A

Fig. 7.1. (a) The NMR structure of D -V5'8,l17,K21melittin in DPC DMPG micelles (Sharon et al. 1999). (b) Wheel structure and calculated average structures of dlastereomeric peptides composed of Lys and Leu, showing the orientation of residues Leu-4, Leu-8, Lys-5 and Lys-9 In DPC micelles (Oren et al. 2002) Fig. 7.1. (a) The NMR structure of D -V5'8,l17,K21melittin in DPC DMPG micelles (Sharon et al. 1999). (b) Wheel structure and calculated average structures of dlastereomeric peptides...

A Non Bilayer Phase

A number of experiments have addressed the question of whether or not the presence of lipids favouring the hexagonal Hn phase is required for proper membrane function. For example, the microorganism Acholeplasma laidlawii contains six amphiphilic lipids, three uncharged and three anionic the major uncharged lipids are monoglucosyldiacylglycerol (MGlcDG) and diglucosyldiacylglycerol (DGlcDG), and the anionic lipid is phosphatidylglycerol. Of these, MGlcDG favours non-lamellar phases (cubic and...

And Subcellular Targeting

Recent structural and biochemical studies of a wide variety of eukaryotic peripheral proteins participating in signal transduction and membrane-trafficking have shown that their molecular activity involves the reversible binding to specific lipid ligands in cell membranes (Hurley and Misra 2000). Importantly, the membrane-binding ability of these proteins is conferred by a reduced number of protein domains, the best known being PKC CI (Hurley et al. 1997), PKC C2 (Rizo and Sudhof 1998), FYVE...

Bilayer Deformation Energies

Changes in bilayer shape incur an energetic cost. This can be important when a lipid bilayer is perturbed around a membrane protein. A lipid bilayer can respond to mechanical deformation by changes in its dimensions, up to a point beyond which the bilayer ruptures (Sackmann 1995). The observed changes in dimension are related to the magnitude of the external force (or stress) by a constant, the elastic constant (or modulus). The elastic properties of a lipid bilayer can be described by four...

Conclusions and Future Prospects

Knowledge of the precise three-dimensional structure of proteins is a requirement for understanding how they work. Nowadays, high-resolution structures of macromolecules can be readily determined by using X-ray crystallography and nuclear magnetic resonance (NMR). However, despite the huge amount of structural information obtained for soluble proteins, only the structures of a few membrane proteins are available. The reasons for their scarcity are mainly derived from their low solubility and...

Crystal Structure Determination

Crystallography up till now has been the most powerful technique for determining the atomic structure of proteins. However, of the 31,000 proteins that have been deposited in the Protein Data Bank (PDB) only 25 X-ray structures of unrelated polytopic membrane proteins from the inner membranes of bacteria and mitochondria, as well as from eukaryotic membranes, are available (for a regularly updated list, see Springer Series in Biophysics c.R.Mateo etal. (Ed.) Protein-Lipid Interactions...

Dimensions ofa Lipid Bilayer

The hydrophobic thickness of a membrane has been taken to be the thickness of the central hydrocarbon slab up to the C2 carbons of the chains (Lewis and Engelman 1983). This definition corresponds closely to the separation between the positions of the carbonyl groups in Fig. 6.1 (Wiener and White 1992). The positions of the carbonyls therefore mark the hypothetical hydrocarbon slab boundaries - hypothetical because the extent of the thermal motions is such that there is a significant overlap...

Domains

There has been increased interest in recent years in the formation of domains in biological membranes. This has resulted from the challenges in defining the structure and properties of raft domains and their possible biological roles (Munro 2003). In October, 2004 the Biophysical Society sponsored a four-day meeting Probing Membrane Microdomains which was largely devoted to discussing the existence and characteristics of rafts (http www.netbriefings.com One characteristic of raft domains in...

Energy Minimization as a Driving Force for Domain Formation

Biological membranes are in a steady state with continual cycling of membrane components and membrane vesicles. Nevertheless, it is likely that these membranes are not far from equilibrium. Their arrangement does not change drastically when the membranes are isolated and there is no longer cycling of membrane materials. With regard to organization of domains in model membranes, this occurs spontaneously when a peptide or protein is added to a liposome. The formation of these domains is thus a...

Extrapolation from Model Systems to Biological Membranes

It is worth considering how well studies of simple model systems relate to real biological membranes. The amount of unperturbed lipid bilayer present in a biological membrane will be low, because of the high concentration of protein in a biological membrane. For example, the molar ratio of phospholipid to protein in the sarcoplasmic reticulum membrane is about 90 1, sufficient to form about three shells of lipid around each Ca2+-ATPase molecule, the major protein in the membrane (Warren et al....

General Background

The fluid mosaic model of biological membranes (Singer and Nicolson 1972) emphasizes membrane fluidity and free lateral diffusion of membrane components. This has led to the generalized idea of biomembranes as solutions of proteins embedded in bilayers of randomly distributed phospholipids. The current view is moving towards the probable equal importance of lipids and proteins in the determination of the constitution, structure and dynamics of membrane domains and the hierarchic structure of...

Glycerol Backbone and Headgroup Structures

Useful information about glycerol backbone and headgroup structures in a liquid crystalline bilayer can be deduced from X-ray diffraction studies of crystalline lipids (Pascher et al. 1992). The crystal structures for dilauroylphosphati-dylethanolamine di(C12 0)PE and dimyristoylphosphatidylcholine di(C14 0)PC are shown in Fig. 6.2 the lipid headgroups lie parallel to the bilayer surface with very similar conformations. These conformations reflect the tendency of the ammonium nitrogen to fold...

Hydrophobic Ligand Binding Proteins

The considerable effort dedicated in recent years to the structural characterization at high resolution of a wide array of diverse proteins (both functionally and at the amino-acid sequence level) endowed with the ability to bind different types of hydrophobic ligands has revealed the existence of an underlying structural simplicity which in turn has permitted the definition of a reduced number of distinct protein families. Due to their biological relevance, we will briefly describe the major...

Hypothetical Nature of Lipid Protein Interactions

Despite the extensive information obtained on the functional and structural dependence of several ionic channels on its surrounding lipids (Lee 2004 Palsdot-tir and Hunte 2004), several aspects of the modulation exerted by the different lipid classes on the membrane proteins still remain unclear. Various hypotheses have been implicated in the modulation of ion channels function by lipids (1) modification of physical properties of the bilayer, such as fluidity, membrane curvature, and or lateral...

In Ion Channel Modulation

Ion channels are at the centre of many complex physiological processes such as the control of the beating rate in the heart or the generation of electrical signals in the brain. Ion channels involved in these processes possess two basic characteristics. The first is that they are selective, either for a range of ions (e.g. the nAChR channel, which is cation-selective) or for specific ions (e.g. K+ channels). Second, the channels have the ability to control movement of these ions, that is, to...

Influence of Lipids on nAChR Structure

Structural knowledge of nAChR and other LGIC has been greatly hindered by the absence of a crystal structure. The most informative structure is from electron micrographs of the tightly packed arrays of nAChR in tubular membranes isolated from the electric organ of Torpedo electric rays (Toyoshima and Unwin 1988 Unwin 1995 Miyazawa et al. 2003). These studies indicate that the transmembrane segments (M1-M4) are basically a helices, although Ml seems to have a distorted helical structure,...

Info

Molecular electronic transitions are characterized by absorption and emission dipoles (and their associated transition moments) fixed with respect to the molecular frame. When an assembly of randomly oriented molecules is illuminated with polarized light of suitable frequency the probability of absorption is proportional to cos2y, where y represents the angle between the absorption transition moment and the exciting electric vector. This anisotropy in the photoabsorption process causes...

Introduction

The assembly of lipid-based membranes was a crucial step for triggering the evolution of living cells in the prebiotic scenario. The hydrophobic barrier imposed by the lipid bilayers was essential for the isolation of different protocel-lular compartments and allowing for an independent and progressively complex evolution of small portions of the primordial soup (Deamer 1986 Szostak et al. 2001 Orgel 2004). These protomembranes were likely to be compositionally complex and functionally...

Labelled carbon atom

About a single C-C bond results in a large bend in the chain. Lower down the chain, lateral displacements resulting from rotations about single C-C bonds are very much smaller, and, therefore, steric restrictions on motion become less important. The order parameter profile for the palmitoyl chain in (C16 0, C18 1)PC is very similar to that observed in di(C16 0)PC, but with order being slightly higher in bilayers of di(C16 0)PC than in bilayers of (C16 0,C18 1)PC, suggesting that introduction of...

Lipid Domainsand Protein Protein Interactions

The study of cellular membrane lateral structure has gained considerable interest in the last few years. It has been proposed that organization of membrane domains could be the basis for the assembly of functional platforms that promote the selective encounter of certain proteins and the subsequent activation of specific processes. A well-known example of these platforms is the raft-like domains. Raft-like domains are supposed to promote accumulation and association of certain transmembrane...

Lipidation

Lipids that are covalently attached to proteins will insert into the membrane and will partition preferentially into certain membrane domains. Many proteins that are acylated with saturated fatty acids, particularly with palmitic acid on cysteine residues or myristic acid on the N-terminal amino group, partition into membrane rafts (Brown and London 2000 Resh 2004). This is not the case with prenylated proteins that are commonly excluded from raft domains (Melkonian et al. 1999). Another form...

Lipid Protein Interactions

Effects of lipids on the function of membrane proteins can be described either at the microscopic or at the macroscopic level. An explanation at the microscopic level is a molecular level interpretation in terms of interactions such as hydrogen bonding, charge-charge interactions, and van der Waals interactions. An explanation at the macroscopic level involves properties of the whole membrane such as membrane viscosity, membrane pressure, and curvature stress. In some cases it is clear that an...

List of Contributors

Centro de Qu mica e Bioqu mica Faculdade de Ciencias de Lisboa 1749-016 Lisbon Portugal The Burnham Institute 10901 North Torrey Pines Road Lajolla, CA 92037 USA Instituto de Biolog a Molecular y Celular Universidad Miguel Hern ndez 03206 Elche (Alicante) Spain Department of Biochemistry McMaster University Hamilton, ON L8N 3Z5 Canada Instituto de Biolog a Molecular y Celular Universidad Miguel Hern ndez 03206 Elche (Alicante) Spain The Burnham Institute 10901 North Torrey Pines Road Lajolla,...

Mixing of Lipids in the Liquid Crystalline Phase

The fluid nature of the liquid crystalline phase means that lipid molecules mix well in the liquid crystalline phase (Lee 1977). Thus there is no evidence for im-miscibility in the liquid crystalline phase, even for mixtures of two lipids with very different fatty acyl chain lengths such as di(C12 0)PC and di(C18 0)PC, where phase separation in the gel phase is very extensive (Shimshick and McConnell 1973). Mixtures of branched chains phosphatidylcholines are also miscible in the liquid...

Mode ofAction ofAMPs

The studies described above indicated the cell membrane as the major target of AMPs. However, internal targets were proposed for some of them (Hancock and Diamond 2000 Hancock and Rozek 2002 Wu et al. 1999). To interact and insert into the target membrane, AMPs undergo conformational changes from water-soluble to membrane-soluble form, but the details of the actual membrane permeation pathways for most AMPs are still not clear. Although several models were proposed in recent years, the majority...

Non Specific Interactions of Proteins with Membranes

Dividing protein-lipid interactions into specific and non-specific is somewhat arbitrary. The degree of specificity can be described quantitatively in terms of affinity constants that can span a range of values. Nevertheless, there is a qualitative difference in affinity and specificity between a specific protein-folding domain that recognizes a lipid structure and proteins or peptides that interact with membranes in a less discriminate manner through electrostatic or hydrophobic interactions....

Peptide Hydrophobicityand Charge

The distribution of the positive charges along the peptide backbone have an effect on the biological function of AMPs. AMPs with their positively charged amino acids, spread along the peptidic chain, act better toward bacteria compared with mammalian cells. Examples include magainin (Matsuzaki 1998), ce-cropins (Steiner et al. 1981 Gazit et al. 1994 Bechinger 1997), dermaseptins (Mor et al. 1991 Pouny et al. 1992), and others (Tossi et al. 2000 Andreu and Rivas 1998). In contrast, AMPs that are...

Positive Curvature

The forces present in a lipid bilayer. At the top Is shown the distribution of lateral pressures and tensions across a lipid monolayer. The repulsive lateral pressure Fc In the chain region Is due to thermally activated bond rotational motion. The Interfacial tension y, tending to minimize the interfacial area, arises from the hydrophobic effect (unfavourable hydrocarbon-water contacts). Finally, the lateral pressure Fh In the headgroup region arises from sterlc, hydrational and...

Preface

Biological membranes have long been identified as key elements in a wide variety of cellular processes, including cell defence, communication, photosynthesis, signal transduction and motility, and thus they emerge as primary targets in both basic and applied research. In order to fully appreciate the role that biological membranes play in these essential biological processes it becomes crucial to get a molecular-level picture of the structure of their main components (i.e. lipids and membrane...

Proteins that Bind Specific Lipids

There are several protein-folding domains that recognize specific lipids. Many of these bind specifically to one or more forms of phosphorylated phosphatidyl-inositol through interactions that are more specific than simple electrostatic interactions. These domains are often found in proteins involved in signal transduction. Their function has been considered largely on the basis of specific subcellular localization (Hurley and Meyer 2001 Misra et al. 2001). These domains, such as the PH, FYVE...

References

Abeytunga DTU, Glick JJ, Gibson NJ, Oland LA, Somogyi A, Wysocki VH, Polt R (2004) Presence of unsaturated sphingomyelins and changes in their composition during the life cycle of the moth Manduca sexta. J Lipid Res 45 1221-1231 Aioia, RC, Tian H, Jensen FC (1993) Lipid composition and fluidity ofthe human immunodeficiency virus envelope and host cell plasma membranes. Proc Natl Acad Sei USA 90 51815185 Anderson, RG (1998) The caveolae membrane system. Annu Rev Biochem 67 199-225 Antes P,...

Serum Albumin

Without doubt, one of the most extensively studied proteins is serum albumin. Recent high-resolution crystallographic studies of human serum albumin (HSA) complexed with different FAs (Curry et al. 1998 Bhattacharya et al. 2000 Petitpas et al. 2001) have permitted the analysis of the molecular details of the interactions involved. The protein is heart-shaped, with a high helical content (67 ) and consists of three repeating domains, each one composed of 10 a-helices, which can be further...

Springer

Reyes Mateo Professor Javier G mez ProfessorJos Villala n Professor Jos M. Gonz lez-Ros Instituto de Biolog a MolecularyCelular Avda. de la Universidad s n, edificio Torregait n ISBN 10 3-540-28400-1 Springer-VerlagBerlinHeidelbergNewYork ISBN 13 978-3-540-28400-0 Libraryof Congress Control Number 2005930633 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of...

Summary

The conclusion appears to be that most, if not all, of the effects of phospholipids on the function of membrane proteins can be explained, at least in principle, in microscopic rather than macroscopic terms. This makes sense from a biological perspective. A biological membrane is a multicomponent system, containing a wide variety of different intrinsic and extrinsic membrane proteins, each with their own particular role to play in the cell. These proteins need to be able to work independently a...

The Role of the Membrane and Peptide Properties in the Biological Function

The majority of AMPs share two properties they are highly cationic and they are composed of approximately 50 hydrophobic amino acids (reviewed in Sab- erwal and Nagaraj 1994 Shai 1999 Tossi et al. 2000 Bulet et al.1999 Kobayashi et al. 2000 Hwang and Vogel 1998 Andreu and Rivas 1998). However, there are exceptions, such as temporins, 13 amino acid AMPs isolated from the skin of frogs, which contain only one positively charged amino acid (Simmaco et al. 1996 Mangoni et al. 2004). Regarding the...

The Target of Most Antimicrobial Peptides

Mode-of-action studies have revealed that most AMPs use the microorganisms' cytoplasmic membrane as their final and lethal target. Note, however, that several studies have pointed out that they have other targets inside the cell as well (Bowdish et al. 2004 Davidson et al. 2004). Lehrer et al. (1989) were the first to demonstrate a membrane permeabilization mechanism in intact bacteria. They showed that human neutrophil peptide defensin HNP -mediated bactericidal activity against E. coli is...

Transmembrane Helices

The nature of the transmembrane helical segment of integral membrane proteins will also contribute to determining the partitioning of the protein between raft and non-raft domains. It is possible that a smooth, uniform surface contour of a transmembrane helix would more readily mix with a rigid cholesterol-rich domain. Another factor is the possible length mismatch between the transmembrane helix and the bilayer thickness. Cholesterol will contribute to increasing the thickness of the bilayer...

Ray versus Neutron Diffraction

Nowadays, the development of synchrotron radiation facilities, associated with progress in detectors and computing, makes it possible to solve very complex structures with a large number of atoms, at atomic or near-atomic resolution, even with small crystals. Thus, X-ray diffraction covers the full range of analysis from structure determination to the understanding of biological function. By contrast neutron diffraction techniques are mostly used to analyse particular aspects of the molecular...

The Structure ofa Lipid Bilayer

High-resolution structures cannot be obtained for bilayers in the liquid crystalline phase because the thermal motion and disorder in the bilayer means that the positions of the atoms are relatively ill-defined. Nevertheless, a picture of the bilayer can be developed, showing the average spatial distribution of atoms or groups of atoms, projected along the direction normal to the bilayer surface (White and Wiener 1995). The structure of a bilayer of dioleoylphosphatidylcho-line (di(C18 l)PE) in...

Contents

From Lipid Phases to Membrane Protein Organizatin Fluorescence Methodologies in the Study C. Reyes Mateo, Rodrigo F.M. de Almeida, Luis M.S. Loura, and Manuel Prieto 1.2 Fluorescence 1.2.2 Anisotropy 1.3.2.2 Introducing and Changing the Intrinsic Fluorescence of 1.4 Relevant Problems in Lipid-Protein Interaction and 1.4.1 Partition to the 1.4.2 Protein Peptide 1.4.3 Lipid Selectivity the Annular 1.4.4 Protein Peptide 1.4.5 Protein Peptide NMR of Membrane Proteins in Lipid Environments the Bcl-2...

Fatty Acyl Chain Region of the Bilayer

One characteristic of the liquid crystalline phase is the considerable intramolecular motion of the lipid fatty acyl chains, due to rotation about C-C bonds in the chains. The change in steric energy that results from rotation about the C6-C7 single bond in dodecane is shown in Fig. 6.3. The minimum potential energy occurs when the two neighbouring methylene groups are related by a dihedral angle of 0 , in the trans conformation. Two other minima are obtained at torsion angles of 115 and 245 ,...

Electrostatic Interactions

Electrostatic interactions can promote domain formation in membranes. In mammalian plasma membranes, anionic lipid components are found almost exclusively on the cytoplasmic leaflet. These lipids can interact with cationic proteins or clusters of cationic residues in a protein sequence. Anionic lipids will attract cations into the electrical double layer of the membrane. When polycationic peptides are bound to the membrane it will reduce the surface charge and hence the extent of the electrical...

Structural Analysis of Protein Lipid Interactions by XRay Crystallography Some General Remarks

The elucidation of high-resolution structures of membrane-spanning proteins has, over the past few years, yielded fascinating insights into the molecular mechanisms of diverse transmembrane processes, although the number of structures solved to atomic resolution is still small. Therefore, it is clear that to gain an understanding of the function of membrane proteins at a molecular level one must consider their contacts with lipids. However, while a high-resolution snapshot of the 3D structure...

The Role of the Amphipatic Structure and its Stability

Most studies were carried out with L-amino acid peptides possessing amphip-athic a-helix or p-sheet structures, which are known to be important for biological activities. In contrast, studies which compared the effect of significantly altering the sequence of an amphipathic a-helical peptide (15aa long) and its diastereomer (composed of both L- and D-aa) did not support the need for a preferred structure or sequence (Papo et al. 2002, Papo and Shai 2004). Table 7.1 lists the sequences of the...

Relevant Problems in Lipid Protein Interaction and Fluorescence

The extent of the partition of a peptide between the lipid and water phases is very variable and is usually given by the partition coefficient, Kp. The determination of Kp is usually the first step in the study of the interaction of a peptide with a given membrane system. It is defined by (for an alternative definition and interconversion see Santos et al. 2003) where Vi are the volumes of the phases, and ws,i are the moles of solute present in each phase (i W, aqueous phase i L, lipid phase)....

Protein Lipid Interactions in 3D Structures

A large number of molecular processes involve protein-lipid interactions occurring in different biological environments. Thus, they are essential in the folding mechanism of membrane proteins (Marsh 1993 Bogdanov and Dowhan 1999), as well as in their activity (Gouaux and White 2003), which includes an astounding range of biological functions respiration, signal transduction and cellular recognition, molecular transport, etc. However, and despite the critical importance of membrane proteins,...

The Role of Peptide Self Association in Solution andor in Membranes

Self-association of AMPs is an important parameter which affects their selectivity toward different cells. Self-association is driven either by the peptidic chain, or by the attachment of extrinsic hydrophobic groups such as fatty acids, resulting in the formation of a-helical bundles that could initiate strong hydrophobic binding to zwitterionic membranes (Oren et al. 1999 Strahilevitz et al. 1994 Ghosh et al. 1997). The role of self-association of AMPs in their selective binding to target...

Lipases

The lipolytic enzymes belong to a large family of enzymes that facilitate the degradation of lipids. Lipases and phospholipases are members of this family that have been investigated extensively (Rubin and Dennis 1997). Lipolytic enzymes are water-soluble enzymes that are characterized by their ability to hydrolize aggregated lipids with a much higher velocity than the same lipid in its monomo-lecular form. This rate enhancement at lipid-water interfaces is the central theme in the study of...

And Lipid Protein Interactions

As suggested from an evolutionary perspective, proteins could have sensed in different ways the lateral structure imposed by the heterogeneous composition of the lipid matrix when interacting with the membrane. Partition of a protein from the aqueous bulk phase into the membrane interface requires the initial establishment of thermodynamically favorable interactions of specific protein regions with groups at the interfacial environment. Structural contributions to membrane partitioning, both at...

Fluorophores in Lipid Protein Studies

The kinds of molecules capable of undergoing electronic transitions that finally result in fluorescence are known as fluorophores. Application of fluorescence spectroscopy to study lipid-protein interactions requires the presence of, at least, one of these molecules in the system under study. In many cases, the fluorophore is an inherent component of the protein or peptide - fluorescent amino acids as tryptophan (Trp) and tyrosine (Tyr) - or an extrinsic probe bonded to the macromolecule, and...

NMR in Micelles

Solution NMR methods rely on rapid molecular reorientation for line narrowing, and can be successfully applied to membrane proteins in micelles (Henry and Sykes 1994 Williams et al. 1996 Almeida and Opella 1997 Gesell et al. 1997 MacKenzie et al. 1997 Arora et al. 2001 Fernandez et al. 2001 Hwang et al. 2002 Ma et al. 2002 Mascioni et al. 2002 Oxenoid et al. 2002 Sorgen et al. 2002 Crowell et al. 2003 Krueger-Koplin et al. 2004 Howell et al. 2005). The size limitation is substantially more...

P

Headgroup conformations ofphosphatidyiethanoiamine di(C12 0)PE and phosphatidylcholine di(C14 0)PC molecule 2 in the unit cell . The three glycerol carbons are marked sn-1 to sn-3. Coordinatesfrom Harlos et al. (1984) and Pearson and Pascher (1979) diester group is such that the initial part of the sn-2 fatty acyl chain extends parallel to the bilayer surface but then bends sharply at the second carbon atom (Fig. 6.2). As a consequence, the sn-1 chain extends further into the bilayer...

Alternative Methods

However, the limited number of membrane protein structures available in the protein data bank also reflects the difficulties in obtaining well-ordered crystalline arrangements. Indeed, the lack of homogeneity in the starting solution is very often the major drawback for several reasons. First the detergent has to provide a homogeneous solubilization without denaturing the protein. The difference in dynamical behaviour of solvent and lipidic membranes certainly influences the dynamical...

Intracellular Lipid Binding Proteins

Since the description of the first cellular fatty acid-binding protein (FABP) (Ockner et al. 1972), different types of iLBPs have been reported from various organisms and tissues (Hanhoff et al. 2002 Zimmerman and Veerkamp 2002 Haunerland and Spener 2004). Although much effort has been spent in elucidating the specific roles of iLBPs, this issue has thus far not been fully explored. Accordingly, various functions have been proposed for iLBPs, including not only uptake and transport of FAs, but...

Protein Expression and Purification

NMR structural studies require milligram quantities of isotopically labeled proteins, and the most versatile and widely used method for obtaining recombinant proteins is by expression in E. coli, since this enables a wide variety of isotopic labeling schemes to be incorporated in the NMR experimental strategy. Smaller peptides can be prepared by solid phase peptide synthesis however, this is impractical for larger proteins and for the preparation of uniformly labeled samples, where efficient...

Juxta Membrane Domains

Juxta-membrane domains can be distinguished from transmembrane helices or lipidation in that these segments do not penetrate deeply into the bilayer. One type of juxta-membrane domain is a cluster of cationic amino-acid residues that can bind to the surface of anionic membranes (Ellena et al. 2004). There are some lipoproteins, such as NAP-22, in which a smaller cationic cluster on the protein is found close to the amino terminus that is post translationally modified by myristoylation. In such...

Oocyte as a Cell Model for the Study of Lipid Protein Interactions

Most of the studies dealing with the functional and structural dependence of nAChR on its surrounding lipids have been carried out on model membrane systems to avoid the complexity of the cell membrane and to prevent the changes that a single variable can make in the whole system. Though these model systems are useful, providing a reductionism approach, we must develop novel methods allowing the study of the lipid-protein interaction in native cell membranes in order to confirm the results...

NAChR Modulation by other Lipophilic Compounds

As indicated above, some of the lipids surrounding the nAChR play an important role in determining its functional activity. Besides, many other hydrophobic molecules, with astonishingly different molecular structures, modulate the nAChR function, including (1) Free fatty acids (Andreasen and McNamee 1980 Villar et al. 1988) (2) Steroid hormones, both glucocorticoids (Bouzat and Barrantes 1996 Nurows-ka and Ruzzier 1996) and sexhormones (Valera et al. 1992) (3) Local anaesthetics (Katz and...

Neutron Diffractionwith Contrast Variation

A three-dimensional crystal diffracting to high resolution does not contain only highly ordered atoms. In many structural models refined from X-ray diffraction data, a few amino-acid side-chains, and N-terminal or C-terminal residues are missing. Also small molecules, ligands, cofactors, individual detergent molecules or lipids are not always easy to locate in electron density maps even if they interact strongly with the protein. In fact, above a certain value of mean-square atomic displacement...

Fatty Acyl Chains

Lipid fatty acyl chain lengths determine the hydrophobic thickness of a lipid bilayer. The hydrophobic thickness of a lipid bilayer is expected to match well the hydrophobic thickness of any protein embedded in the bilayer, because of the high cost of exposing either fatty acyl chains or hydrophobic amino acids to water. The efficiency of hydrophobic matching has been demonstrated experimentally for the potassium channel KcsA where varying the chain length for the surrounding phospholipids from...

PAnAChR Interaction

As stated above, nAChR binds preferentially anionic lipids, which are positive modulators of its function. Among them, PA seems to interact in a special fashion with this protein. In vitro studies with nAChR reconstituted in lipid vesicles of controlled composition show that PA is among those phopholipids that bind the protein with a higher affinity, and it is the most effective lipid in preserving nAChR function (Jones and McNamee 1988 Marsh and Barrantes 1978 Ellena et al. 1983 Esmann and...

NMR in Bilayer Membranes

Solid State Nmr Pisema

When the lipid bilayers are oriented with their surface perpendicular to the magnetic field, the solid-state NMR spectra of the membrane-associated proteins trace out maps of their structure and orientation within the membrane, and thus provide very useful structural information prior to complete structure determination (Marassi and Opella 2000 Wang et al. 2000 Marassi 2001). For example, helices give characteristic solid-state NMR spectra where the resonances from amide sites in the protein...

Lateral Lipid Organization in Membranes

Several processes have been proposed for producing non-homogeneous inplane organization of different lipid species in membranes (Binder et al. 2003). Some of these processes suggest self-organization due to equilibration of the membrane system towards thermodynamically favorable states, without a necessary major participation of specific lipid-lipid interactions. An example of this type of process is the phase separation that spontaneously occurs in bilayers made from a mixture of lipids with...

The Barrel Stave Model

The barrel-stave model was first proposed to describe the formation of transmembrane channels pores by bundles of amphipatic a-helical peptides (Ehrenstein and Lecar 1977 Sansom 1993,1998). Peptides which act via this mechanism are inserted into the membrane such that their hydrophobic surfaces interact with the lipid core of the membrane and their hydrophilic surfaces point inward producing an aqueous pore (Fig. 7.3). Amphipatic a-helical lytic peptides which act on a specific or several types...

General Features of Membrane Protein Structures

Presently, about 160 coordinate files of membrane protein structures are accessible in the PDB. Among them, roughly 80 are unique proteins (see Despite a significant increase in the last couple of years, the growth rate since 1986 is still not comparable to the early days of soluble protein structure determination (White 2004). The large majority of membrane proteins in the PDB span over both sides of the membrane, with a few exceptions (prostaglandin synthase, cyclooxigenase and...

Effects of Internal Bilayer Pressures and Curvature Stress

Although, as described above, lateral pressures in the headgroup and chain regions of a lipid bilayer must balance, the distribution of lateral pressures within the chain region are not uniform (Lindahl and Edholm 2000 Gullingsrud and Schulten 2004). It has been suggested that the lateral pressure profile in a lipid bilayer could be an important factor in determining the conformational equilibrium of a membrane protein (Cantor 1997). For example, if changes in the cross-sectional area of a...

Influence of Lipids on nAChR Function

LGICs are membrane proteins that transiently open a pore through the lipid membrane in response to neurotransmitter binding. The nAChR is one of the best-understood members of this family, principally due to two factors that have aided in its characterization (1) the rich source of nAChR present in the electric organ of some fishes (T. marmorata, T. californica and E. electricus) and (2) the presence of neurotoxins in snake venoms that bind specifically to the nAChR providing the means for...

The Classical Approach

Micelle Diagrams

Integral membrane proteins are embedded in a lipidic membrane. In order to be crystallized in three dimensions, they have to be solubilized and purified from their native environment. Amphiphilic molecules such as detergent form a belt around the membrane proteins, in which the tails of the detergent protect the hydrophobic protein surface whereas the headgroups protrude toward the solvent. The crystallization was described in several reviews or book chapters (Hunte and Michel 2003 Reiss-Husson...

Lipid Transfer Proteins

Lipid-transfer proteins (LTPs) facilitate the transfer of lipids between membranes. They are widely distributed, being produced by both prokaryotic and eu-karyotic organisms bacteria, yeasts, plants and animals (Rueckert and Schmidt 1990). LTPs cover a wide spectrum of ligand specificities FAs, phospholipids, gly-colipids, steroids, acyl-CoA, etc. in this regard, it must be remarked that whereas some LTPs are highly specific (Roderick et al. 2002), others are non-specific (Tsu-jishita and...

The Carpet Model

The barrel-stave mechanism was found to be used by the non-cell-selective AMPs but not by those peptides that are selectively active on bacteria (Strahilevitz et al. 1994 Shai et al. 1990 Rapaport and Shai 1991,1992 Gazit et al. 1994 Pouny and Shai 1992 Shai 1994). Detailed mode-of-action studies suggested an alternative model, termed the carpet model or a detergent-like model (Pouny and Shai 1992 Gazit et al. 1995a,b). Figure 7.4 depicts the four steps proposed to be involved in the carpet...

Lipocalins

Membrane Lipocalin

The lipocalin protein family is a large group of small extracellular proteins which are characterized by common ligand-binding properties they all bind small hydrophobic molecules (retinoids, arachidonic acid and steroids), specific cell-surface receptors and form complexes with soluble macromolecules (Flower 1996). The large degree of variation in amino-acid sequences found within the family may be consistent with the functional diversity found for these proteins. Thus, they participate in...

Pore Forming Protein Toxins

Pore Protein

Many organisms produce polypeptide chains that can exist either as a water-soluble state or as a membrane-embedded species, usually an oligomeric integral membrane protein (Gouaux 1997 Heuck et al. 2001). The conversion between both protein forms is spontaneous, and it is generally accepted that it proceeds through discrete steps water-soluble state, membrane-receptor binding, oli-gomerization, membrane insertion and final formation of the functional pore. The current structural information on...

Non Bilayer Phases

Most, if not all, biological membranes contain lipids that, in isolation, prefer to adopt a curved, hexagonal Hn phase rather than the normal, planar, bilayer phase Cullis and de Kruijff 1979 Rietveld et al. 1993 . Sometimes such lipids can be the major lipids in a membrane for example, monogalactosyl diacylglycerol makes up about 50 of the total lipid in the chloroplast membrane Shipley et al. 1973 and the E. coli inner membrane contains about 70 phosphatidylethanolamine Har-wood and Russell...