Tenchov

College of Pharmacy, Ohio State University,

Columbus, OH, USA

Institute of Biophysics, Bulgarian Academy of

Sciences, Sofia, Bulgaria

Synonyms

Lateral phase separation; Lipid phase equilibria; Lipid

self-organization; Lipids; Polymorphic and mesomor-

phic behavior of lipids

Definition

Lipid polymorphism is the ability of lipids to form

more than one solid structure (phase). Mesomorphic

state (phase) is a state of matter intermediate between

liquid and crystal. Lipid phase transitions are intercon-

versions between various polymorphic and mesomor-

phic lipid phases.

Introduction

Lipids constitute a varied and important group of

biomolecules. Most lipids are amphiphilic and can

behave as lyotropic liquid crystals. In the presence of

water, they self-assemble in a large variety of phases

with different structure and morphology. The lipid

polymorphic and mesomorphic behavior, i.e., their

ability to form various ordered, crystalline, gel, or

liquid-crystalline phases as a function of water content,

Phase Transitions and Phase Behavior of Lipids 1841 P

P

temperature and composition, as well as the mutual

transformations between these phases, is the subject of

this entry.

Lipid Phase Nomenclature

Lipid self-assembly in a variety of different phases is

a function of their chemical structure as well as of

external variables such as water content, temperature,

pressure, and aqueous phase compositions. These

phases are made of aggregates of different architecture

(Fig. 1), with the aggregation process being driven by

the hydrophobic effect (see ▶Lipid Organization,

Aggregation, and Self-assembly).

Lipid polymorphic and mesomorphic phases are

characterized by their (1) symmetry in one, two, or

three dimensions; (2) hydrocarbon chain arrangement

in the ordered gel and crystalline phases; and (3) type

(normal or inverted) for the curved mesomorphic

phases. For more than four decades, the nomenclature

introduced by Luzzati has been used to designate

lipid phases (Luzzati 1968). In this nomenclature,

lattice periodicity is characterized by upper-case

Latin letters: L for one-dimensional lamellar lattice,

H for two-dimensional hexagonal lattice, P for

a

d

b c d

e

i

nIII.

II.

I.

q r s

o p

j k l m

f g h

Im3m Pn3m Ia3d

Phase Transitions andPhase Behavior of Lipids,Fig.1 Structures of

lipid phases. I. Lamellar

phases: (a) subgel, Lc; (b) gel,untilted chains, Lb; (c) gel,tilted chains, Lb0; (d)rippled gel, Pb

0; (e)fully interdigitated gel, Lb

int;

(f) partially interdigitated gel;

(g) mixed interdigitated gel;

(h) liquid crystalline, La. II.

Lipid micellar aggregates: (i)spherical micelles, MI; (j)cylindrical micelles (tubules);

(k) disks; (l) inverted micelles,

MII; (m) liposome. III.

Nonlamellar mesomorphic

(liquid crystalline) phases of

various topology: (n)hexagonal phase, HI; (o)inverted hexagonal phase, HII;

(p) inverted micellar cubic

phase, QIIM; (q) bilayer cubic

(QII) phase, Im3m; (r) bilayercubic phase, Pn3m; (s) bilayercubic phase, Ia3d

P 1842 Phase Transitions and Phase Behavior of Lipids

two-dimensional oblique or rectangular lattice, and T,

R, and Q for the three-dimensional rectangular,

rhombohedral, and cubic lattices, with space groups

specified according to the International

Tables (Kasper and Lonsdale 1985). A Greek or

Latin subscript is used as a descriptor of the

chain conformation: a for disordered (liquid crystal-

line), b for ordered (gel), b0 for ordered tilted,

and C for crystalline (subgel). Roman numerals

are used to designate the aggregate type: I for

oil-in-water (normal) and II for water-in-oil

(inverted) type.

Phase Transition Types

Temperature and water content are primary variables

in lipid–water systems, responsible for their thermo-

tropic and lyotropic phase behaviors. Best known is the

phase behavior of diluted lipid–water systems with

water contents sufficient not only for full hydration of

the lipid molecules (so-called excess water limit) but

also for the transitions into mesomorphic phases

with complicated spatial geometry and large internal

aqueous volumes such as the inverted bicontinuous

cubic phases (Fig. 1q–s), which require water contents

well above the excess water limit for their

development.

A generalized phase sequence of thermotropic

phase transitions for membrane lipids (phospholipids

and glycolipids) may be written as (Tenchov 1991):

Lc $ Lb $ La $ QBII $ HII $ QM

II $ MII: (1)

On heating, a lamellar crystalline (subgel) Lc phase

transforms into lamellar gel Lb phase; the latter phase

undergoes a melting transition into the lamellar liquid-

crystalline La phase. Upon further increase of temper-

ature, a series of mesomorphic phase transitions follow

the sequence bilayer cubic QIIB – inverted hexagonal

HII – inverted micellar cubic QIIM – inverted micellar

MII. Some lipids can form two or more modifications

of a given phase, for example, gel phases of different

structures (interdigitated, noninterdigitated, tilted, etc.,

see Fig. 1I.), mesomorphic cubic phases of different

topology (Im3m, Pn3m, etc., see Fig. 1.IIIq–s). Inter-

mediate lipid phases have been reported as well, for

example, the liquid-ordered phase has attracted atten-

tion in the recent years because of its relevance to the

functional lipid rafts in membranes (Simons and

Ikonen 1997).

With increase of the water content at constant tem-

perature, the mesomorphic lipid phases arrange in the

following sequence (Seddon 1990):

inverted phases MII;QIIM;HII;Q

BII

� �$ La

$ normal phases QBI;HI;QI

M� �

$ micellar solution$ monomers: (2)

Typically, double-chain lipids only form La and

inverted phases, while single-chain lipids can also

form normal phases and micellar solutions. The

phase sequence (2) is rationalized by the effect of

water content on the effective shape of the lipid mol-

ecules. Low hydration levels lead to tighter packing

and smaller surface molecular areas of the lipid polar

head groups, resulting in negative interfacial curvature

and a tendency to formation of inverted phases. With

increase of the water content, the surface molecular

area increases; consequently, the concave interfaces of

the inverted phases sequentially transform into the flat

interface of the La phase and into the convex interfaces

of the normal mesomorphic phases. Further dilution

results in dissipation of the periodic lipid structures and

formation of micellar solutions (and eventually mono-

mer solutions at very high dilutions which bring the

lipid concentration below the critical micellar concen-

tration, CMC).

Gel–Liquid-Crystalline Phase Transition

From a biological viewpoint, of greatest interest are

the transitions involving the physiologically

important lamellar liquid-crystalline phase, namely,

the gel–liquid-crystalline (melting) transition, and the

lamellar–nonlamellar mesomorphic transitions.

The lamellar gel–lamellar liquid-crystalline

(Lb–La) phase transition, also referred to as (chain-)

melting, order–disorder, solid–fluid, or main transi-

tion, is the major energetic event in the lipid bilayers,

taking place with a large enthalpy change. It is associ-

ated with rotameric disordering of the hydrocarbon

chains, increased head group hydration and increased

intermolecular entropy (Nagle 1980). The energy

required to expand the hydrocarbon chain region

against attractive van der Waals interactions (volume

Phase Transitions and Phase Behavior of Lipids 1843 P

P

expansion) and to increase bilayer area (increased

hydrophobic exposure at the polar–apolar interface)

contributes to the large transition enthalpy change.

The melting gel–liquid-crystalline transitions in

fully hydrated lipids are accompanied by large

increases in lipid surface area (�25%) and specific

volume (�4%). In calorimetric measurements, they

manifest as sharp, narrow heat capacity peaks

with enthalpy of �20–40 kJ/mol (Marsh 1990)

(see▶Differential Scanning Calorimetry (DSC), Pres-

sure Perturbation Calorimetry (PPC), and Isothermal

Titration Calorimetry (ITC) of Lipid Bilayers). Also,

large volume fluctuations give rise to a strong increase

of the isothermal bilayer compressibility at the melting

transition temperature (Schrader et al. 2002). As a

result of a dramatic increase of the bending elasticity,

large bilayer undulations (anomalous swelling) have

been observed at the melting transition (Honger et al.

1994; Chu et al. 2005; Pabst et al. 2004).

The temperature of the chain-melting transition is

determined largely by the hydrocarbon chains – the

longer and more saturated they are, the higher the

transition temperature (Fig. 2a). For lipids with unsat-

urated chains, the position and type of the double bond

substantially modulate the melting temperature

(Fig. 2b). Additionally, the melting temperature is

affected also by chain branching and by the chemical

linkage between the chains and the polar head group.

Anhydrous lipids with identical hydrocarbon chains

exhibit melting phase transitions at nearly identical

temperatures. In aqueous dispersions, however, the

head group interactions and the lipid–water interac-

tions largely modify the lipid phase behavior.

A summary of the phase transition temperatures of

the major membrane lipid classes with different chain

lengths is given in Table 1.

Most of the membrane lipids have two different

hydrocarbon chains, usually one saturated and one

unsaturated; most common are the glyceropho-

spholipids with saturated sn-1 chain typically 16–18

carbon atoms long, and unsaturated sn-2 chain typi-

cally 18–20 carbon atoms long. The gel–liquid-

crystalline (Lb ! La) transition temperatures of

mixed-chain phosphatidylcholines are summarized in

Table 2. It is evident from these data that altering the

lipid chain length and unsaturation modulates the lipid

phase state in very broad limits, thus providing the

basis of a mechanism for membrane adaptation to

large fluctuations in the environmental temperatures.

Formation of Nonlamellar Phases inMembrane Lipids

Dispersions of double-chain nonlamellar membrane

lipids most frequently display a lamellar–inverted-

hexagonal, La–HII, phase transition. In some instances,

they can also form inverted phases of cubic symmetry.

Important role in the lamellar–nonlamellar transforma-

tions plays the membrane elastic energy (Siegel 2005;

Gruner 1994).

The La–HII transition may be considered as a result

of competition between the spontaneous tendency of

the lipid layers to bend and the resulting hydrocarbon

0

0

20

40

0

10 12 14 16Chain length

18 20 22

20

40

60

80

100

120a

b

–20

2 4 6 8

Position of double bond

La

La

Lb

Lb

H||

Tem

pera

ture

[°C

]Te

mpe

ratu

re [°

C]

10 12 14 16 18

Phase Transitions and Phase Behavior of Lipids,Fig. 2 Dependence of the phase transitions temperature on

lipid chemical structure: (a) hydrocarbon chain length depen-

dence of the Lb–La (black squares) and La–HII (open circles)phase transition temperatures in saturated diacyl phosphatidyl-

ethanolamines (Seddon et al. 1983; Koynova and Caffrey 1994)

and (b) dependence of the Lb–La phase transition temperature on

the double-bond position for dioctadecenoyl phosphatidylcho-

line bilayers (Koynova and Caffrey 1998)

P 1844 Phase Transitions and Phase Behavior of Lipids

chain packing strain – thus, membranes exist in a state

of frustrated curvature stress (Gruner 1985). Respec-

tively, the La–HII transition is believed to be driven by

the relaxation of the curvature of the lipid monolayers

toward their spontaneous curvature. Oppositely to the

Lb–La transition, the La–HII transition temperature

Phase Transitions and Phase Behavior of Lipids, Table 1 Gel–liquid-crystalline and lamellar–nonlamellar phase transition

temperatures (�C) of fully hydrated lipids as a function of the lipid polar head group and hydrocarbon chain length

Head group PC PE PG PS PA PI CL Glc Gal Mal N-Sph PC N-Sph GalChains sn-1/sn-2

Gel–liquid-crystalline transitions (Lb! La)

10:0/10:0 �5.7 2.0 �6.911:0/11:0 �13.9 16.9 1.9

12:0/12:0 �1.9 31.3 �2 14.2 32 25.4 19.5 26.8

13:0/13:0 13.5 42.1 10.7 32.9

14:0/14:0 23.4 50.4 24 35.4 54 20 40 40.5 48.7 40.9 25

15:0/15:0 33.7 58.4 33 58 50.7 42

16:0/16:0 41.7 64.4 41.5 51.4 65 58 57.2 61.6 56.6 44.5 82

17:0/17:0 48.6 70.5 63.4 46.5

18:0/18:0 54.5 74.2 54 63.7 75.4 70 68.4 73.5 66.7 44.5 84

19:0/19:0 60.2 79.2 73.7a 47.5

20:0/20:0 65.3 83.4 76.8a 80.0a 83

21:0/21:0 70.7

22:0/22:0 73.6 90.0a

23:0/23:0 77.9

24:0/24:0 80.3

16:1c9/16:1c9 �4.0 �33.518:1c9/18:1c9 �18.0 �7.3 �18.3 �11.0 �11.018:2c9,12/18:2c9,12 �55.118:3c9,12,15/18:3c9,12,15 �63.0Lamellar–nonlamellar transition (La! HII, unless otherwise indicated)

14:0/14:0 105b 80.6b

15:0/15:0 82b

16:0/16:0 118.5 79b;

119c80.7b;

82.3d

17:0/17:0 107.6 76.6

18:0/18:0 101 74.5;

73.9d76.0;

85.0d

19:0/19:0 97.8 73.7a

20:0/20:0 94.2 76.8a;

78.9d80.0a;

89.0d

22:0/22:0 90.0a

16:0/18:1c9 70.8

16:1c9/16:1c9 43.4

18:1c9/18:1c9 8.5

18:1 t9/18:1 t9 62.2

18:1c9/20:4c5,8,11,14 32.0

PC, diacylphosphatidylcholines, PE diacylphosphatidylethanolamines, PG diacylphosphatidylglycerols, PS diacylphosphati-

dylserines, PA diacylphosphatidic acids, PI diacylphosphatidylinositols, CL cardiolipins,Glc diacylglucosylglycerols,Gal diacylga-lactosylglycerols, Mal dialkylmaltosylglycerols, N-Sph PC sphingomyelins, N-Sph Gal galactocerebrosides (for the sphingolipids,chain length refers to the single fatty acid chain)aLb! HIIbLa! QIIcQII! HIIdLc! HII transition

Phase Transitions and Phase Behavior of Lipids 1845 P

P

decreases with the hydrocarbon chain length increase

(Fig. 2a). At sufficiently long chains, the La phase is

completely eliminated, and direct Lb–HII transitions

take place on heating. Such direct transitions have

been observed for diacyl PEs of 22-carbon chains and

monoglycosyldiacylglycerols of 19–20-carbon chains

(Table 1). With long-chain glycolipids, a direct Lc–HII

transition is even observed, where both the Lb and La

phases are eliminated from the phase sequence. Inter-

estingly, intermediate phases missing on heating may

intervene in the cooling phase sequence.

Among the seven cubic phases so far identified in

lipids, of significant interest are the inverted

bicontinuous or bilayer cubic phases with space groups

Q229 (Im3m), Q224 (Pn3m), and Q230 (Ia3d) (Lindblom

and Rilfors 1992; Luzzati et al. 1997) (Fig. 1.IIIq, r, s).

Whenever present, the bilayer cubic phases are located

in a temperature range between the La and the HII

phases. However, direct La–QIIB transitions are rare

in membrane lipid dispersions and mainly observed for

short-chain PEs and monoglycosyldiacylglycerols. In

many cases, a QIIB phase can be induced by means of

temperature cycling through the La–HII transition or

by cooling of the HII phase (Shyamsunder et al. 1988;

Tenchov et al. 1998). A transformation from lamellar

into bilayer cubic phase may be considered as cooper-

ative act of multiple fusion events, whereby a set of

initially separate, parallel bilayers fuse into a single

bilayer of specific topology. The lamellar–cubic tran-

sitions have very small, if any, latent heats. Although

energetically inexpensive, these transitions are typi-

cally rather slow. The slow formation, hysteretic

behavior, and extended metastability ranges of the

cubic phases create significant difficulties in their

study and applications.

Inverted micellar cubic phases have been observed

mainly in mixtures of double-chain polar lipids

with fatty acids or diacylglycerols, and also in some

Phase Transitions and Phase Behavior of Lipids,Table 2 Decrease of the gel–liquid-crystalline (Lb! La) tran-

sition temperatures of fully hydrated acyl-chain phosphatidyl-

cholines with increasing sn-2 chain unsaturation. The first cisdouble bond causes the biggest transition temperature drop to

occur, while further increases of chain unsaturation have much

smaller effects

Chains sn-1/sn-2 Temperature (�C)16:0/16:0 41.7

16:0/16:1c9 30.0

16:0/18:0 49.0

16:0/18:1c9 �2.516:0/18:2c9,12 �19.616:0/20:0 51.3

16:0/20:4c5,8,11,14 �22.516:0/22:0 52.8

16:0/22:1c13 11.5

16:0/22:6c4,7,10,13,16,19 �3.018:0/18:0 54.5

18:0/18:1c9 6.9

18:0/18:2c9,12 �14.418:0/18:3c9,12,15 �12.318:0/20:0 60.4

18:0/20:1c11 13.2

18:0/20:2c11,14 �5.418:0/20:3c8,11,14 �9.318:0/20:4c5,8,11,14 �12.918:0/20:5c5,8,11,14,17 �10.418:0/22:0 61.9

18:0/22:1c13 19.6

18:0/22:4c7,10,13,16 �8.518:0/22:5c4,7,10,13,16 �6.418:0/22:6c4,7,10,13,16,19 �3.818:0/24:0 62.7

18:0/24:1c15 31.8

20:0/18:0 57.5

20:0/18:1c9 11.5

20:0/20:0 65.3

20:0/20:1c11 20.5

20:0/20:2c11,14 5.4

20:0/20:3c11,14,17 1.8

20:0/20:4c5,8,11,14 �7.520:0/22:0 69.6

20:0/22:1c13 29.2

20:0/24:0 70.6

20:0/24:1c15 36.6

22:0/18:0 58.6

22:0/18:1c9 15.1

22:0/20:0 67.7

22:0/20:1c11 22.9

22:0/22:0 73.6

(continued)

Phase Transitions and Phase Behavior of Lipids, Table 2(continued)

Chains sn-1/sn-2 Temperature (�C)22:0/22:1c13 32.8

22:0/24:0 77.1

22:0/24:1c15 41.7

24:0/18:0 58.9

24:0/18:1c9 20.7

24:0/20:0 68.4

24:0/20:1c11 24.5

P 1846 Phase Transitions and Phase Behavior of Lipids

single-component dispersions of glycolipids (Seddon

et al. 2000). The most frequently observed inverted

micellar cubic phase in lipids is of space group Q227

(Fd3m). For medium-chain lipids (�16 C atoms), it

typically forms via an HII–QIIM transition.

Polymorphic Transitions Between SolidLipid Phases

At temperatures below the main transition, a basic

equilibrium structure is the subgel (crystalline) Lc

phase. Its formation usually requires prolonged low-

temperature incubation. In addition to the Lc phase,

a large number of intermediate stable, metastable, and

transient lamellar gel structures are adopted by

different lipid classes – with perpendicular or tilted

chains with respect to the bilayer plane, with fully

interdigitated, partially interdigitated, or noninter-

digitated chains, rippled bilayers with various ripple

periods, etc. (Fig. 1). A number of polymorphic phase

transitions between these structures have been

reported. Examples of such polymorphic transitions

are the subtransition (Lc–Lb0) and the pretransition

(Lb0–Pb0) in phosphatidylcholines. A polymorphic

transition including rapid, reversible transformation

of the usual gel phase into a metastable, more ordered

gel phase with orthorhombic hydrocarbon chain pack-

ing (so-called Y-transition) was reported to represent

a common pathway of the bilayer transformation into

subgel (crystalline) Lc phase (Fig. 3) (Tenchov et al.

2001).

a

Lα

LR1 LR1

Lc Lc Lc

Temperature

6 nearest neighbours 4 nearest neighbours 2 nearest neighbours

d-110

d-110

d110

d200

d200a

a

bb d110

b

a

Lc

SGII

Lα Lα Lα

Lβ Lβ Lβ

Lβ�

Pβ�

b c d

ΔH

e f g

Phase Transitions and Phase Behavior of Lipids,Fig. 3 Transition pathways in fully hydrated lipids. Schemes

(a) and (b) are representative for PEs of intermediate chain

length and for protonated DPPG; scheme (c), for short-chainPEs and PGs; and scheme (d), for the PCs. Solid horizontal linesindicate stable phases and dashed lines indicate metastable

phases. The wavy lines represent isothermal relaxation into the

Lc phase. LR1 and SGII are metastable ordered gel phases with

orthorhombic hydrocarbon chain packing. Schemes of the

hydrocarbon chain packing: (e) hexagonal packing, (f) ortho-rhombic packing with four nearest neighbors, and (g) ortho-

rhombic packing with two nearest neighbors (Reproduced from

Tenchov et al. (2001). With permission of the Biophysical

Society)

Phase Transitions and Phase Behavior of Lipids 1847 P

P

Reversibility of the Phase Transitions:Transition Hysteresis and Formation ofMetastable Phases

Due to long relaxation times, especially in the transi-

tion vicinity, lipid phase transitions often display pro-

nounced hysteresis and end up with formation of

metastable phases, which replace the equilibrium

phases in cooling scans. The metastable phases can

be long-lived and display no spontaneous conversion

to the ground state on reasonable time scales.

A large number of rate-limiting factors have been

suggested as physical reasons leading to hysteretic

behavior and formation of metastable phases: long

hydration/dehydration times, slow reformation of

hydrogen-bond networks, restricted molecular motion

in the low-temperature solid–solid transformations,

relative stability of the interfaces between solid and

fluid domains, large spatial rearrangements in lamel-

lar–nonlamellar transitions, low rate of appearance of

critical-size nuclei of the nascent phase, and arrestment

in local free energy minima. A comparison between

heating and cooling phase sequences observed in aque-

ous dispersions of lipids shows the frequent occurrence

of additional, metastable phases, which only form in

the cooling direction (Table 3).

Phase Transitions in Lipid Mixtures: PhaseDiagrams

Composition is another important variable that

strongly modulates the lipid phase behavior. The

molecular interactions in a lipid mixture are reflected

Phase Transitions and Phase Behavior of Lipids, Table 3 Examples of heating, cooling, and isothermal phase sequences in

lipid dispersions (Tenchov 1991)

Lipid Scan direction Phase sequence

DPPC Heating Lc1 �13�C������!Lb0 �35�C������!Pb0 �41:5�C������!La

Cooling

Isothermal equilibration

La �41:5�C������! Pb0(mst)! Lb0 ! Lc2

<8�C Lc2! Lc1

DLPE 1st heating Lc1 �43�C������! La

Cooling La �30�C������!Lb

Isothermal equilibration 2�C, 9 days Lb! Lc1

Isothermal equilibration 26�C, 15 h Lb! Lc2

Isothermal equilibration 32�C, 15 h La! Lc1

DMPE 1st heating Lc �56�C������! La

Cooling La �49�C������! Lb

Isothermal equilibration 2�C, 9 days Lb! Lc

DOPE Heating Lb ��16�C������! La �8�C������!HII

Cycling (n > 100) (La ↔HII)n! QII

Deep cooling (<�20�C) QII! Lb

DOPE-Me Heating (>1�C/h) Lb �<0�C������! La �67�C������!HII

Heating (<1�C/h) La �62�C������!QII �72�79�C������!HII

Isothermal equilibration 55�C, 20 h La! disordered state! QII

Deep cooling (<�20�C) QII! Lb

14-Gal 1st heating Lc1! Lc2 �69�C������!HII

Cooling HII �60�C������! La �47�C������! Lb �38�C������!Lc2

2nd heating Lc2 �69�C������!HII

18-Gal 1st heating Lc1! Lc2 �78�C������!HII

Cooling HII �67�C������! Lb

2nd heating Lb��������������������!exotherm;�43�CLc2 �78�C������!HII

Isothermal equilibration 60–70�C, 1 h Lb! Lc2

DPPC dipalmitoyl PC,DLPE dilauroyl PE,DMPE dimyristoyl PE,DOPE dioleoyl PE,DOPE-Me dioleoyl-N-methylethanolamine,

14-Gal ditetradecylgalactosylglycerol, 18-Gal dioctadecylgalactosylglycerol

P 1848 Phase Transitions and Phase Behavior of Lipids

in its phase behavior and are best presented by means

of a temperature–composition phase diagram (see

▶Thermodynamics of Lipid Interactions). Various

types of lipid phase diagrams reported in the literature

are shown in Fig. 4. The lens-like diagram in Fig. 4a is

characteristic for lipid mixtures which are completely

miscible in both gel and liquid-crystalline phases. In

order to display such complete miscibility, the two

components must be structurally very similar. This

kind of diagram is typical for lipid species with the

same head group, differing by not more than two

methylene groups in their hydrocarbon chains, such

as the DMPC/DPPC binary (Fig. 4a). A usual compli-

cation of the lens-type diagram is the frequently

DMPC / DPPC

a

d ef

ihg

b c

DEPC / DPPE

DEPC / DMPCDMPE / DSPE

L

L+S

25

15

50 20 40

Mole% diC 14PC

Mole% C12C24PC

S1+S2

L

L+S1

L+S2

C18C10PC / diC14PC

S2S1

60 80 100

0 50 100

20

30

40

50DPPC / C12C24PC

Lα

Pβ�

Pβ�+SI

Pβ�+Sc

Lβ�+Sc

Lβ� Pβ�

Lα

XαXα

Lβ�

Lc+ScLc

S

40

35

30

Tem

pera

ture

[°C

]T

empe

ratu

re [°

C]

Tem

pera

ture

[°C

]T

empe

ratu

re [°

C]

Tem

pera

ture

[°C

]

Tem

pera

ture

[°C

]

Tem

pera

ture

[°C

]

Tem

pera

ture

[°C

]

25

0

0

50

60

60

60

50

40

300 50 100

50

45

40

35 so

so - lo

so - ld

ld

ld - lo

lo

0 10 20 30 40

Mole% Palmitic acidMole% Cholesterol

DPPC / Cholesterol

Cholesterol1.0

1.0

0.9Lo+

Chol.crystals

Lo

LoLα + LoLα + Lo

Lα

Lα + Lβ + LoLβ

+Lβ

Lα + Lb

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.90.80.70.60.50.40.30.20.10.0

0.0DOPC DSPC

DPPC / Palmitic acid

C

HII I

25

20

15

10

0 25 50 75 100

L1L2

L1+L2L2+S

L1+S

S

S

40

20

00 50 100

70

25 50 75 100

20 40 60

Mole% DPPC

Mole% DPPE

Mole% DMPC

Mole% DSPE

80 100

Phase Transitions and Phase Behavior of Lipids, Fig. 4 (continued)

Phase Transitions and Phase Behavior of Lipids 1849 P

P

occurring solid-state miscibility gap, where the mix-

ture separates into two solid phases of different com-

position. In mixtures of lipids with sufficiently

different structures, the miscibility gap may overlap

with the region of the solid–liquid-crystalline phase

coexistence, giving rise to eutectic (Fig. 4b, c) or

peritectic phase diagrams (Fig. 4d), in which single

three-phase points exist. Horizontal solidus lines,

reporting for such kind of behavior, have been

observed for numerous lipid mixtures. Miscibility

gaps may also occur in the liquid-crystalline phase of

certain lipid mixtures. A phase diagram with a liquid–

liquid immiscibility region is the monotectic phase

diagram shown in Fig. 4e, with a monotectic triple

point of coexistence of one solid and two liquid phases.

Deviations from ideal mixing may occur not only

with tendency for clustering of the like molecules,

eventually leading to phase separation, but also when

contacts between unlike molecules are preferred, i.e.,

when the nearest-neighbor pairs tend to be made up

of unlike molecules (“chessboard” arrangement)

(see ▶Thermodynamics of Lipid Interactions). Such

mixtures often display phase diagrams with upper

isoconcentration (azeotropic) point – such as the

DPPC/palmitic acid diagram shown in Fig. 4g. Except

for the phosphatidylcholine/fatty acid mixtures, phase

diagrams with upper isoconcentration point are typical

for mixtures containing charged lipid. An example of

a phase diagram with lower isoconcentration point is

shown in Fig. 4f.

The DPPC/cholesterol phase diagram in Fig. 4h

contains a critical point. It is related to the existence

of a peculiar, liquid-ordered (lo) phase in the mixtures,

which is believed to be the prototype of the lipid rafts.

Ternary phase diagrams are another important tool

for characterization of the phase properties of complex

lipid mixtures. These kinds of diagrams have proven

especially useful recently, in the analysis of domains in

model systems (see ▶Lipid Domains). Ternary mix-

tures of one phospholipid having a relatively high

melting temperature and another phospholipid having

a relatively low melting temperature together with

cholesterol are viewed as useful models for the outer

leaflet of animal-cell plasma membranes. An example

of a ternary phase diagram is shown in Fig. 4i. It

illustrates the rich phase behavior displayed by ternary

lipid mixtures represented in this particular case by

four regions of two-phase coexistence and one region

of three-phase coexistence.

Role of the Aqueous Phase Composition

The Hofmeister Effect. The interactions of the lipid

polar groups with water have an important contribution

to the energy balance of a given phase. Many of the

lipid phase transitions take place with large changes in

the lipid surface area and consequently in the amount

of bound water. The lipid hydration is determined by

the chemical structure of the polar groups, but in fully

hydrated systems, with water in excess, the extent of

the polar group hydration depends also on the state

of the bulk water. On the other side, various

low-molecular solutes are known for their ability

��

Phase Transitions and Phase Behavior of Lipids,Fig. 4 Types of lipid phase diagrams: (a) close to ideal phase

diagram of the DMPC/DPPC mixture (Reproduced from Mabrey

and Sturtevant (1976) with author’s permission); (b) eutectic

phase diagram of the 1-stearoyl-2-caprylphosphatidylcholine/

DMPC mixture (Reproduced from Lin and Huang (1988). With

permission from Elsevier); (c) phase diagram of the DPPC/

C12C24PC mixture exhibiting eutectic and peritectic points

(Reproduced from (Gardam and Silvius 1989) with permission

from Elsevier); (d) peritectic phase diagram of the DMPE/DSPE

mixture (Reproduced from (Dorfler 2000) with permission of

Springer Science and Business Media); (e) monotectic phase

diagram of the DEPC/DPPE mixture (Reproduced with permis-

sion from (Wu andMcConnell 1975); copyright (1975) American

Chemical Society); (f) phase diagram with a lower

isoconcentration (azeotropic) point) of the DEPC/DMPCmixture

(Reproduced from (VanDijck et al. 1977) with permission from

Elsevier); (g) phase diagram of the DPPC/palmitic acid mixture,

combining upper isoconcentration (azeotropic) point with eutectic

and peritectic points (Reproduced from (Inoue et al. 2001) with

permission from Elsevier); (h) phase diagram with a critical point

of the DPPC/cholesterol mixture (Reproduced from (Ipsen et al.

1990) with permission of the Biophysical Society); (i) phase

diagram of the ternary mixture distearoylphosphatidylcholine

(DSPC)/dioleoylphosphatidylcholine (DOPC)/cholesterol at

23�C, showing four regions of two-phase coexistence: liquid

crystalline and gel (La + Lb), liquid ordered and gel (Lo + Lb),

liquid crystalline and liquid ordered (La + Lo), and liquid ordered

and crystals of cholesterol monohydrate; there is also one region

of three-phase coexistence, liquid crystalline, gel, and liquid

ordered (La + Lb + Lo) (Reproduced from (Feigenson 2006)

with permission)

P 1850 Phase Transitions and Phase Behavior of Lipids

to strongly modulate the bulk water structure – “water-

structure makers” (kosmotropes) and “water-structure

breakers” (chaotropes). It thus turns possible that, even

without direct interaction with the lipid polar heads,

solutes can largely modulate the properties of the

lipid–water interface and hence the lipid phase behav-

ior. Changes in the aqueous phase composition are able

to shift substantially the temperature regions of stabil-

ity of the different lipid phases as well as to induce or

suppress the formation of certain phases. Indirect sol-

ute effects of such kind on the interfacial properties,

generally termed the Hofmeister effect, have been

found in a large number of lipid–water phases

(Koynova et al. 1997). A great number of studies on

the nature of the Hofmeister effect indicate that it

results from an interplay of electrostatics, dispersion

forces, thermal motion, fluctuations, hydration, polar-

izability, ion size effects, and the impact of interfacial

water (Collins and Washabaugh 1985).

According to their effect on the lipid phase transi-

tions, the Hofmeister solutes fall into two categories:

(1) chaotropic solutes favoring formation of the lamel-

lar liquid-crystalline phase La at the expense of the

neighboring HII and Lb phases and (2) kosmotropic

solutes favoring formation of HII and Lb phase at the

expense of the La phase. Their effects are correctly

described by an equation of Clapeyron–Clausius type

between phase transition temperature and solute con-

centration. The sign and magnitude of the transition

shifts induced by the different solutes depend on the

solute ability to unevenly distribute between

interlamellar and free water. Kosmotropic solutes

tend to minimize the area of the lipid–water contact.

They suppress the La phase, as it has the largest surface

area in contact with water. At high enough concentra-

tion of kosmotropic solutes, the latter phase may

completely disappear from the phase diagram. This is

Phase Transitions and Phase Behavior of Lipids,Table 4 Effect of solutes on the phase transition temperatures

of hydrated PE (Koynova et al. 1997)

Lipid Solute Slope (dTtr/dc) [K/M]

Lb-La La-HII

Salts

DMPE NaCl 1.5 (1–4 M) �8.0 (3–6 M)

DPPE NaCl 2.0 �7.4CaCl2 3.4

POPE Na2SO4 �16.0NaCl �7.3NaBr �3.5NaI 6.0

GuHCl 8.0

NaSCN 27.0

DSPE NaCl 1.3 �4.3DEPE Na2SO4 1.7 �9.9

NaOAc 1.5 �9.1NaCl 1.3 �5.6NaSCN �3.3 20.0

GuHCl �4.6 5.0

GuHSCN �6.5 50.0

DTPE NaCl 1.2 �3.8DHPE NaCl 1.3 �2.5

Monosaccharides

DEPE Glucose �0.2Galactose �0.6Fructose �2.5Sugar alcohols

POPE Sorbitol �9.0DEPE Sorbitol �5.4

Myo-inositol �5.5Other polyols

POPE Glycerol �3.3DSPE Glycerol 0.9 �1.0DEPE Glycerol �2.8

PEG 0.2

Disaccharides

POPE Sucrose �16.5Raffinose �30.0

DEPE Sucrose �12.6Trehalose �10.8Lactose �10.0Maltose �9.0

DMPE Sucrose 0.3

DSPE Sucrose 1.4 �8.4Trehalose 1.1 �8.7Amino acids

DHPE Proline 0.9 �1.8(continued)

Phase Transitions and Phase Behavior of Lipids, Table 4(continued)

Lipid Solute Slope (dTtr/dc) [K/M]

Lb-La La-HII

Others

POPE Urea 1.3

DPPE dipalmitoyl PE, POPE palmitoyl-oleoyl PE, DSPEdistearoyl PE, DEPE dielaidoyl PE, DTPE ditetradecyl PE,

DHPE dihexadecyl PE

Phase Transitions and Phase Behavior of Lipids 1851 P

P

precisely what is seen with sucrose, trehalose, proline,

and some salts and is consistent with the opposite

effect caused by chaotropic solutes (Table 4) (Tenchov

and Koynova 1996). Addition of chaotropic solutes

can also induce the appearance of missing liquid-

crystalline phases from the general lipid phase

sequence (1).

Effect of pH. Changes in pH modulate the lipid

phase behavior as a consequence of protonation/

deprotonation of the lipid head groups, resulting in

a change of the surface charge of the membrane

(Trauble et al. 1976). They also modify the

surface polarity and hydration. Typically, protonation

decreases lipid hydration and increases the main

transition temperature (Cevc and Marsh 1987). The

effects of pH titration on the chain-melting transition

temperature Tm of dimyristoyl phospholipids is illus-

trated in Fig. 5a, showing that single protonation

increases the melting transition temperature by about

5–15�C.The shifts of the lamellar–hexagonal transition

upon titration are greater and in the opposite direction

relative to changes of Tm. Thus, for didodecyl PE, the

lamellar–hexagonal transition decreases by 41�C upon

phosphate protonation (pK�1.9) and by 50�C upon

amine protonation (pK�9.3), while for Tm, these shifts

are 6�C and 15�C, respectively, in the opposite direc-

tion (Fig. 5b) (Seddon et al. 1983).

Biological Relevance of Lipid Phase Behavior

Biological roles of lipids are varied – in energy storage

and fat digestion, as enzyme cofactors, electron car-

riers, light-absorbing pigments, intracellular messen-

gers, hormones, as constituents of the pulmonary

surfactant and the skin stratum corneum (see

▶ Skin Lipids), etc. The issue of their phase behavior

in water is particularly relevant to their major struc-

tural function – as building blocks of the biological

membranes (see ▶ Functional Roles of Lipids in

Membranes).

The major discovery in the field of biological mem-

branes is undoubtedly the finding that the

biomembrane is a liquid-crystalline lipid bilayer with

embedded proteins (Singer and Nicolson 1972;

Shimshick and McConnel 1973; Trauble and

Sackmann 1972). This so-called fluid-mosaic model

has been a central paradigm in membrane biophysics

for more than three decades, and it has been very

successful in rationalizing a large body of experimen-

tal observations. The model includes two basic refer-

ences to the lipid phase state – liquid crystalline and

bilayer – both of which are of vital importance for the

PA, MPAa

b

PS+, PE+

PG, PC(+), PS+ –60

50

40

30

20

Tem

pera

ture

[°C

]T

empe

ratu

re [°

C]

10

160

120

80

40

0

0pH

pH

DDPE

HII

Lα

Lβ

2 4 6 8 10 12 14

0 2 4 6 8 10 12 14

PA–

PE+ –

PS–

MPA–

PG–

PC+ –

1,2-dimyristoylglycerophospholipids, 0.1M salt

PG–, PC+ –PS– –, PE–PA– –, MPA–

PG–, PC+ –

Phase Transitions and Phase Behavior of Lipids,Fig. 5 pH. TIF. Dependence of the phase transitions tempera-

ture on pH: (a) chain-melting transition temperature of

dimyristoyl glycerophospholipid bilayers (superscripts give the

lipid charge; abbreviations as in Table 1; MPA, methylpho-

sphatidic acid) (Cevc and Marsh 1987); (b) gel-fluid (circles)and lamellar–hexagonal (squares) phase transition temperatures

of didodecylphosphatidylethanolamine; the dashed line indi-

cates the appearance of additional lines in the region of the

lamellar–hexagonal transition (Reproduced with permission

from (Seddon et al. 1983); copyright (1983) American Chemical

Society)

P 1852 Phase Transitions and Phase Behavior of Lipids

proper functioning of membranes. The liquid-

crystalline bilayers can arrange in stacks with

interbilayer aqueous spaces, thus building up the

lamellar liquid-crystalline phase La. However, in addi-

tion to the La phase, the membrane lipids are also able

to form a large variety of other phases (subgel, gel,

cubic, hexagonal, micellar). All these phases are inter-

related and transform into each other via different

kinds of phase transitions. On the temperature scale,

the existence range of the La phase is typically limited

from above by lamellar–nonlamellar transitions into

cubic, hexagonal, and micellar phases, and limited

from below by fluid–solid transitions into gel and

subgel phases. A remarkable from biological view-

point property of the lipid dispersions is that the tran-

sition temperatures limiting the stability ranges of the

different phases can be altered by tens of degrees by

varying the composition of the lipid–water system.

The possibility to modulate the lipid phase behavior

in very broad limits by varying the lipid composition

appears to represent the basis of important regulatory

mechanisms involved in the biomembrane responses

to external stimuli such as changes in the environmen-

tal conditions, as well as for the regulation of various

membrane-associated processes.

Summary

Lipids constitute a varied group of biological mole-

cules of diverse biological roles. Their major function

is as building blocks of the biological membranes.

According to the fluid-mosaic model, biomembranes

are liquid-crystalline lipid bilayers with embedded

proteins. This model includes two references to the

lipid phase state – liquid crystalline and bilayer –

both of which are of vital importance for the proper

membrane operation. Except for the lamellar liquid-

crystalline “membrane” phase, the amphiphilic mem-

brane lipids are able to form also a large variety of

other phases, which transform into each other via dif-

ferent kinds of phase transitions. The impressive vari-

ety of lipids in the biomembranes includes a large

fraction of species that, in isolation, prefer to adopt

curved, hexagonal, cubic, or micellar phases, rather

than the lamellar phase. The physiological importance

of lipid diversity and mesomorphism stems from the

possibility to finely tune and optimize the collective

properties of the biomembranes.

Cross-References

▶Atomic Force Microscopy of Lipid Membranes

▶Critical Fluctuations in Lipid Mixtures

▶Differential Scanning Calorimetry (DSC), Pressure

Perturbation Calorimetry (PPC), and Isothermal

Titration Calorimetry (ITC) of Lipid Bilayers

▶ Functional Roles of Lipids in Membranes

▶Hierarchically Structured Lipid Systems

▶ Infrared Spectroscopy of Membrane Lipids

▶Lipid Domains

▶Lipid Organization, Aggregation, and Self-

assembly

▶Micropipette Manipulation of Lipid Bilayer

Membranes

▶ Skin Lipids

▶Thermodynamics and Thermodynamic Nonideality

▶Thermodynamics of Lipid Interactions

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Phoborhodopsin

▶ Sensory Rhodopsin II: Signal Development and

Transduction

Phosphatases – Computational Studies

Marc Willem van der Kamp

Centre for Computational Chemistry, School of

Chemistry, University of Bristol, Bristol, UK

Definition

Enzymes that catalyze the hydrolysis of phosphate

esters from a variety of phosphate-containing substrates.

Basic Characteristics

Phosphatases are a large group of diverse enzymes.

One important role is their involvement in the

most common form of reversible posttranslational

P 1854 Phoborhodopsin