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Introduction
The cell membrane (also known as the plasma membrane or cytoplasmic membrane) biological membrane that separates the interior of all cells from the outside environment.1,2 The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells.3 It consists of the phospholipid bilayer with embedded proteins. Cell membranes are involved in a variety of cellular processes such as cell adhesion, conductivity and cell signalling and serve as the attachment surface for several extracellular structures, including the cell wall, glycocalyx, and intracellular cytoskeleton.
2. Structure of cell membrane
The most satisfactory model of membrane structure is the Fluid mosaic model postulated by S.J. Singer and Nicolson in 1972.According to tis model the phospholipids of the membranes are arranged in a bilayer with their ionic polar head groups facing the aqueous phase to form a fluid and crystalline matrix core and the nonpolar groups largely buried in the hydrophobic interior of the membrane. These globular molecules are partially embedded in a matrix of phospholipid.The bulk of the phospholipid is organized as a fluid bilayeralthough a small fraction of the lipid may interact specifically with the membrane proteins. The fluid mosaic structure is therefore formally analogous to a two-dimensional oriented solution of integral proteins (or lipoproteins) in the viscous phospholipid bilayer solvent.The mosaic is dynamic since the proteins are free to diffuse laterally in two dimension. Recent experiments with a wide variety of techniques and several different membrane systems are described, all of which abet consistent with, and add much detail to, the fluid mosaic model.But certain aspects of it have been revised. It has been found that proteins diffuse in a complicated way that indicates lateral heterogeneity in the membrane structure.The coexistence of multiple modes of diffusion and directed transport indicates a pronounced lateral heterogeneity in the membrane(Jacobson,et.al.,1995)
Until 1982, it was widely accepted that phospholipids and membrane proteins were randomly distributed in cell membranes, according to the Singer-Nicolson fluid mosaic model, published in 1972.4,5 However, membrane microdomains were postulated in the 1970s using biophysical approaches by Stier& Sackmann6 and Klausner& Karnovsky.7These microdomains were attributed to the physical properties and organization of lipid mixtures by Stier&Sackmann and Israelachvili et al.8 In 1974, the effects of temperature on membrane behavior had led to the proposal of "clusters of lipids" in membranes and by 1975, data suggested that these clusters could be "quasicrystalline" regions within the more freely dispersed liquid crystalline lipid molecule. In 1978, X-Ray diffraction studies led to further development of the "cluster" idea defining the microdomains as "lipids in a more ordered state".One type of microdomain is constituted by cholesterol and sphingolipids.These microdomains (‘rafts’) were shown to exist also in cell membranes.9The plasma membranes of cells contain combinations of glycosphingolipids and protein receptorsorganized in glycolipoproteinmicrodomains termed lipid rafts.10 Research has shown that lipid rafts generally contain 3 to 5-fold the amount of cholesterol found in the surrounding bilayer. Also, lipid rafts are enriched in sphingolipids such as sphingomyelin, which is typically elevated by 50% compared to the plasma membrane.Cholesterol interacts preferentially, although not exclusively, with sphingolipids due to their structure and the saturation of the hydrocarbon chains. Although not all of the phospholipids within the raft are fully saturated, the hydrophobic chains of the lipids contained in the rafts are more saturated and tightly packed than the surrounding bilayer.4Cholesterol is the dynamic "glue" that holds the raft together.12These specialized membrane microdomains compartmentalize cellular processes by serving as organizing centers for the assembly of signaling molecules, influencing membrane fluidity and membrane protein trafficking, and regulating neurotransmission and receptor trafficking. Lipid rafts are more ordered and tightly packed than the surrounding bilayer, but float freely in the membrane bilayer.11
. Membrane Components
Cell membranes contain a variety of biological molecules, notably lipids and proteins. Carbohydrate may comprise as much as 10% of the weight of some membranes, but the carbohydrate is invariably in the form of glycolipid or glycoprotein.
The cell membrane consists of three classes of amphipathic lipids: phospholipids, glycolipids, and cholesterols.The amount of each depends upon the type of cell, but in the majority of cases phospholipids are the most abundant.In RBC studies, 30% of the plasma membrane is lipid.
Phospholipids
Biological bilayers are usually composed of amphiphilic phospholipidsthat have a hydrophilic phosphate head and a hydrophobic tail consisting of two fatty acid chains. Phospholipids are the most abundant class of lipids present in the membrane.Theyare derived from either glycerol (phosphoglycerides) or from sphingosine (sphigomyelins). Phospholipids with certain head groups can alter the surface chemistry of a bilayer and can, for example, serve as signals as well as "anchors" for other molecules in the membranes of cells.13 Just like the heads, the tails of lipids can also affect membrane properties, for instance by determining the phaseof the bilayer. The bilayer can adopt a solid gelphase state at lower temperatures but undergo phase transition to a fluid stateat higher temperatures, and the chemical properties of the lipids' tails influence at which temperature this happen. Of the phospholipids, the most common headgroup is phosphatidylcholine(PC), accounting for about half the phospholipids in most mammalian cells.14 PC is a zwitterionicheadgroup, as it has a negative charge on the phosphate group and a positive charge on the amine but, because these local charges balance, no net charge.Otherheadgroups are also present to varying degrees and can include phosphatidylserine(PS) phosphatidylethanolamine(PE) and phosphatidylglycerol(PG). These alternate headgroups often confer specific biological functionality.Unlike PC, some of the other headgroups carry a net charge, which can alter the electrostatic interactions of small molecules with the bilayer.
Glycolipids
Glycolipids are lipids with a carbohydrate attached by a glycosidic bond. Their role is to serve as markers for cellular recognition and also to provide energy. The carbohydrates are found on the outer surface of all eukaryotic cell membranes. They extend from the phospholipid bilayer into the aqueous environment outside the cell where it acts as a recognition site for specific chemicals as well as helping to maintain the stability of the membrane and attaching cells to one another to form tissues.
Protiens
Proteins within the membrane are key to the functioning of the overall membrane. These proteins mainly transport chemicals and information across the membrane.Proteins can be in the form of peripheral or integral.
Transmembrane proteins
A transmembrane protein (TP) is a protein that goes from one side of a membrane through to the other side of the membrane.Its function is to deny or permit the transport of specific substances across the biological membrane, to get into the cell, or out of the cell as in the case of waste byproducts.TPs may have special ways of folding up or bending that will move a substance through the biological membrane.Atransmembrane protein is a polytopic protein that spans an entire biological membrane.There are two basic types of transmembrane proteins: Alpha-helical,Beta-barrels.
Peripheral membrane proteins
Peripheral membrane proteins are proteins that adhere only temporarily to the biological membrane with which they are associated. These molecules attach to integral membrane proteins, or penetrate the peripheral regions of the lipid bilayer. The regulatory protein subunits of many ion channels and transmembrane receptors, for example, may be defined as peripheral membrane proteins.The reversible attachment of proteins to biological membranes has shown to regulate cell signaling and many other important cellular events.
Integral membrane proteins
An integral membrane protein (IMP) is a type of membrane protein that is permanently attached to the biological membrane. All transmembrane proteins are IMPs, but not all IMPs are transmembrane proteins.IMPs comprise a significant fraction of the proteins encoded in an organism's genome.IMPs include transporters, linkers, channels, receptors, enzymes, structural membrane-anchoring domains, proteins involved in accumulation and transduction of energy, and proteins responsible for cell adhesion.
4.Artificial / Model membrane
Model membranes are a useful tool for studying the behavior of proteins and lipids in a membrane under different conditions. They can be used to study things like the functions of membrane protein complexes, ion channels, and membrane receptor proteins. 15Due to their simplicity and decreased amount of confounding factors compared to biological membranes, model membranes are easier to manipulate and to study. Commonly referred to as liposomes, model membranes are synthetic lipid bodies. They can be composed entirely of one type of lipid (e.g. phosphatidyl choline), a mixture of lipids of the same class (e.g. phosphatidyl choline and phosphatidyl serine), and can also have proteins and protein complexes integrated into them for studies of lipid-protein interactions and their effects on protein function.16
Depending on the type (s) of lipid molecules that is/are used to construct liposomes, different structures such as unilamellar vesicles, multilamellar vesicles, and bilayers can be produced. Among the variety of liposome structures that can be produced, the most biologically relevant are lipid bilayers because they are the most structurally similar to cell membranes.
Liposomes can be used to study a variety of topics such as lipid behavior at varying temperatures, protein behavior in particular lipid environments, membrane transport processes, enzyme activities of membrane proteins.
Preparation
The physical forces that drive the formation of a liposome and maintain its structure are the same as those that drive the formation of biological membranes.Membrane lipids typically have hydrophobic tails and polar head groups. When these molecules are introduced into an aqueous environment, the association of non-polar lipid tails with one another rather than with polar water molecules is driven by what is called the hydrophobic effect .17 The self-assembly of bilayers leads to a net increase in the entropy (disorder) of the system: the increased entropy of water molecules is greater than the decreased entropy (i.e., increased order) of the lipid molecules of the bilayer. This phenomenon is also what drives the self-assembly of liposomes.
Two methods of preparation of liposomes are given below:
Reverse-phase evaporation: The starting material can be a single type of phospholipid or a mixture of phospholipids. This is then added to an organic solvent, homogenized, and the solvent is evaporated from the mixture at low pressure. The resulting lipid film is then hydrated with a solution containing molecules and/or ions one is interested in studying in terms of membrane properties/ associations, and this process leads to the formation of multilamellar vesicles (MLVs). These are then sonicated to yield a more homogeneous mixture of liposomes without multiple membrane layers. A final evaporation of any remaining organic solvent followed by purification by dialysis, centrifugation, or column chromatography should yield relatively pure liposomes. Liposomes produced with this method are able to trap the aqueous phase and its components, such as macromolecular structures, with high efficiency compared to other methods of preparing liposomes.
Another method commonly used for the preparation of liposomes is the extrusion of multilamellar vesicles through pores in a polycarbonate membrane.18The principle is that repeated extrusions of MLVs through these pores act to slowly filter the sample until a preparation containing only liposomes of similar sizes is obtained. In practice, MLVs can be prepared and treated as described in the reverse-phase separation method to yield single membrane liposomes of varying sizes. Samples can then be extruded through pores in a polycarbonate membrane under an applied pressure. After several extrusions and dialyses to remove debris, liposomes of relatively homogeneous size distributions can be obtained.
5.Peptide-lipid interaction and its importance (chemical & biological)
The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated cellular processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes.The biological action of pharmaceutical agents is dependent on the binding of peptides to lipid-bilayers.
Membrane interacting peptides can be classified according to their structure or their interaction with lipid membranes. They can assume different structures such as helical, β-stranded, mixed (containing both helices and strands) and cyclic, which are fundamental for modulating their membrane function. They usually contain amino acids with marked hydrophilic (Asn, Gln, Pro) or relatively hydrophilic (Phe, Trp, Tyr, Met) character, according to the Wimley and White hydrophobicity scale, which influences their position in membranes. The presence of positively (Arg, Lys, His) or negatively (Asp, Glu) charged residues is also a key feature, determining their interaction with target membranes.
Two main approaches are considered to study peptide-lipid interaction., centered either on the peptide or on the membraneOn the first, we consider mainly the methodologies based on the intrinsic fluorescence of the aminoacid residues tryptophan and tyrosine.
Determination of the partition coefficient of the peptide by intrinsic fluorescence parameter
The partition coefficient of a molecule between a lipid and an aqueous phase can be evaluated by fluorescence spectroscopy as long as: (i) there is a difference in a fluorescence parameter (e.g., quantum yield, fluorescence anisotropy or fluorescence lifetime) of the partitioning molecule when in aqueous solution and after incorporation in the membrane.The intrinsic fluorescence of peptides containing tryptophan (Trp) or tyrosine (Tyr) residues is a valuable tool to quantify their insertionon lipid membranes. Due to the strong dependence of the fluorescence of these aromatic amino acids (specially Trp) on the physicalproperties of their micro-environment, the insertion of the peptide on a lipid membrane can lead to substantial changes on the quantum yield, wavelength of the emission maximum, fluorescence anisotropy and fluorescence lifetime.19As long as there is a significant difference between the quantum yield of the peptide in the aqueous environment and in the lipid membrane, its partition constant can be determined by conducting fluorescence intensity,measurements with a fixed concentration of peptide and increasing lipid concentrations ([L]). This usually yields a hyperbolic-like I vs. [L] variation profile,which can be fitted using
I=(I_w+K_p γ_L [L] I_L)/(1+K_p γ_L [L])(1)
Whereγi is the molar volume of water (i=W) or lipid (i=L). Ideally, Ishould be the integrated fluorescence emission intensity.
The steady-state fluorescence anisotropy ® can be used to determine the partition coefficient of a fluorescent molecule,molecule, it is not only necessarythat this parameter has a significant change between the lipid and aqueous phases, but also the fluorescence intensity from both phases must be comparable.Using an experimental design identical to that indicated for the fluorescence intensity-based methodology, the data should be fitted using
r=(r_w ((γ_L [L]^(-1) )-1)+(r_L K_p ε_L ϕ_L)⁄((ε_w ϕ_w)))/((γ_L [L]^(-1) )-1+(K_p ε_L ϕ_L)⁄((ε_w ϕ_w)))(2)
whereϕ is the fluorescence quantum yield and ε is the molar absorption.In addition to the steady-state fluorescence spectroscopy methodologies, partition coefficients can also be obtained by time-resolved fluorescence spectroscopy. When carrying out a time-resolved fluorescence spectroscopic study of the interaction of a fluorescent molecule with a membrane system, ideally two exponentials would describe the experimental fluorescence intensity decay, one corresponding to the molecules in aqueous media and the other to the molecules in the lipid environment
I(t)=a_w exp(-t⁄(τ_w)+a_L exp(t⁄(τ_L)))(3)
(where ist time and τ is the fluorescence lifetime of the molecules in each phase). In this ideal case, the relative concentration of each specie could be calculated from the pre-exponential factors ratio(a_L⁄a_w ), if the radiative rate constants and absorption coefficient ratios in both phases are known. However, in most of the cases (including intrinsically fluorescent peptides), the decays both in the aqueous phase and on the membrane are complex. Thus, the total decay must be described by a sum of exponentials, mixing up all the contributions.
Therefore, an average of the fluorescent lifetime should be used. The average fluorescence lifetime of a fluorophore ,〈τ〉, is given by,
〈τ〉=(∑▒〖a_(i ) τ¬_i^2 〗)/(∑▒a_i τ_i )(4)
However, if 〈τ〉is used for K_p determination, a complex equation would be attained, where steady-state and transient-state data must be combined,
〈τ〉=〈τ〉_w+(〈τ〉_L-〈τ〉_w)(K_p γ_L [L])/(K_p γ_L [L]+(ε_w ϕ_w)/(ε_L ϕ_L ))(5)
Therefore, it is more convenient to calculate the variation of the fluorescence lifetime averaged by the pre-exponentials,τ ̅ ,
τ ̅=(∑▒〖a_i τ_i 〗)/(∑▒a_i )(6)
leading to the formalism for the determination of K_p
τ ̅=(τ ̅_w+K_p γ_L [L] τ ̅¬_L)/(1+K_p γ_L [L] )(7)
This equation is similar to Eq. (1) because both τ ̅ and Iare additive parameters.