Table of Contents
Cover
Title Page
Copyright
Dedication
Preface
Chapter 1: Biological Membranes
1.1 Introduction
1.2 The Biological Membranes
1.3 The Proteins
1.4 The Membrane Functions
References
Chapter 2: Electrostatics of Biomembranes
2.1 The Electric Field
2.2 Electric Field Created by a Uniform, Infinitely Extended Planar Charge Distribution
2.3 The Parallel-plate Capacitor
2.4 The Dielectric Constant
2.5 The Electric Potential Across a Membrane
2.6 Poisson–Boltzmann Equation and Gouy–Chapman Theory
2.7 Measurement of the Charge Density of the Polar Heads of a Charged Lipid
2.8 Electropermeabilization of Lipid Bilayers
References
Chapter 3: Thermodynamics
3.1 Some Concepts of Chemical Thermodynamics
3.2 The Electrochemical Potential
3.3 Thermodynamics of Irreversible Processes
3.4 Coupling of Primary and Secondary Active Transport in Biomembranes
References
Chapter 4: Passive Transport
4.1 How do Ion Channels Look Like?
4.2 The Nernst Equation and the Resting Potential
4.3 A First Approach to the Action Potential
4.4 Single-channel Open Probability
4.5 The Goldman–Hodgkin–Katz Equation
4.6 Open Probability and Gating Charge of Ion Channels
4.7 Rate Theory of Membrane Transport
4.8 Action Potential Revisited
References
Chapter 5: Active Transport
5.1 The Ion Pumps
5.2 Electromotive Force and Inversion Potential of Ion Pumps
5.3 Energy Levels of the Enzymatic Cycle of Ion Pumps
5.4 Kinetics of Ion Pumps Under Steady-State Conditions
5.5 Electrogenicity of the Ion Pumps
5.6 Kinetics of Ion Pumps Under Pre-Steady-State Conditions
5.7 Transporters
References
Chapter 6: Biomimetic Membranes
6.1 The Various Types of Biomimetic Membranes
6.2 Electrochemical Techniques for the Investigation of Biomimetic Membranes
6.3 Lipid Bilayers Interposed Between Two Aqueous Phases
6.4 Biomimetic Membranes Noncovalently Supported by Metals
6.5 Biomimetic Membranes Covalently Supported by Metals
6.6 Conclusions
References
Chapter 7: Auxiliary Techniques
7.1 Physical Properties of Electromagnetic Waves
7.2 Surface Plasmon Resonance
7.3 Infrared Spectroscopy
7.4 Neutron Reflectivity
7.5 Fluorescence Microscopy
7.6 Scanning Probe Microscopy
7.7 Langmuir–Blodgett and Langmuir–Schaefer Transfers
7.8 Quartz-Crystal Microbalance
References
Index
End User License Agreement
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Guide
Cover
Table of Contents
Preface
Begin Reading
List of Illustrations
Chapter 1: Biological Membranes
Figure 1.1 Schematic picture of a plasma membrane, showing the bimolecular layer of lipid molecules (including cholesterol), integral proteins spanning the lipid bilayer, peripheral proteins, filaments of cytoskeleton (the cellular “scaffolding” present in the cytoplasm), as well as glycoproteins, which expose their covalently attached oligosaccharide chains (glycans) to the extracellular fluid.
Figure 1.2 Structures of dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and diphytanoylphosphatidylcholine (DPhPC).
Figure 1.3 (A) Basic building blocks of sphingolipids; ceramide consists of the combination of the two differently highlighted portions, with . (B) Structure of the predominant form of egg sphingomyelin.
Figure 1.4 Structures of cholesterol and ergosterol.
Figure 1.5 Sections of a unilamellar vesicle, a micelle, and a lipid bilayer.
Figure 1.6 Schematic picture of a lipid raft (2), with a predominance of sphingolipids (8) and cholesterol (7), surrounded by the liquid-disordered matrix (1). (A) denotes the cytosolic side, (B) the extracellular side.
Figure 1.7 Cross section of an animal cell.
Figure 1.8 Cross section of a chloroplast.
Figure 1.9 Formation of a peptide bond.
Chapter 2: Electrostatics of Biomembranes
Figure 2.1 Lines of force generated by a positive point charge .
Figure 2.2 The three steps of the thought experiment; the dark area is the original charge distribution and the irregularly shaped curve is the original closed surface enclosing it.
Figure 2.3 Portion of a uniform, infinitely extended planar charge distribution of surface charge density , with an ideal cylinder of unit cross-sectional area normal to it. The arrows are unit vectors directed outward.
Figure 2.4 A parallel-plate capacitor. The thick line is the electric potential profile and the arrows are the partial electric fields created by the corresponding uniform charge distributions.
Figure 2.5 Scheme of molecular dipoles aligned along the direction of the electric field created by the true surface charge densities, and , on the plates of a parallel-plate capacitor. The corresponding surface polarization charge densities are denoted by and .
Figure 2.6 Electric potential profile across a neutral bilayer lipid membrane, for a negative transmembrane potential .
Figure 2.7 Fragment of a bilayer lipid membrane, with two ideal cylinders of unit cross-sectional area protruding from its two surfaces toward the respective bulk phases. The fading halftones mimic the fading local charge densities as we move away from the membrane surface.
Figure 2.8 Electric potential profile across a neutral bilayer lipid membrane, for a negative transmembrane potential , according to the linearized Gouy–Chapman theory.
Figure 2.9 Electric potential profile across a charged bilayer lipid membrane, for a zero transmembrane potential , according to the linearized Gouy–Chapman theory.
Figure 2.10 Electric potential profile (thick lines) across a symmetrical BLM at before (A) and after an increase in the electrolyte concentration, , in the external bulk solution (B), and subsequent adjustment of to attain a new minimum in the versus curve (C). Downward arrows denote negative quantities and upward arrows positive quantities.
Figure 2.11 Typical stages of a current versus time curve during the irreversible electrical breakdown of a BLM: decaying capacitive current from to ; low steady-state current from to ; random fluctuations from to ; and tendency toward the maximum limiting current corresponding to the absence of the BLM after .
Figure 2.12 Dependence of the total work of pore formation upon pore radius for absolute values of increasing progressively from 0 to 0.06 V, as calculated from Eq. 2.53 for , , and .
Figure 2.13 Schematic vertical cross section of (A) a hydrophobic pore and (B) a hydrophilic pore in a BLM.
Figure 2.14 Representation of a spherical cell and the relative fluorescence change along its surface, for . The electric field is directed from left to right. The arrows measure the transmembrane potential , positive in the direction of the arrow, at different points of the cell surface. At the cell equator , only the resting transmembrane potential is present, negative toward the interior of the cell.
Chapter 3: Thermodynamics
Figure 3.1 Plot of against for different values of the degree of coupling .
Figure 3.2 Scheme of the coupling of the to secondary active transport across the plasma membrane of animal cells.
Figure 3.3 Scheme of the respiratory electron-transport chain.
Figure 3.4 Scheme of the coupling of the proton-pumping oxidoreductases to secondary active transport across the inner mitochondrial membrane.
Figure 3.5 Scheme of the photosynthetic electron-transport system.
Chapter 4: Passive Transport
Figure 4.1 The four domains of a sodium channel. G, P, S, and I denote sites of glycosylation, phosphorylation, ion selectivity, and inactivation, respectively. Positive (+) charges in S4 are important for transmembrane voltage sensing.
Figure 4.2 Selectivity filter of the KcsA potassium channel. Only two of the four subunits of the tetramer are displayed for the sake of clarity. Potassium ions occupy the S2 and S4 sites, whereas the oxygen atoms of water molecules occupy the S1 and S3 sites. The tips of the P-loops that interact with these species are carbonyl groups. (Zhou et al., 2001).
Figure 4.3 Scheme of a closed, open, and inactive ion channel, with the ball-and-chain model of the inactivation gate.
Figure 4.4 Scheme of the sliding helix model of voltage sensor. The arginine residues of the S4 segment determine a helix of positive charges, which form ion pairs with an array of negative charges on other segments.
Figure 4.5 Plot of the transmembrane potential against time during a hypothetical action potential determined by a single sodium channel and a single potassium channel.
Figure 4.6 Plot of the transmembrane potential (V ) against time, as a neuron fires an action potential (right-hand scale). The Figure also shows the time dependence of the specific conductances, g Na and g K , of Na+ and K+ channels during the action potential (left-hand scale).
Figure 4.7 Plot of ψ(N , N 0 , p ) against N 0 for N = 30 and p = 0.2 (a), 0.5 (b), and 0.8 (c).
Figure 4.8 From top to bottom: Six successive records of I Na from a node of Ranvier during potential steps from a resting potential of −75 to −5 mV, their deviations from the mean, and the resulting variance against time. The variance attains a broad peak when I Na reaches a peak.
Figure 4.9 Plot of the variance, , against the Na+ current, I , from the same voltage clamp records as in Figure 4.8. The solid curve is the best fit of the variance experimental data by Eq. 4.35. If all Na channels in this node opened simultaneously, they would determine a current I of 15 nA. Insets show the time course of variance (upper trace) and mean current (lower trace), with scale factors of 2 × 10−22 Å2 and 1 nA per small division, respectively.
Figure 4.10 Curves of jh /(Ph F ) against Δφ calculated from Eq. 4.51 for ch (0) = 0.1 M and ch (d ) = 0.1 M (a ), 0.05 M (b ), 0.01 M (c ), and for ch (d ) = 0.1 M and ch (0) = 0.05 M (d ), 0.01 M (e ).
Figure 4.11 Electric potential profile (thick line) across a bilayer lipid membrane, showing the position of the gating charge q ′ in the closed and open states.
Figure 4.12 Plot of ih /γh (c ) and of the probability p (b ) against Δφ for a Na channel, calculated as described in the text. Curve a , which coincides with curve c for Δφ > −40 mV, is the ih /γh value without the inclusion of the probability p .
Figure 4.13 Scheme of a gap junction between two plasma membranes. The connexons decrease the intercellular space between the membranes and are open when their connexins are inclined with respect to the normal to the membrane planes.
Figure 4.14 Plots of p 2 against Δφ for q /kT = 0.3 (a ), 0.5 (b ), 1 (c ), 2 (d ) and for Δφ1/2 = −30 mV (a ), −20 mV (b ), −10 mV (c ), −5 mV (d ). The positive slope of the left sigmoidal branch of the curve increases with increasing q /kT , just as the absolute value of the negative slope of the right branch.
Figure 4.15 Scheme of the voltage clamp technique for studying the potentiostatic membrane current of a squid axon.
Figure 4.16 Ionic current in a squid axon in response to a depolarization of 56 mV, with the axon in sea water (A ) and in a solution comprising 10% sea water and 90% isotonic choline chloride (B ), which makes the selected transmembrane potential equal to the sodium Nernst potential, ΔφNa . Curve C is the difference between curves A and B .
Figure 4.17 Curves of the specific conductances, G Na and G K , of sodium and potassium against time, obtained with a squid giant axon at different depolarization potentials reported near each curve. The open circles are experimental values, while the solid curves were calculated from Eqs. 4.90 and 4.92–4.97.
Figure 4.18 Curves of n , m , and h against the transmembrane potential.
Figure 4.19 Schematic representation of the section of an axon.
Figure 4.20 Calculated (A and B) and experimental (C and D) propagated action potentials in a squid axon at 18.5 °C.
Figure 4.21 Scheme of the position of a positive gating charge in a membrane, before and after its hyperpolarization and depolarization, together with the corresponding currents. The sum of the two currents (dashed curve) yields the gating current.
Chapter 5: Active Transport
Figure 5.1 Scheme of ATP synthase, including the H+ channel through which protons flow down into the matrix, resulting in ATP production.
Figure 5.2 Enzymatic cycle of a light-driven proton pump. The intracellular space is on the left side, the extracellular space on the right side. The dashed step is kinetically inhibited.
Figure 5.3 Scheme of bacteriorhodopsin and its enzymatic cycle.
Figure 5.4 Enzymatic cycle of a P-type proton ATPase. The intracellular space is on the left side, the extracellular space on the right side. The dashed steps are kinetically inhibited.
Figure 5.5 Enzymatic cycle of a P-type proton ATPase (A) and the corresponding Gibbs energy levels (B).
Figure 5.6 Enzymatic cycle in which only the initial and final states and a generic intermediate step of the cycle are shown.
Figure 5.7 Movement of a charge qi in a membrane over a distance (x 2 − x 1 ) at constant transmembrane potential, as induced by an electrogenic step, and the resulting movement of a charge Qi along the external circuit.
Figure 5.8 Movement of a charge qi in a membrane fragment over a distance (x 2 − x 1 ), as induced by an electrogenic step. The membrane fragment is adsorbed on a mixed bilayer anchored to a metal support and is inserted into an electric circuit that maintains the potential difference across the whole electrified interface constant, causing the flow of a charge Qi along the external circuit.
Figure 5.9 Equivalent circuit simulating a membrane fragment adsorbed on a lipid bilayer and closed with an external applied potential E .
Figure 5.10 Experimental (black curve) and calculated current transient (gray curve) following a 100 µM ATP concentration jump on proteoliposomes incorporating Ca2+ -ATPase and adsorbed on an octadecanethiol/DOPC mixed bilayer anchored to gold, in the presence of 100 µM free Ca2+ at pH 7.
Figure 5.11 Current transients on proteoliposomes containing Ca2+ -ATPase and adsorbed on an octadecanethiol/DOPC bilayer anchored to gold: (a) following a 28.2 µM Ca2+ concentration jump in the absence of ATP; (b) following a 100 µM ATP concentration jump in the presence of 28.2 µM Ca2+ . The inset shows a current transient caused by a 28.2 µM Ca2+ concentration jump in the absence of ATP (on-current ), as well as the subsequent off-current following a rapid displacement of the Ca2+ -containing solution by an inactivating solution differing from the activating one by the absence of Ca2+ .
Figure 5.12 Current transients following 0.225 mM ATP concentration jumps on membrane fragments containing Na+ , K+ -ATPase and adsorbed on a BLM, in a pH 6.2 solution of 0.13 M NaCl and 3 mM MgCl2 , both in the absence and presence of monensin and of the protonophore 1799.
Figure 5.13 Scheme of a single proteoliposome incorporating Ca2+ -ATPase and adsorbed on a thiol/lipid bilayer anchored to a gold slide (the SSM). The Figure illustrates the instant in which a rapid flux of ATP solution impinges upon the proteoliposome, inducing the calcium pumps to translocate Ca2+ ions (the positive charges) from the solution into the vesicle. The positive charge accumulating on the SSM determines a simultaneous flux of electrons (the negative charges) along the external circuit connecting the reference electrode (RE) to the SSM. A personal computer (PC) records the resulting negative on-current.
Figure 5.14 Current transients following jumps of increasing ATP concentrations on proteoliposomes of Ca2+ -ATPase of the sarcoplasmic reticulum in the presence of 1 mM CaCl2 at pH 7. ATP concentrations: 100 (solid curve), 50 (dashed curve), 25 (up triangles), 10 (dotted curve), 5 (open circles), and 1.5 µM (crosses). The inset shows the ATP dependence of the normalized peak currents. The experimental points are fitted by the Michaelis–Menten equation, yielding a K S value of 2.9 ± 0.3 µM.
Figure 5.15 Eight-state enzymatic cycle of SERCA.
Figure 5.16 Structural model of SERCA.
Figure 5.17 Pictorial scheme of the enzymatic cycle of SERCA.
Figure 5.18 Current transients following 100 µM ATP concentration jumps on proteoliposomes of Ca2+ -ATPase of the sarcoplasmic reticulum in 100 mM free calcium and 0.15 M choline chloride, at different pH values: 6.55 (stars), 6.78 (crosses), 7.03 (open up-triangles), 7.35 (solid up-triangles), 7.58 (open circles), and 8.13 (solid circles). Calcium ionophore A23187 and protonophore 1799 (1.25 µM) were also used. The inset shows the dependence of the normalized charge under the peaks upon pH. The solid curve is the best fit of the experimental points to the phenomenological Hill function.
Figure 5.19 Schematic picture of the enzymatic cycle of Na+ , K+ -ATPase, with a representation of nowadays superseded large vestibula to justify nonelectrogenic ion-binding and ion-release steps.
Chapter 6: Biomimetic Membranes
Figure 6.1 Schematic picture of different biomimetic membranes: (A) solid-supported BLM (sBLM); (B) tethered BLM (tBLM) consisting of a thiolipid monolayer with a lipid monolayer on top; (C) polymer-cushioned BLM (pBLM); (D) S-layer stabilized BLM (ssBLM) consisting of an S-layer with a lipid bilayer on top; (E) protein-tethered BLM (ptBLM).
Figure 6.2 Space-filling model and structure of 2,3,di-O -phytanyl-sn -glycerol-1-tetraethylene-glycol-d,l-α lipoic acid ester lipid (DPTL) and of diphytanoylphosphatidylcholine (DPhPC), in a tail-to-tail configuration; l , s , and m denote the lipoic acid residue, the tetraethyleneoxy spacer, and the hydrocarbon tail region, forming the monomeric unit of a DPTL/DPhPC-tethered bilayer lipid membrane.
Figure 6.3 Equivalent circuit for a BLM of dioleoylphosphatidylcholine.
Figure 6.4 Plot of log|Z | (solid circles) and ϕ (solid triangles) against log(f ) (Bode plot) for a mercury-supported DPTL/DPhPC bilayer incorporating valinomycin from its 0.15 µM solution in aqueous 0.1 M KCl at −0.375 V versus Ag/AgCl(0.1 M KCl). The solid curve is the best fit of the impedance spectrum by the equivalent circuit shown in the figure, with R lar = 2.8 MΩ cm2 , C lar = 4.2 µF cm−2 , R s = 48 kΩ cm2 , C s = 3.3 µF cm−2 , R m = 3.4 kΩ cm2 , C m = 0.9 µF cm−2 , R Ω = 3.7 Ω cm2 , and C Ω = 43 nF cm−2 .
Figure 6.5 Plot of Z ″ against Z ′ (Nyquist plot) for the same tBLM as in Figure 6.4. The solid curve is the best fit of the impedance spectrum by the equivalent circuit shown in Figure 6.4, obtained by using the same R and C values. The semicircle in the Figure corresponds to the mesh of highest time constant and highest resistance, ascribable to the lipoic acid residue. The inset shows an enlargement of the initial portion of the Nyquist plot.
Figure 6.6 Plot of ωZ ′ (solid circles) against −ωZ ″ for the same tBLM as in Figure 6.4. The solid black curve is the best fit of the impedance spectrum by the equivalent circuit shown in Figure 6.4. From left to right, the distorted semicircles of increasing radius refer to ωZ ′ contributions from the lipoic acid residue, the spacer, the lipid bilayer, and the solution.
Figure 6.7 Schematic pictures of the barrel-stave model (A) and toroidal model (B) of an ion channel, and of the gradual passage from a “flat cluster” to an “embedded cluster” (i.e., an ion channel) via a “flat monomer” and an “embedded monomer” (C).
Figure 6.8 (A) Proceeding upward, current versus time curves at a BLM in aqueous solution of 1.0 M NaCl and 5 × 10−7 g mL−1 alamethicin, following transmembrane potential steps from 0 to −40, −42, −50, −55, and −58 mV. Source: Eisenberg (1973). Reproduced with permission of Springer. (B) Current versus time curves at a BLM in aqueous solution of 1.0 M NaCl and 4 × 10−8 g mL−1 alamethicin, at a pressure of 100 MPa, following transmembrane potential steps of the height (in mV) reported in the Figure In both cases, alamethicin was added only on the cis side of the membrane. Source: Bruner (1983). Reproduced with permission of Elsevier.
Figure 6.9 The solid curves are three successive current–time curves on the same BLM following transmembrane potential steps from 0 to −60 mV (a ), −55 mV (b ), and −50 mV (c ), in aqueous 0.1 M KCl containing 0.625 µM monazomycin (Muller et al., 1981). The corresponding dashed curves were calculated by the nucleation-and-growth model outlined in the text using the parameters θ0 = 0.1, n = 2, and for all three curves; p and were given the values: (a ) 1 and 15 s−3 ; (b ) 0.438 and 130 s−3 ; (c ) 0.163 and 1250 s−3 . The three calculated currents were matched to the experimental ones by multiplying them by the same factor 80.
Figure 6.10 Markers are conductance values in squid giant axon at 6–7 °C brought about by the following depolarizations: 32 (a ), 38 (b ), 51 (c ), 63 (d ), 76 (e), 88 (f ), and 100 mV (g ). The solid curves were calculated for θ0 = 0.03, n = 2, , p = 0.47 (a ), 0.55 (b ), 0.67 (c ), 0.74 (d ), 0.83 (e ), 0.946 (f ), 1 (g ), and for a normalizing factor of 697 mS cm−2 .
Figure 6.11 Schematic pictures of a DPTL/DOPC tBLM incorporating a gramicidin channel in the closed state (top left) and in the open state (bottom left). The typical shape of charge versus time and current versus potential curves at a closed ion channel (or in the absence of ion channels) and at an open ion channel is depicted on the right-hand side of the corresponding schematic pictures.
Figure 6.12 Charge transients recorded in 0.1 M KCl and 4 µg mL−1 DCD-1L at a DPTL/DOPC tBLM in an unbuffered solution (curve a ) and at a DPTL/DOPS tBLM in a pH 7 buffer solution (curve b ), by stepping the applied potential from −0.15 to −1.00 V(SCE); the curves are corrected for the background current and normalized to unity. The corresponding dashed curves are the best fits by the model outlined in the text. The inset shows charge transients at a DPTL/DOPS bilayer in the pH 7 buffer solution recorded by stepping the potential from −0.15 to −0.75 V (a ), −0.85 V (b ), and −0.95 V (c ).
Figure 6.13 Current–voltage curves at a phosphatidylethanolamine BLM in aqueous 0.1 M NaCl, with the cis side solution containing different alamethicin concentrations: (a ) 0.05, (b ) 0.1, (c ) 0.2, (d ) 0.5, (e ) 1, and (f ) 1.5 µg mL−1 . Scan rate = 12 mV/s. The baseline current is the current through the bare membrane. The dashed line intersects the curves at a current whose ratio to the corresponding voltage is constant and measures the average conductance G .
Figure 6.14 Current–voltage curve at a dibromostearylphosphatidylcholine BLM in a pH 5.5 unbuffered solution of 1 M KCl and 0.3 µg mL−1 alamethicin, added on the cis side of the membrane (right upper quadrant).
Figure 6.15 Plots of ln G versus V for different values of a and for Δμ = 70 D, θ0 = 0.1, , and n = 1. The curves shift toward progressively lower ln G values with decreasing a .
Figure 6.16 Experimental I–V curve for 0.2 µg mL−1 alamethicin in bacterial PE, taken from curve 2 in Figure 5 of Vodyanoy et al. (1983) (solid curve) and curve calculated for a = 1 × 10−2 , Δμ = 70 D, θ0 = 0.1, , and n = 1 (dashed curve). The height of the calculated curve was normalized to that of the experimental one.
Figure 6.17 Experimental I–V curve for 0.4 µg mL−1 melittin in DOPC, taken directly from Figure 7 of Pawlak et al. (1991) (curve with small fluctuations), and curve calculated for a = 1 × 10−4 , Δμ = 70 D, θ0 = 0.1, , and n = 1 (smooth curve). The height of the calculated curve was normalized to that of the experimental one.
Figure 6.18 The solid curves are experimental cyclic voltammograms at a DPTL/DOPC tBLM in aqueous solution of 0.1 M KCl and 0.1 µM gramicidin A at pH 6.8, 5.4, and 3. Scan rate = 50 mV s−1 . The corresponding dashed curves were calculated as outlined in the text.
Figure 6.19 CV at a DPTL/DOPC tBLM in a pH 6.8 buffer solution of 0.1 M KCl and 1 µg mL−1 SP25A, recorded between −0.20 and −1.20 V at a scan rate of 50 mV s−1 . The solid curve is the experimental CV, the dashed curve is the best fit by the model. The inset shows the CV calculated as outlined in the text (solid curve), as well as the corresponding charge densities of the monovalent cation (dashed curve) and anion (dotted curve), against potential.
Figure 6.20 Cyclic voltammograms at a DPTL/DOPC tBLM in aqueous 0.1 M KCl at a scan rate of 10 mV s−1 , in the absence (dashed curve) and presence of 0.4 µM alamethicin (solid curve).
Figure 6.21 AC voltammograms at 75 Hz of a DOPC monolayer in aqueous 0.1 M KCl (a ), and in aqueous solution of 0.1 M KCl and 2 µg mL−1 DCD either unbuffered (c ) or buffered at pH 7 (b ).
Figure 6.22 Schematic picture of a cell consisting of two compartments separated by a septum, with an enlarged picture of a lipid bilayer spanning a small hole in the septum, according to the method of BLM formation by Mueller et al. (1962).
Figure 6.23 Schematic picture of a cell containing a solution whose surface is separated by a movable septum, with two different lipids spread on the two portions of the solution surface. The solvent-free BLM is formed by lowering the septum so as to bring the hole in the septum below the level of the solution surface.
Figure 6.24 The three stages of BLM formation at the tip of a micropipette: (i) immersion before spreading the lipid at the solution/air interface; (ii) emersion after spreading the lipid; (iii) reimmersion.
Figure 6.25 Different configurations of the patch-clamp technique, showing the micropipette tip and a cell. First, a seal in the cell-attached configuration is formed. From this, whole-cell, perforated-patch, and inside-out configurations can be generated. The outside-out configuration requires starting from the whole-cell configuration.
Figure 6.26 Schematic picture of a hanging mercury drop before its gradual expansion in contact with a lipid film spread on the surface of an aqueous solution (left); hanging mercury drop almost completely immersed in the solution, in such a way as to prevent the solution from wetting the capillary tip (right). The thickness of the lipid monolayer is enormously enlarged with respect to the mercury drop radius, for clarity.
Figure 6.27 The left-hand side of the Figure shows Q (t ) versus t curves for the reduction of 1 mol% UQ in a mercury-supported DOPC self-assembled monolayer immersed in aqueous 0.075 M borate buffer of pH 9.4, as obtained by stepping the potential from a fixed initial value E i = −0.200 V to final values E varying from −0.225 to −0.700 V by 25 mV increments. The right-hand side shows the corresponding Q (t = 50 mV) versus E curve.
Figure 6.28 Scheme of the splitting and spreading of a vesicle onto a metal-supported self-assembled hydrophobic thiolipid monolayer (A), and of its unrolling and spreading on a self-assembled monolayer of a thiolated hydrophilic spacer tethered to the metal (B).
Figure 6.29 Schematic picture of a gold-supported tBLM consisting of a mixture of mercaptoalcohol and thiolipid molecules, with a patch of lipid monolayer on top of the thiolipids and a patch of lipid bilayer on top of the mercaptoalcohols. The space between the mercaptoalcohols and the lipid bilayer is occupied by water molecules and is the site over which proteins are preferentially incorporated.
Figure 6.30 Schematic picture of a gold electrode coated with short spacer molecules (dithiobis propionic acid, DTP) and longer molecules with the nitrilotriacetic (NTA) functionality. A Ni2+ ion is coordinated by both this functionality and the hystidine tag of a cytochrome c oxidase (COX) protein, thus anchoring the latter to the gold surface. A lipid bilayer is formed around the protein, leaving a water layer between the bilayer and the spacer.
Figure 6.31 Schematic picture of a tethered bilayer lipid membrane (tBLM) array. (A) An optical microscope image of the probe pad and the tungsten electrode tip. (B) Graphical representation of the tBLM array. The lower left corner shows the gold sensor pad covered with a tBLMs that incorporates ion channels. The inset shows a tBLM formed at the gold surface of the sensor pad.
Chapter 7: Auxiliary Techniques
Figure 7.1 Incident, reflected, and transmitted radiations at the interface between two phases of different refractive index.
Figure 7.2 Unpolarized, linearly polarized, and left-handed circularly polarized light.
Figure 7.3 The p- and s-polarized components of circularly polarized light.
Figure 7.4 Fresnel layer model of a multilayer interface. Lighter halftones simulate lower optical densities.
Figure 7.5 Dispersion relations at metal/prism and metal/air interfaces and prism coupling to surface plasmon polaritons.
Figure 7.6 Excitation of surface plasmon polaritons in the Otto and Kretschmann geometries.
Figure 7.7 Reflectivity against the angle of incidence for four different thickness values, q , of the gold film. Prism (n d = 1.51); gold film (ϵw = −25 + 1.44i); and water . Wavelength = 800 nm.
Figure 7.8 Plots of reflectivity (solid curve) and resistance (dotted curve and squares) at a gold-supported hexaethyleneoxy-based thiolipid monolayer, following addition of DPhPC vesicles, rinsing and further addition of 180 nM α-hemolysin.
Figure 7.9 Phase shift of the electric field vector for s-polarized (top) and p-polarized (bottom) radiation at an isotropic metal surface.
Figure 7.10 PM FTIRRA spectra in the ν(CO) stretching region of a DOPC bilayer at an Au(111) electrode in 0.1 M NaF/D2 O solution at potentials indicated in the Figure Thick lines are spectra calculated for a 4.5 nm thick DOPC bilayer in different environments: (solid line) D2 O; (dashed line) CCl4 solution.
Figure 7.11 Schematic diagram of the DOPC molecule and the directions of the transition dipole moments estimated from PM IRRAS.
Figure 7.12 Incident plane wave, circular wave scattered by a scattering center at r = 0, and a scattering triangle formed by the wave vectors of an incident and a scattered neutron and by the scattering vector Q .
Figure 7.13 Constructive interference between waves reflected from two adjacent scattering planes.
Figure 7.14 (A) versus Qz plots for a DPTL SAM at various potentials against a reference electrode having a potential of 288 mV versus the NHE. The solid lines are the reflectivity fits of the experimental data by a box model. (B) SLD profiles of the models that are best fits to the data shown in (A). The box model consisted of the following layers: Si, SiOx , Cr, Au, tetraethyleneoxy spacer, lipid monolayer, electrolyte.
Figure 7.15 Schematic picture of a WC14/DPhPC tBLM reconstituted with α-hemolysin (B) and of the corresponding SLD profiles (A), resulting from the simultaneous fitting of a molecular model to five neutron reflectivity data sets. The SLD profiles contain the calculated contribution of the α-hemolysin X-ray crystal structure at a lateral density and insertion depth derived from the model fit.
Figure 7.16 Simplified electronic-state diagram, where the energies of some vibrational levels of the two electronic states S0 and S1 are sketched.
Figure 7.17 Lipid bilayer uniformly labeled with a fluorescent tag (A), selectively photobleached by a small and intense light pulse (B), monitored as the bleached dye diffuses out of the beached area and new dye diffuses into it (C), and monitored after uniform intensity is ultimately restored (D).
Figure 7.18 TCSPC measurement principle.
Figure 7.19 Two-photon (λ excitation = 760 nm) fluorescence lifetime 21 × 21 µm image (left panel) and fluorescence lifetime distribution histogram (right panel) of a distal monolayer of DOPC/PSM/Chol (47:47:6) mixture, labeled with 1 mol% LAURDAN, at room temperature. The mixture is the distal monolayer of a micro tBLM. The color code in the FLIM image is that indicated in the histogram on the right. Source: Becucci et al. (2010). Reproduced with permission of Royal Society of Chemistry.
Figure 7.20 Scheme of a system for converting the deflection of an AFM cantilever into an electrical signal.
Figure 7.21 (A–D) Force measurements of template stripped gold, modified with EO3-cholesterol/6-mercaptohexanol (30:70) before (A) and after (B–D) incubation with B. subtilis membrane vesicles. Panel B is recorded in a region free of vesicles, and panels C and D are force curves at vesicles. The arrow indicates a typical signal obtained when the AFM tip protrudes through the phospholipid bilayer. Solid lines are for the tips approaching the surface, dashed lines for receding tips. (E) Tapping mode AFM image, prior to force measurements, indicating the positions where force curves were acquired. (F) AFM pictures before and after the force measurements.
Figure 7.22 Electrochemical cell for EC-STM with bipotentiostatic setup.
Figure 7.23 AC-STM image of flower-like structures at a Au(111)-supported DMPC monolayer incorporating alamethicin, as acquired using a constant tunneling current of 1.16 nA (A). Schematic arrangement of pores formed by alamethicin molecules (B).
Figure 7.24 (A) Scheme of the Langmuir balance method. (B) Schematic π–A isotherm of a long-chain phospholipid.
Figure 7.25 Langmuir trough for vertical transfer onto a solid substrate (A); Langmuir–Blodgett deposition onto a hydrophobic (B, C) and a hydrophilic (D) substrate; Langmuir–Schaefer deposition (E, F).
Figure 7.26 (A) Quartz crystal with alternating current applied across two gold electrodes, schematically depicted under shear stress in opposite directions. (B) Oscillatory decay of the QCM-D as it comes to rest, both before (dashed curve) and after deposition of the adsorbed material (solid curve).
Figure 7.27 Lipid deposition pathways measured by QCM-D on silica. The legends indicate the lipids used: dioleoyltrimethylammonium-propane (DOTAP), dioleoylphosphatidylcholine (DOPC), and dioleoylphosphatidylserine (DOPS), with their molar mixing ratios.
Bioelectrochemistry of Biomembranes and Biomimetic Membranes
Copyright © 2017 by John Wiley & Sons, Inc. All rights reserved
Published by John Wiley & Sons, Inc., Hoboken, New Jersey
Published simultaneously in Canada
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Library of Congress Cataloging-in-Publication Data:
Names: Guidelli, R. (Rolando)
Title: Bioelectrochemistry of biomembranes and biomimetic membranes / Rolando
Guidelli.
Description: Hoboken, New Jersey : John Wiley & Sons, Inc., [2017] | Includes
bibliographical references and index.
Identifiers: LCCN 2016026090| ISBN 9781119271055 (cloth) | ISBN 9781119278405
(epub)
Subjects: LCSH: Biomimetic materials. | Bioelectrochemistry. | Membranes
(Biology)
Classification: LCC R857.M3 G86 2017 | DDC 610.28–dc23 LC record available at https://lccn.loc.gov/2016026090
To my grandchildren Neri and Petra
This book is largely based on the lecture notes for the course of Bioelectrochemistry, which I have been holding for students of the “biennio di laurea specialistica” (roughly equivalent to the last year of a UK college) at the Department of Chemistry of Florence University, from 2005 to 2010. It is accessible to graduate students and final year undergraduate students in chemistry and biology, as well as researchers in related disciplines including biology, physics, physiology, and pharmacology. It is particularly suitable for students attracted by the fascinating area of biological membranes and their functions and interested in reading a supplemental text that takes practically no argument for granted and leads the reader, step by step, through gradually more fundamental aspects of this area. To this end, the manuscript describes the essential electrochemical basics required to understand why and how electrochemical and electrophysiological tools are fundamental in elucidating the mode of ion transport across biomembranes. In this respect, it is also of interest to many electrochemists attracted by the biological realm (as it happened to me 20 years ago) and wishing to get a better view of the potentialities of the their own background and tools to move more closely into this area. On the other hand, it is of interest to biophysicists and biochemists willing to get an exhaustive overview of the potential of biomimetic membranes for the investigation of the function of membrane peptides and proteins.
The book deals with the bioelectrochemistry of biological membranes and their mimics in a homogeneous and thematically unified way. It is not a collection of selected, separate topics on the bioelectrochemistry of membranes. To understand in depth the structure and function of biological membranes, it is also essential to understand and apply principles of physical chemistry. In particular, the fundamental role played by the transmembrane potential in modulating the function of biomolecules incorporated in the membrane allows us to regard and treat the membrane as an outright electrified interface. Hence, to understand the function of biological membranes and the properties of their experimental models, called biomimetic membranes, a knowledge of some basic principles of electrochemistry and of the most significant electrochemical techniques is required. The purpose of this manuscript is to construct a coherent thermodynamic and electrochemical framework to achieve this goal.
The book is composed of seven chapters. In Chapter 1, some basic concepts of membrane biochemistry and the relative terminology are briefly outlined. Chapter 2 deals with the electrostatics of biomembranes; here, the relation between electric field and electric potential is briefly touched upon, in order to introduce a simplified derivation of the Poisson equation and, subsequently, the derivation of the Poisson–Boltzmann equation and the Gouy–Chapman theory, which are required to determine the profile of the electric potential across a bilayer lipid membrane. In this chapter, thermodynamic concepts are still not adopted, except for Boltzmann's factor, which is used to illustrate the competition between the ordering effect of the electric field and the disordering effect of temperature. Chapter 3 is devoted to thermodynamics; after a brief introduction to some basic concepts of chemical thermodynamics (including the concept of electrochemical potential, which is not clear to many students), the thermodynamics of irreversible processes is explained and its importance in providing the basis for the function of all ion pumps is pointed out. This chapter ends with a number of examples of coupling of chemical reactions to the vectorial transport of molecules across different types of membranes. The first three chapters serve to introduce the reader to the heart of the problem of passive and active transport across membranes, as carried out by embedded proteins. Passive transport by ion channels is dealt with in Chapter 4, whereas primary active transport by ion pumps and secondary active transport by transporters are discussed in Chapter 5. Particular emphasis is placed on the role of the transmembrane potential in modulating the function of ion channels and pumps. In Chapter 4, the channel current is discussed at two levels: at the first level, it is considered to be proportional to the transmembrane potential according to Ohm's law, with the necessary inclusion of the open probability; at the second level, it is obtained by derivation of the Goldman–Hodgkin–Katz equation, and the outcomes of the two approaches are compared. Two levels are also adopted for explaining the transmission of nerve impulses; the second level leads to the derivation of the equation for the action potential. Chapter 5 deals with the energy levels of the enzymatic cycle of ion pumps and describes in detail how this cycle can be investigated under both steady-state and pre-steady-state conditions by means of electrochemical/electrophysiological techniques. Chapter 6 describes the various biomimetic membranes used to incorporate peptides and proteins in order to investigate their functional activity by electrochemical means, starting from the traditional “bilayer lipid membrane” (BLM) and considering the evolution toward more robust and sophisticated experimental models of biomembranes. Before classifying them in detail on the basis of their structural features and before scrutinizing both their advantages and drawbacks, a number of electrochemical techniques used for their investigation is described and illustrated by selected examples. Possible future developments and applications are foreseen. Finally, Chapter 7 describes the salient features of a number of nonelectrochemical, auxiliary techniques that are frequently employed to characterize solid-supported biomimetic membranes, either at a macroscopic or a molecular level; they are complemented by a few illustrative applications.
I am very grateful to those publishers who have allowed me to reproduce figures that appeared in their own publications, and to Wikipedia for the nice images that I downloaded from Wikimedia Commons. The sources of each of these are indicated in the legends. I would also like to acknowledge the constant collaboration of Lucia Becucci, who started working with me on mercury-supported biomimetic membranes as a student in 1992 and has been conducting research in this area until now, demonstrating notable skillfulness in organizing and carrying out research work and in devising stimulating experiments. It is a pleasure to thank my colleague Jacek Lipkowski for his invaluable suggestions and helpful criticism on Chapter 7. Special thanks go to Ms. Anita Lekhwani for her cheerful assistance in the preparation of this book proposal and to Ms. Sumathi Elangovan, the Project Editor, for her helpful advice and excellent editorial suggestions. Recognition should also be accorded to my daughter Ilaria and her husband, Niccolò Amerini, for managing my computer's performance.