What is the best description of the selective permeability of a cell?

8.2 Controlling the Ion Selectivity of U60

Although U60 demonstrate selective permeability for Na+ and K+, making it further differentiate between Na+ and K+, which are almost identical in size, remains challenging. As the shape and size of the U60 surface pore are fixed, the possibility of tuning its selective permeability may only come from controlling the hydration shell, which can be simply achieved though regulating the solution temperature.

Small amounts of NaCl/KCl/RbCl/CsCl (5 mM) were titrated into U60 solution (1 mg/mL, 3 mL, same volume, and concentration were used in all groups) at room temperature. DLS and SLS confirmed the formation of blackberry, and the trend of blackberry size was consistent with the previous results. U60/NaCl and U60/CsCl precipitate in solution, while the blackberry size of U60/KCl (ca. 36 nm) is larger than U60/RbCl (ca. 15 nm), as shown in Fig. 15, group 1. Subsequent to the completion of blackberry formation, the group 1 solutions were kept at 70°C. Two weeks later, the following phenomenon can be observed. (1) The precipitates in both U60/NaCl and U60/CsCl solution were disappeared. While the scattering intensity level of U60/CsCl solution remained high, the U60/NaCl solution demonstrated a very low scattering intensity. (2) The blackberry size of U60/KCl had an obvious drop however the blackberry size of U60/RbCl remained stable. From the above observations, several conclusions can be drawn. First, the precipitation in U60/NaCl solution is due to the aggregation of discrete U60 clusters, while the precipitation of U60/CsCl solution is a result of blackberry aggregation. The U60/CsCl blackberry aggregations redissolved when heated up to 70°C. Second, K+ ions migrate out of U60 cage after incubating at 70°C, leading to a higher surface charge density of U60 thus a smaller blackberry size. This is due to two reasons: (1) the higher kinetic energy makes the trapped K+ ions less willingly to stay inside the cage and (2) the thinner hydration layer of U60 facilitates the escape of K+ ions (65).

To clarify the role of hydration layer, another three sets of experiments were conducted (Fig. 14). Group 2 solutions were titrated directly with different counterions at room temperature, and group 3 solutions were preincubated at 50°C before titration. Both groups 2 and 3 were held at 50°C to form blackberries. For U60/NaCl, the solution intensities in both groups 2 and 3 remained relatively low indicating the Na+ cannot enter the U60 cage at this temperature due to the change of hydration shells. Blackberry sizes of U60/RbCl and U60/CsCl in groups 2 and 3 did not vary a lot from group 1. However, while U60/KCl blackberry sizes of group 2 were still similar to group 1, the initial blackberry sizes in group 3 demonstrated a noticeable increase comparing to group 1. Those results clearly showed the effect of hydration layers in self-assembly process. U60 clusters in group 3 were preincubated in 50°C in order to have a thinner hydration shell before contacting with the additional counterions. Therefore, they were more permeable to the diffusion of K+ ions thus more K+ ions will enter the cage after preincubation, which will make the blackberries larger (Fig. 15). Moreover, compared to group 3, group 2 was immediately placed at 50°C after titration. However, the blackberry size did not vary from group 1. This suggested that the transport of K+ ions had already been finished before blackberry formation. Combined with the fact that the self-assembly process of U60 happened immediately after salts titration, it was obvious that the K+ diffusion must be extremely fast, so that the transport can finish even before the hydration shells being impaired by high temperature. This is another important feature U60 clusters shared with K+ ion channels. Groups 2 and 3 were then heated up to 70°C. U60/KCl solution in both groups 2 and 3 showed an obvious blackberry size drop at 70°C, while U60/RbCl and U60/CsCl blackberry sizes remained, which confirmed our previous conclusion (65).

Those conclusions were further proved by ITC studies. One set of U60 solutions were titrated directly at 50°C, while the other set of solutions were preincubated at 50°C for 3 days, before ITC measurements. First, the results showed that no noticeable interaction between U60 and Na+ existed after preincubation, while the binding strength between U60 and K+ were still strong, suggesting that indeed U60 can distinguish between Na+ and K+ at 50°C. Second, the entropy change (△S) became much more positive after preincubation. Although the thicknesses of hydration shells were reduced after preincubation, the entropy loss can be compensated by the excess K+ ions migrating out of U60 cages (65).

In group 4, the preincubation temperature was further increased to 70°C in order to further dehydrate U60 clusters. The U60/NaCl solution still showed a low scattered intensity. While the U60/CsCl blackberry size remained similar as in group 1, the sizes of both U60/KCl and U60/RbCl blackberries clearly increased comparing to group 1, suggesting that both K+ and Rb+ entered the cage of U60 clusters after a 70°C preincubation because of a thinner hydration shell (65).

In general, U60 clusters show tunable ion selectivity in respond to different temperatures. U60 allowed Na+ and K+ to enter the cage at room temperature. After preincubation at 50°C, only K+ can migrate across the surface pores. When the preincubation temperature reaches 70°C, both K+ and Rb+ can diffuse into U60 cages (65).

1 Introduction

Biomembranes are complex colloidal systems forming enclosed compartments with selective permeability to molecules and mediate numerous crucial biological processes including receptor signaling, transport of molecules, translocation of polypeptides, formation of ion channels, membrane fission–fusion, cellular communication, and immune regulation [1–5]. These processes are generally facilitated by modulating the activity of membrane-associated proteins [2,6]. The membranes are also targets for many pore-forming toxins, antimicrobial, and cytolytic peptides which disrupt membrane integrity [7,8]. These membrane-interacting peptides/proteins with diverse compositions and structures are also valuable candidates or targets for therapeutic intervention [9–11]. As such, understanding the mechanisms of peptide/protein–membrane interaction is of prime importance in developing peptides/proteins with well-controlled functionalities for therapeutic purposes. However, the role of the complex composition and physicochemical properties of biomembranes in mediating the specific molecular interactions with the membrane-active peptides and proteins remains elusive [2,12–14].

Peptide/protein–membrane interactions involve complex conformational changes of the peptides/proteins and membrane lipids. A series of changes in secondary structure, orientation, degree of insertion/embedding relative to a bilayer and assembly (oligomerization) of peptides/proteins are important structural features of peptide/protein function [6,15]. Various spectroscopic and computational studies have provided structural data in an attempt to establish the relationship between peptide/protein structures and the molecular details of the interaction mechanisms [16–19]. In addition, studying the role of different physicochemical parameters in mediating the binding affinity, kinetics, and thermodynamics is also critical in understanding the membrane selectivity and destabilization processes. The parameters associated with peptides/proteins include charge, hydrophobicity, amphipathicity, hydrophobic moment, and polar angle, while those associated with membranes include charge and charge density/distribution, lipid domains, membrane thickness, curvature, fluidity, and lipid packing order [9,15,20]. These physicochemical parameters also vary with pH, temperature, ionic strength, and salt and divalent ion concentration [21–23]. To fully understand the mechanisms of membrane interaction, methods for systematic analysis with well-controlled experimental conditions need to be established for the correlation of each physicochemical parameter in mediating peptide/protein–membrane interactions to structure–function relationships. Various analytical systems have been established for structural and functional characterization of peptides/protein–membrane interactions. Recent advances in high-resolution surface scanning and sensitive spectroscopic techniques have also provided the molecular structural basis to fully quantitate the active concentration, affinity and kinetics of membrane interaction mechanisms [17,19]. This vast information has been collectively used to establish molecular models for the global landscape of peptide/protein–membrane interactions. In spite of various models proposed for membrane-mediated processes, there is an enormous gap in our understanding of the role of lipid molecular organization on the selective binding and the effect of peptide/protein–lipid binding on membranes in the physiological and pathological states [24].

Optical biosensors are among the highly sensitive biophysical techniques used to obtain both qualitative information and quantitative measurement of real-time biomolecular interactions [25–30]. The microinjection and microfluidic systems of the biosensor allow high-throughput screening of the structural and compositional factors that mediate peptide/protein binding to the membrane [31–34]. However, the use of biosensors in studying the membrane interaction is still relatively low compared to their application in other nonmembrane molecular-binding studies [26,27]. This is partly due to the lack of robust protocols to create stable, reliable membrane systems on the biosensor chip in situ, and inability to provide information on the overall geometrical properties of the membrane. Several waveguide-based optical biosensors have been developed to study the formation of biomembranes and to determine the geometrical properties, including mass per unit area, thickness and density, and molecular packing of membranes either immobilized or physisorbed onto the sensor chip [26]. One of these waveguide techniques, dual polarization interferometry (DPI), is based on an integrated planar optical waveguide interferometer that combines evanescent field sensing and optical phase difference measurement methods and provides the advantage of multiparameter measurements in a single-binding assay which yields the optogeometrical properties of density and thickness of the adsorbed layer [35]. The significance of these features lies in the ability to now analyze the impact of membrane-active peptides and proteins on the structure of the bilayers simultaneously with the mass changes associated with the binding event.

Biological membranes are highly heterogeneous in their molecular composition and distribution and exhibit broad dynamic physical states [36,37]. The mutual effects of biomembrane composition and physical properties on membrane-mediated binding processes are therefore highly complex and difficult to reproduce in a stable and reproducible manner, with very few examples of complex lipid extracts from, e.g., Escherichia coli outer membrane that have been deposited on a solid support [38–42]. Although the use of natural lipids or membranes is more representative of the native cell membrane, the undefined lipid compositions make it difficult to understand the exact role of specific lipid components and membrane properties on the activity of peptides/proteins and other molecules. Therefore, a wide variety of simpler model membrane systems with well-controlled and finely tuned physicochemical properties are commonly used to quantitate the effect of defined lipid components on the membrane structure and organization and to correlate these with peptide/protein binding and functional activity [14,43–46].

The design of model membrane systems for characterizing membrane interactions also depends on the instrumental methods being applied. Vastly different configurations of biomembrane systems have been developed for applications ranging from drug development and delivery, energy/biofuels production, biomolecular sensors, and tissue engineering [47–49]. Among numerous types of solid support materials with different structural/topological configurations and chemical/physical properties [46,50,51], biomembranes formed on an optically transparent planar solid support are ideal for the design of a model membrane platform for optical biosensing technology and provides the most promise for fabricating a broad range of functional biomaterials mimicking the cell surface [52,53]. Preparing a solid-supported biomembrane with stable, reproducible structural, and physical properties is a prerequisite for biophysical characterization of the interaction of molecules with membranes. The binding, location, and orientation of a peptide relative to a lipid bilayer, as well as the rearrangement of lipid molecules, are all critical features of peptide–lipid interactions. Hence, the accurate analysis of the membrane-mediated interaction demands robust methods in preparing the membranes with selected structural and physical properties for specific activity.

This chapter provides an overview of the methods to prepare supported lipid bilayers (SLBs) for DPI analysis. In particular, we outline the key parameters that mediate the formation of SLBs via vesicle fusion and with specific examples of in situ characterization of the formation of SLBs using DPI. An example of the data analysis procedure is then described to illustrate how the experimental outputs allow mass-binding data to be directly correlated with the structural changes in the bilayer, which overall, provides novel information on the sequence of interactive events that determines the mutual activity of peptides/proteins and biomembranes.

Introduction

A biomembrane can be considered as a barrier of highly selective permeability that allows and regulates the traffic of a myriad of different molecular species between the interior of the cell and its surrounding environment (as, e.g., in a plasmatic membrane), or between different compartments within the same cell (e.g., in the membrane of the different organelles). Its basic structure is formed by a bilayer of amphiphilic molecules, the most common of which are three different classes of lipids (glycerolipids, sterols, and sphingolipids). On the extracellular side, this bilayer couples to a glycocalix, a carbohydrate network composed of oligosaccharides believed to be responsible for cell–cell recognition and adhesion. On the intracellular side, the bilayer couples to the cytoskeleton, that contributes to the mechanical properties of the overall composite structure.

The main functions exerted by a biomembrane can be summarized as follows: (1) the membrane is a selective filter which controls the transport and the permeation of ions, molecular aggregates, and even large particles between the extracellular medium and the cytosol; (2) the membrane is the site for energy producing processes and for hormone signal transduction; (3) the membrane acts as receptor for extracellular signals and mediates the communications between intra- and extra-cellular media; and (4) the membrane can perform mechanical tasks as in cellular motion or in eso- or endo-cytosis processes.

These demanding tasks are fulfilled thanks to a very complex, yet in principle, surprisingly simple, basic structure based on two-layered sheets of lipid molecules held together by a delicate balance between hydrophobic and hydrophilic interactions (Figure 1). The first important fact is that this is a self-assembling structure. The balance between a favorable interaction of the hydrophilic polar heads of the lipids with the aqueous solvent and the unfavorable hydrophobic interactions of their aliphatic chains, represents the driving force to the spontaneous formation in an organized closed structure. Due to hydrophobic/hydrophilic interactions, the lipid molecules arrange themselves into two-faced leaflets (the double layer) with the hydrophobic moiety in the inside and “polar heads” on the outside, representing the interface with the aqueous environment. The presence of lipids of different shapes (different volume ratios of the hydrophobic/hydrophilic moieties) in the two leaflets stabilizes the radius of curvature of the double layer.

The second important aspect is that, being mainly stabilized by “structure-solvent” effects, without strong “links” between different molecules, the bilayer is a highly dynamic structure, where lipids (and proteins) can flex, rotate, and diffuse laterally, as in a “two-dimensional” fluid. The lipid distribution results from a continuous inward and outward movement between the two monolayers, the lipid asymmetry being maintained by specific mechanisms that counterbalance the concentration-driven transbilayer permeation (Figure 2). The lipid bilayer has often been considered, in the past, as a passive structure and only recently, more attention is being directed towards its implications in a variety of membrane biological functions.

Due to its peculiar structure, the overall shape of the membrane during the biological functionality can be easily changed, for example, during pseudopodia movement or eso-/endo-cytosis processes. In endocytosis, in particular, after a region of the membrane has surrounded the particle to be introduced within the cell, it detaches from the rest, forming a vacuum that can move within the cytoplasm. The whole process is reversed in eso-cytosis, when a substance has to be expelled from the cell. These processes well illustrate the “modularity” of the membrane structure, that can easily lose or acquire parts to accomplish particular tasks.

Among the enormous variety of possible lipids, only a few classes are used to build up a biomembrane. Lipids that exert a predominantly structural role can be grouped into three categories: sterols (e.g., cholesterol), phospholipids (e.g., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SPHM)), and sphingolipids (cerebrosides, glycolipids). Lipids with a predominantly functional role are: phosphatidylinositol, phosphatidic acid, and many types of gangliosides.

The lipid composition of different cell types shows great differences and within the same cell, the plasma membrane, the nuclear envelope, mitochondria, and other membranous organelles and structures have different lipid compositions. Depending on the cell type, proteins constitute 20–80% of the membrane mass. Nevertheless, by comparing the average molecular weight of lipid molecules (in the range 700–1000 Da) and the typical molecular weight of a protein (in the range of the order of tens of thousands Da), lipids are the more abundant components in terms of molar concentration.

Membrane proteins are usually divided into two classes, “intrinsic” and “extrinsic” ones. This differentiation is mainly based on the greater or lesser difficulty in separating the fraction of the protein of the two classes from the other membrane components by organic solvent extraction. However, intrinsic proteins are generally considered more deeply embedded into the nonpolar environment (the interior of the lipid double layer), while extrinsic proteins are assumed more “at the surface” or even simply adsorbed at the double-layer interface. However, most intrinsic proteins possess specific sites that are exposed to the aqueous environment on one, or on both the sides of the membrane.

The thickness of a lipid bilayer can be estimated in the range of 4–6 nm. However, in estimating the thickness of a real biological membrane, the fact that proteins and many lipids (glycolipids) bear groups that extend into the aqueous medium, has to be taken into account. This region (glycocalix), at the outer surface of the plasmatic membrane, is typically 10–100 Å thick.

Membranes of cells and organelles are sometimes corrugated and folded or show protrusions of different shape and size. For example, the needle-like protrusions of the plasmatic membrane of some cells (microvilli) typically measure 50–200 nm in diameter and may extend for several hundreds of nanometers and even more.

Publisher Summary

This chapter discusses the membrane-based separation processes that make use of selective permeability. A considerable number of different membrane processes have found industrial applications. This discussion deals mainly with four: microfiltration (MF), ultrafiltration (UF), nanofiltration (NF), and reverse osmosis (RO). The driving force for material transport through the membrane in those four processes is a pressure difference. These processes are therefore called pressure-driven membrane processes. Electrodialysis, in which the driving force is an electric field, has a number of interesting applications in food processing and is briefly discussed at the end of the chapter. The importance of biological membranes as selective barriers in cells and tissues is well known. The property of selective permeability (perm-selectivity) of natural membranes was discovered by the French physicist Abbé Nollet. The industrial application of membranes, however, is a fairly recent field, rapidly expanding thanks to the continuing development of new membranes with improved properties. MF and UF are genuine filtration processes where particle size is practically the sole criterion for permeation or rejection. In contrast, RO membranes separate particles at molecular level and their selectivity is based on the chemical nature of the particles. NF is, in essence, a membrane process similar to reverse osmosis.

Outer Membrane

The outer membrane is an integral component of the cell envelope of Gram-negative bacteria, where it is thought to act as a selective permeability barrier mainly. It is composed of (lipo)proteins, phospholipids, and lipopolysaccharides (LPSs). The arrangement of these components in the outer membrane is essentially asymmetric (Figure 17(c)). The chemical asymmetry is clearly seen in freeze-fractured membranes at the ultrastructural level.

The LPSs are located in the outer leaflet of the outer membrane, whereas the phospholipids mainly reside in the inner leaflet (i. e., the leaflet pointing to the interior of the cell). LPS consists of three regions: lipid A, which is anchored to the outer leaflet of the outer membrane, core oligosaccharide, and O-specific polysaccharides, also called O-antigen. The composition of the latter can be extremely variable and the genes that express O-antigen are grouped in gene clusters.

Outer membrane proteins tend to be organized into trimers to allow their function as hydrophilic transmembrane channels. As such, the channel proteins are referred to as porins. Several porins may occur in one and the same cell and they have been studied in many Gram-negative organisms. A monomeric porin has a ß-barrel structure, which traverses the outer membrane. Three ß-barrel structures constitute a pore as shown for the osmoporin OmpC (Figure 18).

5.6.2.10 Porosity

Exponentially growing PEMs allow for ion exchange and even for the incorporation of nanoparticles [180–182] in contrast to linearly growing films. These ones display nevertheless some selective permeability for small molecules and small weakly charged ions [218,219]. This is directly related to the presence of pores with diameters in the nanometer range.

To conclude this section, a nice application of the Gibbs–Thomson equation for the determination of the pore size distribution of PEM films will be presented. The PEM films may also contain some small electrolytes (e.g., Na+ and Cl− ions from the solution) when the pairing between the oppositely charged groups of the PEM film is not perfect and when counterions are required to ensure the electroneutrality of the medium. When small counterions are incorporated in the film, the mole fraction of water will increase and then film will swell due to an osmotic pressure effect, as already described.

The fusion temperature of a liquid depends on the size of the liquid clusters (of size R) to be molten according to the Gibbs–Thomson equation:

(5.8)Tm−T0=KR

where K is a constant depending on the melting enthalpy of the considered liquid and T0 is the melting temperature of the bulk liquid. As long as water is in the solid state, it cannot be detected in 1H spin echo experiments. As soon as it melts, i.e., as soon temperature Tm is reached for a pore with radius R, a spin echo signal is measured. Hence by progressively increasing the temperature, all the clusters of increasing size progressively melt and the distribution of pore sizes can be determined (Fig. 5.37). This method is called NMR cryoporosimetry. As can be seen in Fig. 2.37, PDADMAC-(PAH-PSS)4-PAH films are characterized by a pore size distribution of about 1–3 nm [220].

5.2.1 Ion Channels

An ion channel is a protein embedded in the cell membrane. The protein consists of an ion filter and a gate. Channels are usually described in terms of their selectivity and gating mechanism. Due to the selective permeability to ions, the cell membrane may be described as an electrochemical membrane. An open channel is highly selective; the permeability for K+ may, for instance, be 1000–10,000 times higher than for Na+. The anion channel macroprotein for Cl− is very different from the cation channel protein of Na+ or K+, for example (Jentsch, 2002). The selectivity is not due to ion size but to the charge spatial distribution in the filter part of the protein. Channel capacity is high; a K+ channel can let through 200 million ions per second. If the channel is open only 1 ms, then 0.2 million ions are let through in each trigger event. As a monovalent ion corresponds to an electric charge of 0.16 aC [attocoulomb = 10−18 C], this corresponds to a charge of 32 fC. If one cell membrane has 10 million channels, a charge of 0.32 μC flows out of the cell into the extracellular gel.

11.6.1.3 Membrane separation

Membrane separation is a technology often used to treat textile dyeing effluent. In this process, the membrane filter is used for filtration. It consists of micropores and separates organic substances from the effluent using membrane-selective permeability. This methodology is classified into three categories: (1) UF, (2) NF and (3) RO.

Ultrafiltration (UF) is a promising methodology for separation. UF works at low transmembrane pressure to remove dissolved substances. UF membranes may be made from polyelectrolyte complexes, cellulose acetate and inert polymers. These membranes are capable of handling high flux and are free from microbes. Pore size ranges between 0.001 and 0.02 μm.

NF is an advanced membrane technology that is effective in removing heavy metals. NF membranes have a nonporous thin skin layer which provides high permeability. NF possesses the property of intermediate UF and RO. NF processes consume less energy and result in the high removal of pollutants.

RO technology has many applications in the treatment of wastewater. It gives a clear permeate and leaves an RO concentrate. This RO concentrate will have a high level of organic concentration. Because RO membranes have fine pores, they are prone to fouling by suspended particles. To prevent this fouling, the feed water must undergo a pretreatment process to control the turbidity level.

3.1.3 Size and shape

For a chemical to be degraded it will usually have come into contact with an enzyme. Often, but not always, the degradative enzymes are intracellular, i.e. they are located inside a microbial cell and therefore within the cytoplasmic membrane – a selective permeability barrier that controls access to the cell. Some chemicals, by dint of their size, cannot pass across the membrane and therefore cannot come into contact with the enzymes therein. Thus, if they cannot be attacked by extracellular enzymes they will remain undegraded.

For an enzymic reaction to occur the substrate must enter into intimate contact with the enzyme and form an enzyme/substrate complex. Some recalcitrant compounds may be unable to complex with the active site of the enzyme due to their size, and probably more so, to their shape.

Permeability Properties