P2X Receptors for ATP: Molecular Properties and

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P2X Receptors for ATP: Molecular Properties and Functional Roles Richard J. Evans Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK

Synonyms Purinergic receptors

are several ways in which ATP levels in the extracellular space can increase. For example, ATP is costored and co-released with classical neurotransmitters like noradrenaline, and it is released following shear stress in blood vessels as well as from damaged cells. The extracellular ATP acts at cell surface purinergic P2 receptors to mediate physiological responses ranging from muscle contraction to sensory function (Khakh and North 2006). The P2 receptors can be divided into G-protein-coupled P2Y receptors and ligand-gated P2X receptor ion channels (Burnstock 2006).

Definition The P2X Receptor Family P2X receptors for ATP comprise a family of ligand-gated cation channels that are distinct from the cys-loop and glutamate channel families. They are expressed in one form or another by almost all cell types and play physiological roles ranging from pain sensation to blood clotting. This entry gives an overview of purinergic transmission, the basic properties of P2X receptors, and finally summarizes the main features and roles of molecularly defined receptor subtypes.

Introduction to Purinergic Transmission Adenosine triphosphate (ATP) is widely thought of as an important intracellular energy carrier, but in addition it also acts as an extracellular signaling molecule. This concept of purinergic signaling was initially championed by Prof Burnstock in the 1970s (for a historical review see (Burnstock 2006)). There

The P2X receptors were initially identified at the molecular levels by expression cloning in 1994, and subsequently a total of seven mammalian P2X receptor subunits (P2X1-7) have been described (Roberts et al. 2006; Stojilkovic et al. 2005). P2X receptor genes have been isolated from a range of other organisms, including algae, indicating a long evolutionary history. However, interestingly no homologues have been found in Caenorhabditis elegans or Drosophila (Fountain and Burnstock 2009). The crystal structure of a zebrafish P2X4 (zP2X4) receptor in an ATP-free closed state (Kawate et al. 2009) shows the receptor forms as a trimer from individual subunits that have two transmembrane domains, intracellular amino and carboxy termini, and a large extracellular ligand-binding loop (Fig. 1). This membrane topology and trimeric assembly is similar to that of the acid-sensing ion channels but the receptors share no homology at the amino acid level. Thus, the stoichiometry and topology of these

G.C.K. Roberts (ed.), Encyclopedia of Biophysics, DOI 10.1007/978-3-642-16712-6, # European Biophysical Societies’ Association (EBSA) 2013

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P2X Receptors for ATP: Molecular Properties and Functional Roles

P2X Receptors for ATP: Molecular Properties and Functional Roles, Fig. 1 P2X receptor structure shows the trimeric nature of the protein and sites associated with ATP and antagonist binding. Homology models of the P2X1 receptor based on the zP2X4 receptor structure (Kawate et al. 2009) are shown. Left panel shows the three P2X receptor subunits, two are shown as surface representations (white and gray) and the third subunit

(blue) is shown in cartoon format. The subunits each have two transmembrane (TM) spanning helices with the second (TM2) lining the pore region of the channel. Right panel shows a surface rendering of the P2X receptor mapping residues that when mutated had an effect on ATP potency (red) or the sensitivity to the antagonist PPADS (magenta). The proposed ATP-binding site (red) forms at the interface between two adjacent subunits

receptors are distinct from the pentameric nicotinic/cys loop, and tetrameric glutamate families of ionotropic receptors.

ATP-binding sites within the receptor. This is consistent with modeling and analysis of the Hill slope of the initial part of the ATP concentration response curves indicating that three molecules of ATP bind to activate the receptor. It has been suggested that conserved positively charged lysine residues (K68, K70 from one subunit and K309 from the adjacent subunit, P2X1 receptor numbering) coordinate the binding of the negatively charged phosphate tail of ATP and a conserved aromatic phenylalanine residue (within the conserved N290F291R292 motif, P2X1 receptor numbering) may coordinate the binding of the adenine ring. Mutations around the proposed ATP-binding site have also been shown to regulate the binding of the P2 receptor antagonists suramin and PPADS (Fig. 1) (Evans 2010). The crystal structure provides a snapshot of the receptor and it remains to be determined the extent of conformational changes in the extracellular domain that are associated with agonist binding and gating of the channel. The pore region of the P2X receptors is formed by the second transmembrane domains (TM2) from each

Molecular Properties/Features of P2X Receptors ATP binding to P2X receptors induces a conformational change in the protein that leads to the opening/ gating of the ionic pore. Mutagenesis-based studies and the zP2X4 receptor structure have provided significant insight into the molecular basis of the receptor’s properties (Evans 2010). P2X receptors do not have commonly found consensus sequences for ATP recognition like the Walker motif, and so mutagenesis-based approaches have been used to identify amino acids involved in ATP action at the receptor. These studies identified conserved amino acids that are likely to be involved in agonist binding. Mapping of these residues onto the zP2X4 receptor structure shows that they form as a cluster at the interface between two adjacent P2X receptor subunits (Fig. 1). This would give rise to three

P2X Receptors for ATP: Molecular Properties and Functional Roles

of the three constituent subunits. The crystal structure of the zP2X4 receptor in the agonist-free closed conformation shows the narrowest region of the channel forms where the TM2 domains cross each other about half way through the channel (Kawate et al. 2009). Mutagenesis studies have shown that this region of overlap plays a key role in the regulation of ionic permeation and is likely to form the channel gate (Surprenant and North 2009). P2X receptors are cation-selective, and under normal physiological conditions have a reversal potential of 0 mV and their activation leads to membrane depolarization. The channels also show appreciable calcium permeability. For example, calcium influx accounts for 10% of the current flow through smooth muscle P2X1 receptors on arteries, and is sufficient to mediate contraction of arterial smooth muscle, and in neurons presynaptic P2X receptors can regulate transmitter release. P2X receptors can therefore have direct signaling roles independent of the activation of voltage-dependent calcium channels. Interestingly, the P2X2,4,7 also show a second form of permeability following prolonged activation (tens of seconds to minutes) leading to an increase in permeability to larger cations. However, there is still controversy over whether this results directly from dilation of the P2X receptor channel pore or results in the recruitment of additional channels, for example, pannexins. One of the key distinguishing features between different P2X receptors is the time-course of current responses to applied agonist. P2X2 and P2X7 receptors, for example, show robust sustained currents in response to 5 s applications of ATP. In contrast, P2X1 and P2X3 receptor currents decay monoexponentially with time constants of 60% esterified) form transparent, thermo-irreversible gels when hot solutions are cooled, usually in the presence of sugar and acid. This reaction is familiar as the basis for the “setting” of jams and fruit jellies. The presence of sugar (usually sucrose) and the acidic conditions (usually citric acid) contrive to make the polysaccharides increasingly insoluble on cooling. It is considered that cooling causes the polysaccharide chains to form threefold helices that associate to form a fibrous

Pectin Biophysics Victor J. Morris Institute of Food Research, Norwich Research Park, Colney, Norwich, UK

Synonyms Gelling agent; Pectin biophysics

Pectin Biophysics Homogalacturonan (smooth) region

1833 Rhamnogalacturonan I (hairy) region

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Pectin Biophysics, Fig. 1 Major structural components of pectin extracts. The hairy (rhamnogalacturonan 1) regions are branched and the major types of side chains are linear or branched galactans, arabinans, or arabinogalactans

network as the basis of the gel structure. Low methoxyl (LM) pectins (typically 30–35% esterified) gel in the presence of certain divalent cations (usually calcium) and/or under acidic conditions. Most investigations have been made on calcium-induced gels. The gels are transparent, thermo-irreversible, and are formed at room temperature when calcium ions are diffused into or released within pectin solutions. Gelation is considered to depend on the presence of several blocks of acid residues along the pectin chain. When the chain length in a block exceeds a certain length (15–20 residues) cooperative binding of calcium occurs, forming junction zones between chains that stick the chains together. The junction zones have been called “egg-box” junctions (Fig. 2a) because the calcium ions (eggs) are contained within the buckled ribbon structures of the pectin chains (egg-box). LM pectins are prepared by de-esterification of HM pectins either enzymatically or under alkaline conditions. The type and duration of the treatment results in different ester distributions along the pectin chain which, in turn, determines the optimum calcium concentration at which gelation occurs: a factor that can compromise commercial applications where the hardness (calcium content) of the water will affect gelation. Alkaline treatment in the presence of ammonia introduces amide groups onto the pectin chain and this leads to thermoreversibility and broader calcium sensitivity on gelation. In the food industry, LM and amidated pectins can be used to produce a range of low sugar jams and jellies. Under acidic conditions, when the charge on the acid regions is neutralized, the chains can also be induced to associate and form gels. In this

Pectin Biophysics, Fig. 2 LM pectin gels. (a) Schematic picture of an “egg-box” junction zone. (b) Atomic force microscopic image of part of a LM pectin gel showing the fibrous network

case, association may not be entirely dependent on the association of the acid blocks. Gels formed under acidic conditions can be thermoreversible and the properties of gels can be manipulated if calcium-set gels are prepared under acidic conditions. Although there have been extensive studies of the formation and nature of the junction zones, less is known about the long-range structure of the gels. Electron and atomic force microscopy (Morris 2007) images of gel precursors, gels, and gel fragments suggest that the junction zones act as sticky patches gluing the pectin chains together into thicker fibers forming a branched fibrous network (Fig. 2b).

Pectin in Plant Cell Walls The gel structure and gelation of isolated pectin is considered to be a good model for aspects of the selfassembly and structure of the pectin-based networks in plant cell walls. Plant cell walls are complex composite structures (O’Neill and York 2003) based on two interpenetrating networks. The load-bearing structure is at present considered to consist of planes of cellulose fibers interconnected by molecular tethers called xyloglucans. Dynamic spectroscopic studies on stretched cell walls suggest that the pectin-based network is independent and not interconnected to the

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cellulose-xyloglucan network (Morris et al. 2003). The pectin-based network is considered to hold water and contribute to the swelling and de-swelling of the plant cell wall. For most fruits and vegetables the removal of calcium ions leads to cell separation and the breakdown of the tissue structure. Isolation of pectin from the intercellular (middle lamellae) region of the cell walls yields a HM pectin, which contains sufficient suitable length acid blocks, and gels in the presence of calcium. Enzymatic de-esterification of pectin in the presence of calcium leads to gelation, and this has been suggested as a possible early stage in the self-assembly of plant cell wall structure (O’Brien et al. 2009). The residual acid residues on the pectin chains, that are not complexed with calcium, are considered responsible for the swelling of the cell wall, in response to changed ionic conditions. In the much more highly concentrated environment of the plant cell wall, it is possible that other divalent cations, monovalent cations, or even complexation with basic proteins may contribute to the association of pectin chains (Morris et al. 2003).

Molecular Structure and Function Labeling of cell wall structures with antibodies to methyl and non-methyl-esterified smooth regions (Verhertbruggen et al. 2009) and to hairy regions has shown the selective distribution of different types of pectin structure within the plant cell wall. Because of the importance of the ester distribution on smooth regions of the pectin chains in controlling the functional properties of pectin extracts, and within the plant cell during growth and development, there is considerable interest in developing chemical and physical methods for characterizing this heterogeneity of ester substitution within populations of pectin molecules (Winnig et al. 2009). These types of approaches largely involve chemical or physical characterization of complex extracts, their fractionation or fragmentation, and the simulation or modeling of the data to predict the composition. Direct visualization of individual pectin molecules has revealed unusual features such as limited branching of the polygalacturonic acid backbone and, in pectin extracts from sugar beet pulp, the presence of a high proportion of pectin-protein complexes (Morris et al. 2003; Morris 2007). The latter observation, and direct studies of the interfacial properties of sugar beet pectin extracts, has demonstrated the

Pectin Biophysics

importance of the complexes in explaining the unique behavior of sugar beet pectin extracts as emulsifying agents. Proteins are known to associate and form elastic interfacial networks that stabilize emulsions, and the presence of the pectin chains forms a steric barrier around the oil droplets, which inhibits droplet-droplet collisions and flocculation (Gromer et al. 2010).

Bioactivity Consumption of fruit and vegetables is considered to be an important aspect of a healthy diet. These foods contain many compounds that are considered to promote health and prevent the incidence and progression of chronic disease. An important aspect is the role for pectin as a source of dietary fiber. Dietary fiber is fermented by colonic microflora contributing to gut health. Pectin is also one of a number of dietary polysaccharides that induce immunomodulatory effects following oral consumption (Ramberg et al. 2010). There is growing evidence from cellular, animal, and some clinical studies that modified forms of pectin may have anticancer properties (Glinsky and Raz 2009). It has been proposed that modification leads to the release of fragments that can bind to the important regulatory protein galectin 3, which controls a series of stages in the initiation, development, and spread of cancers. There is direct evidence that linear galactan chains, present in the hairy regions of the pectin extract, will bind specifically to galectin 3 (Gunning et al. 2009) in a fashion that should inhibit its role as a regulator, suggesting that these fragments may be responsible for the anticancer bioactivity.

Cross-References ▶ Polysaccharides: Biophysical Properties

References Glinsky VV, Raz A. Modified citrus pectin anti-metastatic properties: one bullet, multiple targets. Carbohydr Res. 2009;344:1788–91. Gromer A, Penfold R, Gunning AP, Kirby AR, Morris VJ. Molecular basis for the emulsifying properties of sugar beet pectin studied by atomic force microscopy and force spectroscopy. Soft Matter. 2010;6:3957–69.

Peptide Synthesis on the Ribosome – Computational Studies Gunning AP, Bongaerts RJM, Morris VJ. Recognition of galactan components of pectin by galectin-3. FASEB J. 2009;23:415–24. Morris VJ. Gelation of polysaccharides. In: Hill SE, Ledward DA, Mitchell JR, editors. Functional properties of food macromolecules. 2nd ed. Gaithersburg: Aspen; 1998. p. 143–226. Morris VJ. Atomic force microscopy (AFM) techniques for characterizing food structure. In: McClements DJ, editor. Understanding and controlling the microstructure of complex foods. Cambridge: Woodhead; 2007. p. 209–31. Morris VJ, Ring SG, MacDougall AJ, Wilson RH. Biophysical characterization of plant cell walls. In: Rose JKC, editor. The plant cell wall, Annual plant reviews, vol. 8. Oxford: Blackwell; 2003. p. 55–91. O’Brien AB, Philp K, Morris ER. Gelation of high-methoxy pectin by enzymic de-esterification in the presence of calcium ions: a preliminary evaluation. Carbohydr Res. 2009;344:1818–23. O’Neill MA, York WS. The composition and structure of plant primary cell walls. In: Rose JKC, editor. The plant cell wall, Annual plant reviews, vol. 8. Oxford: Blackwell; 2003. p. 1–54. Ramberg JE, Nelson ED, Sinnott RA. Immunomodulatory dietary polysaccharides: a systematic review of the literature. Nutr J. 2010;9:54. doi:10.1186/1475-2891-9-54. Round AN, Rigby NM, MacDougall AJ, Morris VJ. A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy. Carbohydr Res. 2010;345: 487–97. Verhertbruggen Y, Marcus SE, Haeger A, Ordaz-Ortiz JJ, Knox JP. An extended set of monoclonal antibodies to pectic homogalacturonan. Carbohydr Res. 2009;344:1858–62. Winnig H, Viereck N, Salomonsen T, Larsen J, Engelsen SB. Quantification of blockiness in pectins-A comparative study using vibrational spectroscopy and chemometrics. Carbohydr Res. 2009;344:1779–83.

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Peptide Backbone – Amide Backbone ▶ Far UV Protein Circular Dichroism ▶ Protein Secondary Structure Prediction in 2012 ▶ Ultraviolet Resonance Raman (UVRR) Spectroscopy Studies of Structure and Dynamics of Proteins

Peptide Micro-array ▶ Protein and Peptide Arrays

Peptide Motifs ▶ Linear Motifs in Protein-Protein Interactions

Peptide Synthesis on the Ribosome – Computational Studies Marc Willem van der Kamp Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol, UK

Synonyms Ribosomal peptide synthesis

PEG ▶ Surface Passivation for Single Molecule Detection

Definition The ribosome acts as a catalyst to form peptide bonds in protein synthesis.

PELDOR and DEER ▶ DEER of Metalloproteins ▶ Interspin Distance Determination by EPR

PEO ▶ Surface Passivation for Single Molecule Detection

Basic Characteristics The translation of genetic code into proteins through the specific catalysis of peptide bond formation by the ribosome is one of the most important achievements of evolution. The ribosome is a very large molecular structure which contains RNA molecules as well as proteins. Different from normal protein enzymes,

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however, the catalytic part is based on RNA. With the emergence of structural data on the large subunit of the (bacterial) ribosome responsible for peptide bond synthesis, computational studies into the mechanism ˚ qvist first used could be conducted. Trobro and A molecular dynamics and free energy perturbation simulations with ▶ empirical valence bond methods to examine possible catalytic mechanisms (Trobro and Aqvist 2005). They concluded that the most favorable mechanism does not involve general acid-base catalysis by ribosomal groups but rather proton shuttling between reactants. As in protein enzymes, preorganization of the active site appeared to be crucial for catalysis, even though catalysis is apparently limited to a reduction in activation entropy (and not enthalpy). The same authors later used similar methods to propose a way in which ribosomal release factors can induce the peptidyl-tRNA cleavage in the termination of protein synthesis (Trobro and Aqvist 2007, 2009). Other studies, including QM/MM simulations, have verified the importance of the electrostatic environment provided by the ribosome for catalysis (e.g., K€astner and Sherwood (2010)). New structural data made possible further detailed computational simulations of the interactions of release factors with the ribosome, which revealed how specificity of release factors is achieved (Sund et al. 2010). Reading stop codons was shown to involve interactions and switches that are distinctly different from normal tRNA interactions during peptide synthesis. Overall, computer simulations have been crucial in turning structural information on the ribosome into a detailed mechanistic picture, as also has been the case with ribozymes (enzymes composed of RNA) (Banas et al. 2009).

Peptide Transporter (PTR) Sund J, Ander M, et al. Principles of stop-codon reading on the ribosome. Nature. 2010;465(7300):947–50. Trobro S, Aqvist J. Mechanism of peptide bond synthesis on the ribosome. Proc Natl Acad Sci USA. 2005;102(35): 12395–400. Trobro S, Aqvist J. A model for how ribosomal release factors induce peptidyl-tRNA cleavage in termination of protein synthesis. Mol Cell. 2007;27(5):758–66. Trobro S, Aqvist J. Mechanism of the translation termination reaction on the ribosome. Biochemistry. 2009;48(47): 11296–303.

Peptide Transporter (PTR) ▶ Membrane Transport Proteins: The ProtonDependent Oligopeptide Transporter Family

Peroxidases ▶ Heme Peroxidases

Persistent Chain Model ▶ Worm-Like Chain (WLC) Model

Perturbation Methods ▶ Kinetics: Relaxation Methods

Cross-References ▶ Empirical Valence Bond Methods ▶ Molecular Dynamics Simulations of Lipids ▶ QM/MM Methods

PET ▶ Positron Emission Tomography Methodology

References Banas P, Jurecka P, et al. Theoretical studies of RNA catalysis: hybrid QM/MM methods and their comparison with MD and QM. Methods. 2009;49(2):202–16. K€astner J, Sherwood P. The ribosome catalyzes peptide bond formation by providing high ionic strength. Mol Phys. 2010;108(3–4):293–306.

PF00854 ▶ Membrane Transport Proteins: The Proton-Dependent Oligopeptide Transporter Family

Phase Contrast Electron Microscopy

PFG ▶ Pulsed Field Gradient NMR

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Cut-on frequency: Lowest bound of spatial frequency until which Zernike phase contrast can be recovered, which is determined by the size of the central hole in a Zernike phase plate. Defocus phase contrast: A phase contrast using an interference between an unscattered wave and defocus-modulated scattered waves.

Phase Analysis Light Scattering (PALS) Introduction ▶ Electrophoretic Light Scattering

Phase Contrast Electron Microscopy Kuniaki Nagayama National Institute for Physiological Sciences, 5-1 Higashiyama, Myodaiji-cho, Okazaki, Aichi, Japan

Synonyms Defocus Phase Contrast (DPC); Electron microscopy; Electron cryo-microscopy; Hilbert Phase Contrast (HPC); Phase plate; Zernike Phase Plate (ZPC)

Definitions Phase object: An object that is transparent like glass or water but capable of changing the phase of transmitting electron waves. When the phase change is much smaller than 90 (p/2), the object is called a weak phase object. Phase contrast: A contrast representing phase information carried in imaged objects using interference between an unscattered incident wave representing a zeroth order diffraction and scattered incident waves representing higher-order diffractions. Phase plate: An optical device to visualize transparent objects by converting a phase retardation in the incidence wave induced by objects to an amplitude. Zernike phase plate: A phase plate to generate a Zernike phase contrast. Hilbert phase plate: A phase plate to generate a Hilbert phase contrast.

Phase plate electron microscopy is an emerging biophysics technique for imaging ultrastructures in unstained biological samples with a contrast comparable to that of stained samples (Danev and Nagayama 2001; Nagayama 2005; Danev et al. 2009). Advantages of the phase plate electron microscopic imaging technique are that it can visualize biological systems with a high contrast without invoking heavy metal stains and can be naturally combined with electron cryo-microscopy, which is advantageous in preserving intact structures in biosystems in the physiological condition (Fukuda et al. 2009). All the lengthy treatments imposed to the traditional sample preparation, chemical fixation, dehydration, resin embedding, and staining, can be omitted. A tiny phase plate, which is made of a thin film made of amorphous carbon with a width of about 100 mm and thickness of 20–40 nm, is installed into EM at the portion immediately below the objective lens, where usually an aperture is settled. Practically, two kinds of phase plate of thin film type are proposed; Zernike phase plate (Danev et al. 2001) and Hilbert phase plate (Danev et al. 2002). The former is used for generating a Zernike phase contrast (ZPC) and the latter for generating a Hilbert phase contrast (HPC). Both methods bring about an improved contrast for unstained biological specimens compared with that obtained with a conventional defocus phase contrast (DPC method).

Phase Plates A phase plate is a kind of spatial filter that functions to modify the optical information transferred through a microscopic tool by modulating the phase of an incident wave. Lenses are one of the most typical spatial filter and function to converge or diverge the incident wave in a manner depending on the lens shape

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with no change in the wave amplitude. Phase plates work similarly as like lenses by positionally altering the wave phase but in a much simpler manner. Its function is illustrated in Fig. 1 together with a schematical view of the image formation process. Two types of phase plates, Zernike phase plate and Hilbert phase plate, both made of thin-film amorphous carbon, are shown. The first idea of phase plate electron microscopy was given by Boersch more than 60 years ago (Boersch 1947) and the thinfilm type phase plate is one of the three proposed by him and unique by its simple design to be manufactured. Nevertheless, phase plates of thin-film type had never been put to practical use before the report published in 2001 (Danev and Nagayama 2001). The major hurdle of making workable phase plates has been a hardship inherent in electron microscopy, the charging of phase plates. This difficult issue was partially solved in 2005 by adopting a treatment of conductive coating to wrap charges in the finish stage of the phase plate fabrication (Nagayama 2005). The complete settlement, however, has to be waited until 2012 (Nagayama 2012). Various types of phase plates including the Einzel lens using electrostatic potential generated with a specific design of stacked electrodes, named as Boersch phase plate, have been proposed and are under development (summarized in a review (Danev and Nagayama 2010a)).

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Phase Contrast Electron Microscopy, Fig. 1 Schematics of phase contrast microscopy. (a) A spatial filter schematic for defocus phase contrast (DPC). (b) A schematic for Zernike phase contrast (ZPC). (c) A schematic for Hilbert phase contrast (HPC). The bottom traces represents corresponding phase plates


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Biological Applications of Phase Plate EM The phase plate prepared with a combination of several techniques to neutralize electron-irradiation-induced charges enables us to obtain qualified images for various kinds of ice-embedded biological specimens. Examples of applications of functional phase plates to biological systems are shown. Zernike phase contrast cryo-EM: High-resolution applications to smaller biological systems such as protein molecules, protein molecular machines, and viruses have been obtained by using anti-charging phase plates. In Fig. 2, examples of ZPC images for a protein molecule and two viruses are shown. The first example is a single particle analysis (SPA) for an EM-standard protein, GroEL (Danev and Nagayama 2008). The advantage of ZPC cryo-EM is illustrated as a comparison of the DPC and ZPC cryoEM image of GroEL. With the conventional approach, the most challenging aspect of SPA is the first step: selecting the images that correspond to protein molecules. This is due to the fact that individual protein molecules must be identified for noisy raw images, as shown in Fig. 2aI. The handling of ZPC images with higher contrast, as shown in Fig. 2aII, becomes less prone to human bias. Once a sufficient number of particles have been sampled and particle selection is completed, analysis becomes more computer-based ˚ resolution 3D map and straightforward. A 13 A (Fig. 2aIII) was reconstructed from approximately


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Phase Contrast Electron Microscopy, Fig. 2 Examples of biological applications of thin-film phase contrast microscopy. (a) A protein, GroEL (I DPC image (300 kV), II ZPC image (300 kV), III 3D model obtained from single particle analysis with ZPC images) (Danev and Nagayama 2008). (b) A virus capsid, herpes simplex virus type I (I DPC image (200 kV), II ZPC image (200 kV), III 3D model) (Rochat et al. 2011). (c) Projection images of T4 phage at near zero tilt (I DPC projection image (200 kV), II ZPC projection image (200 kV)).

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Tomographic images sliced from a 3D reconstructed T4 phage (III DPC slice image, IV ZPC slice image) (Danev and Nagayama 2010b). (d) Comparison of TEM images of cyanobacterial cells (Kaneko et al. 2005). I A 300 kV DPCTEM image of an ice-embedded unstained whole cell (15 mm defocus). II A 300 kV HPC-TEM image of the same iceembedded unstained whole cell as shown in (a). (near-focus). III A 100 kV DPC-TEM image of a resin-embedded, sectioned, and stained cell

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1,500 raw particles that were picked directly from raw ZPC images shown in the photograph of Fig. 2aII. The second example is a SPA for a virus capsid, herpes simplex virus type 1 B-capsid (HSV-1B) (Rochat et al. 2011). The ZPC cryo-EM image (Fig. 2bII) was targeted in a near defocus condition to obtain maximum contrast enhancement. The contrast of the resulting images is substantially higher than that of the DPC image (Fig. 2bI). The 2,308 single particle images were used for reconstructing the final asymmetric map (Fig. 2bIII). What is most remarkable in the asymmetric 3D model is the exact positioning of the genome packaging apparatus, which has been a long-standing research target in virology. It has been finally fixed inside the capsid as shown in Fig. 2bIII. Zernike phase contrast cryo-tomography: Phase plate application to Zernike phase contrast cryotomography has been technically more challenging than that to SPA for several reasons followed: (1) a tedious procedure of realignment of the phase plate in association with the sample tilting, (2) a need to write a software to cope semiautomatically with the realignment, and (3) occasional phase plate alternation to a fresh one to manage the charging issue which is unavoidable during the lengthy data acquisition in tomography. Overcoming these difficulties, the first cryo-tomographic data recorded with ZPC-TEM was published for the case of a virus, phage T4 (Danev and Nagayama 2010b). An example of ZPC cryo-ET for the case of T4 phage is shown in Fig. 2c. Figures 2cI and II show T4 phage images sampled from DPC and ZPC tilt series of ice-embedded T4 phage (close to zero-tilt). It is easy to see the overall contrast improvement produced by the phase plate. In the ZPC image, disturbing fringes are appearing around strong-contrast edge; the heavy dark fringe just inside the viral capsid, the thin light fringe outside the capsid, and the light fringe close to the edge of the carbon film in the upper-right comer. All these features arise from the finite size of the phase plate central hole, which corresponds to a finite cut-on frequency. Comparison of tomograms of bacteriophage T4, acquired under identical experimental conditions, except for defocus and the presence of a phase plate, are shown in Figs. 2cIII and IV. The significant advantage with ZPC in contrast and overall details over the DPC image has to be noted.


Hilbert phase contrast cryo-EM: An example of HPC image is shown for a kind of cyanobacteria, which is one of the well-studied bacteria having a cylindrical geometry of about 3 mm (length) 1 mm (diameter). Figure 2d shows a comparison of images obtained for an unstained ice-embedded whole cell and a stained sectioned cell (Kaneko et al. 2005). A characteristic feature of a HPC cryo-EM image, together with the high contrast, is demonstrated in Fig. 2dII for an ice-embedded whole cell. Counterexamples obtained with DPC or a scattering contrast using staining is shown in Figs. 2dI or III. An obscure structureless image is observed in the DPC cryo-EM image, which was taken under the same experimental conditions as for the HPC image in Fig. 2dI, except for the phase plate and the defocus setting. Comparing the pair of images, the ice-embedded whole cell (Fig. 2dII) and the resin-embedded sectioned cell (Fig. 2dIII), a large difference in the image appearance can be observed, which may be attributable to the difference in specimen treatments. In the sectioned cell the ragged cell wall implies some shrinkage of the cell. Many aggregates and associated voids are also recognized, which are inevitably induced by chemical treatment, such as dehydration and selective staining of cellular organelles. On the other hand, the images of the iceembedded cell are smoothly round and recognizably space-filled everywhere. The preserved roundness of cyanobacterial cells allows us to estimate the specimen thickness to be about 1 mm.

Summary The first proposal of the idea of phase contrast electron microscopy using phase plates goes back to 1947 but its practical utilization belongs to a recent topic. The first successful realization using thin carbon film for the phase plate idea was reported in 2001 and various proposals followed to improve the performance and overcome the weakness intrinsic to the technology, the phase plate charging. Among them the thin-film type of phase plates, which is yet a device unique to bring about informative biological results, is focused in this entry. Three phase contrast methods, defocus, Zernike, and Hilbert phase contrast, are compared in their design and in the image characteristics, particularly the image contrast revealed in the micrograms of biological specimens such as proteins, viruses, and

Phase Transitions and Phase Behavior of Lipids

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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.

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.

Cross-References

Phase Plate

▶ 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

▶ Phase Contrast Electron Microscopy

Phase Transitions and Phase Behavior of Lipids Rumiana Koynova and Boris Tenchov College of Pharmacy, Ohio State University, Columbus, OH, USA Institute of Biophysics, Bulgarian Academy of Sciences, Sofia, Bulgaria

References € Boersch H. Uber die kontraste von atomen in elektronmikroskop. 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.

Synonyms Lateral phase separation; Lipid phase equilibria; Lipid self-organization; Lipids; Polymorphic and mesomorphic 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 interconversions between various polymorphic and mesomorphic 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,

P

P

1842

Phase Transitions and Phase 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, Pb0 ; (e) fully interdigitated gel, Lbint; (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) bilayer cubic phase, Pn3m; (s) bilayer cubic phase, Ia3d


I.

a

b

c

d

d

f

e

II.

i

j

g

k

h

l

m

III. o

n

q

p

r

Im3m

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

s

Pn3m

Ia3d

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


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 crystalline), 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 thermotropic 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 $ QB II $ 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 liquidcrystalline La phase. Upon further increase of temperature, 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). Intermediate lipid phases have been reported as well, for example, the liquid-ordered phase has attracted attention in the recent years because of its relevance to the

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P

functional lipid rafts in membranes (Simons and Ikonen 1997). With increase of the water content at constant temperature, the mesomorphic lipid phases arrange in the following sequence (Seddon 1990): inverted phases MII ; QII M ; HII ; QB II $ La $ normal phases QB I ; 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 molecules. 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 monomer solutions at very high dilutions which bring the lipid concentration below the critical micellar concentration, 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 transition, is the major energetic event in the lipid bilayers, taking place with a large enthalpy change. It is associated 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

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a 120

H||

100 Temperature [°C]

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), Pressure 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 unsaturated 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 interactions 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 glycerophospholipids with saturated sn-1 chain typically 16–18 carbon atoms long, and unsaturated sn-2 chain typically 18–20 carbon atoms long. The gel–liquidcrystalline (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.


80

La

60 40 Lb

20 0 10

12

14 16 18 Chain length

20

22

b 40 Temperature [°C]

P

20 La 0

–20

Lb 0

2

4

10 12 14 6 8 Position of double bond

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 dependence of the Lb–La (black squares) and La–HII (open circles) phase transition temperatures in saturated diacyl phosphatidylethanolamines (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 phosphatidylcholine bilayers (Koynova and Caffrey 1998)

Formation of Nonlamellar Phases in Membrane Lipids Dispersions of double-chain nonlamellar membrane lipids most frequently display a lamellar–invertedhexagonal, La–HII, phase transition. In some instances, they can also form inverted phases of cubic symmetry. Important role in the lamellar–nonlamellar transformations 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


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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 Chains sn-1/sn-2 Gel–liquid-crystalline transitions (Lb ! La) 10:0/10:0 5.7 2.0 11:0/11:0 13.9 16.9 12:0/12:0 1.9 31.3 2 14.2 32 13:0/13:0 13.5 42.1 10.7 14:0/14:0 23.4 50.4 24 35.4 54 20 15:0/15:0 33.7 58.4 33 58 16:0/16:0 41.7 64.4 41.5 51.4 65 17:0/17:0 48.6 70.5 18:0/18:0 54.5 74.2 54 63.7 75.4 19:0/19:0 60.2 79.2 20:0/20:0 65.3 83.4 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 33.5 18:1c9/18:1c9 18.0 7.3 18.3 11.0 11.0 18:2c9,12/18:2c9,12 55.1 18:3c9,12,15/18:3c9,12,15 63.0 Lamellar–nonlamellar transition (La ! HII, unless otherwise indicated) 14:0/14:0 15:0/15:0 16:0/16:0 118.5 17:0/17:0 18:0/18:0

107.6 101

19:0/19:0 20:0/20:0

97.8 94.2

22:0/22:0 16:0/18:1c9 16:1c9/16:1c9 18:1c9/18:1c9 18:1 t9/18:1 t9 18:1c9/20:4c5,8,11,14

90.0a 70.8 43.4 8.5 62.2 32.0

CL

Glc

Gal

Mal N-Sph PC N-Sph Gal

6.9 1.9 25.4 19.5 32.9 40 40.5 50.7 58 57.2 63.4 70 68.4 73.7a 76.8a

105b 82b 79b; 119c 76.6 74.5; 73.9d 73.7a 76.8a; 78.9d

26.8 48.7 61.6 73.5 80.0a

40.9 25 42 56.6 44.5 46.5 66.7 44.5 47.5

82 84 83

80.6b 80.7b; 82.3d 76.0; 85.0d a

80.0 ; 89.0d

PC, diacylphosphatidylcholines, PE diacylphosphatidylethanolamines, PG diacylphosphatidylglycerols, PS diacylphosphatidylserines, PA diacylphosphatidic acids, PI diacylphosphatidylinositols, CL cardiolipins, Glc diacylglucosylglycerols, Gal diacylgalactosylglycerols, Mal dialkylmaltosylglycerols, N-Sph PC sphingomyelins, N-Sph Gal galactocerebrosides (for the sphingolipids, chain length refers to the single fatty acid chain) a Lb ! HII b La ! QII c QII ! HII d Lc ! HII transition

chain packing strain – thus, membranes exist in a state of frustrated curvature stress (Gruner 1985). Respectively, 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

P

P

1846


Phase Transitions and Phase Behavior of Lipids, Table 2 Decrease of the gel–liquid-crystalline (Lb ! La) transition temperatures of fully hydrated acyl-chain phosphatidylcholines with increasing sn-2 chain unsaturation. The first cis double bond causes the biggest transition temperature drop to occur, while further increases of chain unsaturation have much smaller effects Chains sn-1/sn-2 16:0/16:0 16:0/16:1c9 16:0/18:0 16:0/18:1c9 16:0/18:2c9,12 16:0/20:0 16:0/20:4c5,8,11,14 16:0/22:0 16:0/22:1c13 16:0/22:6c4,7,10,13,16,19 18:0/18:0 18:0/18:1c9 18:0/18:2c9,12 18:0/18:3c9,12,15 18:0/20:0 18:0/20:1c11 18:0/20:2c11,14 18:0/20:3c8,11,14 18:0/20:4c5,8,11,14 18:0/20:5c5,8,11,14,17 18:0/22:0 18:0/22:1c13 18:0/22:4c7,10,13,16 18:0/22:5c4,7,10,13,16 18:0/22:6c4,7,10,13,16,19 18:0/24:0 18:0/24:1c15 20:0/18:0 20:0/18:1c9 20:0/20:0 20:0/20:1c11 20:0/20:2c11,14 20:0/20:3c11,14,17 20:0/20:4c5,8,11,14 20:0/22:0 20:0/22:1c13 20:0/24:0 20:0/24:1c15 22:0/18:0 22:0/18:1c9 22:0/20:0 22:0/20:1c11 22:0/22:0

Temperature ( C) 41.7 30.0 49.0 2.5 19.6 51.3 22.5 52.8 11.5 3.0 54.5 6.9 14.4 12.3 60.4 13.2 5.4 9.3 12.9 10.4 61.9 19.6 8.5 6.4 3.8 62.7 31.8 57.5 11.5 65.3 20.5 5.4 1.8 7.5 69.6 29.2 70.6 36.6 58.6 15.1 67.7 22.9 73.6 (continued)

Phase Transitions and Phase Behavior of Lipids, Table 2 (continued) Chains sn-1/sn-2 22:0/22:1c13 22:0/24:0 22:0/24:1c15 24:0/18:0 24:0/18:1c9 24:0/20:0 24:0/20:1c11

Temperature ( C) 32.8 77.1 41.7 58.9 20.7 68.4 24.5

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. Interestingly, 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 cooperative act of multiple fusion events, whereby a set of initially separate, parallel bilayers fuse into a single bilayer of specific topology. The lamellar–cubic transitions have very small, if any, latent heats. Although energetically inexpensive, these transitions are typically 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


a

1847

c

b

d

Lα

Lα

Lα

ΔH

Lα

P

Lβ

LR1

LR1

Lβ

Lβ Lβ⬘

SGII

Lc

Lc

Lc

Pβ⬘

Lc

Temperature

e

6 nearest neighbours

f 4 nearest neighbours

g

2 nearest neighbours

d-110 d-110 b

b a

a

b

d110 a

d110 d200

d200

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-chain PEs and PGs; and scheme (d), for the PCs. Solid horizontal lines indicate 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) orthorhombic packing with four nearest neighbors, and (g) orthorhombic packing with two nearest neighbors (Reproduced from Tenchov et al. (2001). With permission of the Biophysical Society)

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.

different lipid classes – with perpendicular or tilted chains with respect to the bilayer plane, with fully interdigitated, partially interdigitated, or noninterdigitated 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 packing (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).

Polymorphic Transitions Between Solid Lipid Phases At temperatures below the main transition, a basic equilibrium structure is the subgel (crystalline) Lc phase. Its formation usually requires prolonged lowtemperature incubation. In addition to the Lc phase, a large number of intermediate stable, metastable, and transient lamellar gel structures are adopted by

P

P

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Phase Transitions and Phase Behavior of Lipids, Table 3 Examples of heating, cooling, and isothermal phase sequences in lipid dispersions (Tenchov 1991) Lipid DPPC

DLPE

DMPE

DOPE

DOPE-Me

14-Gal

18-Gal

Scan direction Heating Cooling Isothermal equilibration 100) Deep cooling (1 C/h) Heating (