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Membrane reconstitution and functional analysis of a sugar transport system
Knol, Jan
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Chapter 1
GENERAL INTRODUCTION
INTRODUCTION
Cells from all kingdoms of life are surrounded by a membrane that forms the barrier between the
cytoplasm and the external environment. The major components of biological membranes are lipids,
proteins and carbohydrates that are associated with them. The fluid mosaic model of Singer and Nicolson
describes the membrane as a fluid phospholipid bilayer, wherein proteins and lipids can freely diffuse
within the plane of the membrane (Singer and Nicolson, 1972). The structure of the membrane is
dependent on complex interactions between these lipids and membrane proteins, and the major role of
membrane lipids is to form the hydrophobic matrix in which the membrane proteins can be fully functional
(see also chapter 7). A striking feature of membrane lipids is their enormous diversity, but also the lipid
composition of different membranes can vary considerably (Gennis, 1989).
MEMBRANES
The most common membrane lipids are the glycerophospholipids, which consist of a glycerol
backbone, two fatty acid acyl chains, and a polar head group at the sn-3 position (Table I).
Table I. Most common phospholipids
Phospholipid
Polar Head
Phosphatidic acid (PA)
R-OH
R-OCH2CHNH2COO
Phosphatidyl serine (PS)
Phosphatidyl glycerol (PG)
R-OCH2CHOHCH2OH
Phosphatidyl inositol (PI)
R-OHC6H5(OH)5
Cardiolipin (CL)
R-OCH2CHOHCH2
Phosphatidyl choline (PC)
R-OCH2CH2N+(CH3)3
Phosphatidyl ethanolamine (PE)
R-OCH2CH2NH3+
Charge
Negative
Negative
Negative
Negative
Negative
Zwitterionic
Zwitterionic
The acyl chains are attached to the sn-1 and sn-2 positions of glycerol by ester or ether linkages and
they can vary widely in length (C14 to C24), branching, and degree of unsaturation (Table II). Fatty acid
esters of glycerol are the predominant type of lipids in most eukaryotic and prokaryotic membranes, of
which phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the major species in bacterial
membranes and phosphatidylcholine (PC) is the most abundant in animal cell membranes. The most
frequently occurring saturated fatty acids in natural membranes are palmitic, stearic, and miristic. Oleic
acid, which is a stearic acid with one double bond in cis configuration in the middle of the chain, i.e., c 918:1, is the most abundant unsaturated fatty acid. Saturated fatty acid chains are very flexible, and, in the
liquid crystalline phase of the membrane, each single bond has complete freedom of rotation. The most
probable conformation is the fully extended chain. Unsaturated fatty acids have one or more rigid kinks
due to double bonds that are nonrotating. The cis double bond produces a bend of nearly 30o, while the
-2-
Chapter 1
trans configuration, which is rare in naturally occurring unsaturated fatty acids, resembles the extended
form of saturated fatty acids (Lasic, 1988)
In archaea the stereoconfiguration of the glycerophospholipids (and glycoglycerolipids) is different
from that in bacteria with the phosphoryl groups on the sn-1 position of the glycerol, and the hydrophobic
constituents are isopranyl glycerol ethers rather than fatty acid esters. The ether linkage (C-O-C) is
chemically less susceptible to hydrolysis and, thus, more stable. Tetraether lipids with C40 isoprenoid
chains contain two polar headgroups linked at opposite sites of the molecule. These molecules span the
entire membrane, resulting in a monolayer organization (De Rosa et al., 1986).
Cardiolipids are essentially dimeric phospholipids, and these molecules are important constituents of
the mitochondrial inner membrane and also of some bacterial membranes. Phosphosphingolipids, rarely
found in plants or bacteria, have similar polar substituents as the glycerophospholipids, but the
hydrophobic group is in this case a ceramide (e.g., sphingomyelin). Another important class of lipids is
the glycoglycerolipids in which the sn-3 position of glycerol forms a glycosidic link to a carbohydrate such
as galactose. Gram-positive bacteria, in particular, have glycoglycerolipids with a wide variety of sugars.
The lipid composition of Streptococcus thermophilus, the organism from which the lactose transport
protein was isolated and reconstituted as described in this thesis, is given in Table II.
Tabel II. Fatty acids commonly found in lipids of biological membranes and the
fatty acid composition of the Gram-positive bacterium Streptococcus thermophilus
Fatty acid
Lauric
Myristic
Palmitic
Palmitoleic
Stearic
Oleic
Vaccenic
Linoleic
γ-Linolenic
α-Linolenic
Arachidic
Eicosenoic
Behenic
Arachidonic
Others
1
Chain length : unsaturation
Percentage of acids in
12 : 0
14 : 0
16 : 0
16 : 1 (9-cis)
18 : 0
18 : 1 (9-cis)
18 : 1 (11-cis)
18 : 2 (9-cis, 12-cis)
18 : 3 (6-cis, 9-cis, 12-cis)
18 : 3 (9-cis, 12-cis, 15-cis)
20 : 0
20 : 1
22 : 0
20 : 4 (5-cis, 8-cis, 11-cis, 14-cis)
S. thermophilus
Trace
2
16
5
3
56
ND1
ND
ND
ND
0
15
ND
ND
3
ND, not determined
Lipid molecules can move from one side of the bilayer to the other (flip-flop), but the rate of this
process is rather low due to the polarity of the headgroups. This helps in maintaining a lipid asymmetry
between the inner and outer leaflets. For instance, in the erythrocyte membrane, PC and sphingomyelin
are most abundant in the outer half of the bilayer, whereas PE and phosphatidylserine (PS) are largely
found in the inner half (Gordesky and Marinetti, 1973). The transmembrane lipid asymmetry is controlled
by ATP-dependent translocators (flippase), e.g., the aminophospholipid transporter plays a primary role
in maintenance of phosphatidylserine asymmetry in the erythrocyte membrane (Zachowski et al., 1989;
Loh and Huestis, 1993). Also proteins known as multidrug transporters can function as flippases; besides
pumping xenotoxins out of cells they may serve a function in organizing natural lipids. MDR1 Pglycoprotein is a translocase with only specificity for short chain fatty acids, but MDR3 P-glycoprotein
General Introduction - 3 specifically translocates PC (Helvoort et al., 1996).
There are a number of other membrane components (e.g., sterols and hopanoids) or other lipids which
can be considered minor in terms of the amount present that are not discussed here, but which might be
important for the functionality of membranes and proteins therein. Sterols, for example, of which
cholesterol is best known, increase the lipid ordering above the gel-liquid crystal phase transition
temperature and reduces it below. In the biologically relevant liquid crystalline phase, the presence of
sterols stabilizes the membrane, which results in a decreased permeability for ions and other solutes
(Gennis, 1989).
Cells or organelles can either have one (Gram-positive bacteria, archaea, fungi, peroxisomes) or two
membranes (Gram-negative bacteria, mitochondria). The membrane of an organism that is in contact with
the cytoplasm is called the cytoplasmic or plasma membrane and this membrane represents the major
barrier of the cell for hydrophilic compounds. The thick cell wall of Gram-positive bacteria is composed
primarily of peptidoglycan, but can also contain large amounts of teichoic acids that are not present in
Gram-negative bacteria. The envelope of Gram-negative bacteria is more complex than that of Grampositive bacteria and consists of three layers. The outer membrane and the plasma membrane enclose the
periplasmic space, in which a thin layer of peptidoglycan is present that gives the cell its structure and
stability. The outer membrane functions as a molecular sieve and harbors, amongst others, various channel
proteins (porins) that allow the passage down the concentration gradient of molecules with masses up to
about 600 Da. The periplasmic space contains, amongst others, the binding proteins of ATP-driven uptake
systems, which, in case of Gram-positive bacteria, are attached to the outer surface of the cytoplasmic
membrane via a lipid modification (Lugtenberg and Van Alphen, 1983; Nikaido and Vaara, 1985).
Although proteins in the outer membrane lack direct access to either ATP or the proton motive force,
Gram-negative bacteria have evolved systems whereby the proton motive force across the cytoplasmic
membrane (see below) is transduced across the periplasmic space to, for instance, high-affinity outer
membrane receptors like FepA. FepA is the receptor for ferrichrome-iron, and the uptake of the
siderophore-iron complex is coupled to the proton motive force by conformational changes in the TonB
protein, which is anchored in the cytoplasmic membrane by its uncleaved amino terminus. TonB spans the
periplasmic space and interacts with proteins in the cytoplasmic membrane and in the outer membrane.
Details of the interactions with the outer membrane receptor and the nature of the energy transduction
event are unknown. The recent crystal structures of FepA (and also FhuA) suggest a novel mechanism of
active transport (Postle, 1999).
MEMBRANE TRANSPORT
In order to survive it is essential that cells are able to control the fluxes of all kinds of substances
across the cytoplasmic membrane. Growth substrates, for example, have to be taken up, whereas metabolic
end-products must be excreted, and toxic molecules need to be kept outside the cell. To achieve this the
cytoplasmic membrane harbors many proteins that actively translocate these solutes, either from out to in
or in to out or in special cases from the lipid bilayer into the medium. These so-called transport systems
are, unlike the outer membrane porins, generally quite specific for one or a few substrates. Exceptions are
the multidrug efflux systems that transport a wide variety of chemically unrelated compounds, and thereby
offer the cells protection against unwanted and toxic compounds in the environment. Cells are able to
express different membrane proteins depending on the chemical composition and physical conditions of
the environment. Although many different types of transporters exist, all bacterial transport systems can
be subdivided in only three categories on the basis of their energetics.
(i) Primary transport systems convert chemical or light energy into electrochemical energy like solute
gradients. Examples are the linear electron transfer chains (e.g., cytochrome oxidase), the light driven ion
-4-
Chapter 1
pumps (e.g., bacteriorhodopsin), the sodium-ion translocating decarboxylases, and the ATP-driven
transporters (e.g., F0F1-ATPase, members of the ABC super family of binding protein dependent transport
systems and others) (for reviews Poolman et al., 1992; Higgins, 1992).
(ii) Secondary transport systems use the free energy that is stored in the electrochemical gradients of
protons, sodium ions or other solutes across the membrane (See below). Generally, these systems are
composed of a single polypeptide that binds and translocates the solutes, but recently it has been shown
that some secondary transport systems employ substrate binding proteins similar to that of binding protein
dependent uptake systems that belong to the ABC superfamily (Jacobs et al., 1996).
(iii) Group translocation systems couple the translocation to a chemical modification of the substrate.
The only group translocation systems found in bacteria are the phosphoenolpyruvate-dependent sugar
phosphotransferase systems (PEP-PTS), which phosphorylate the sugar concomitant with the translocation.
The energy-rich phosphate bond of phosphoenolpyruvate is transferred to the membrane embedded
translocator via cytoplasmic and/or membrane bound phosphoryl carrier proteins (for review Postma et
al., 1993). The phosphorylation state of the phosphoryl carrier proteins also plays an important role in
regulatory processes such as catabolite repression. In some cases, like LacS of Streptococcus thermophilus,
secondary transporters carry a domain that is homologous to a component of PTS systems, through which
the transport activity is regulated (Poolman et al., 1989; Poolman et al., 1995; See also chapter 2).
At the energetic level, the different transport reactions can be linked to each other as postulated by
Mitchell in the chemiosmotic theory (Mitchell, 1961; 1963). For example, in the lactic acid bacterium S.
thermophilus, ATP is generated by substrate level phosphorylation when lactose is broken down. A large
fraction of the ATP is hydrolyzed by the F0F1-ATPase, a membrane protein that pumps protons from the
cytoplasm to the external medium. As a result, the internal pH increases relative to the outside, whereas
the charge translocation leads to the formation of a membrane potential. This inward force on the protons
can, subsequently, be used by secondary transport systems to selectively take up or excrete solutes.
Alternatively, the ATP can be used directly to translocate solutes via primary transport systems.
SECONDARY TRANSPORT
Secondary transporters, like the lactose transport protein (LacS) of S. thermophilus, are a class of
transport proteins that is found in all living cells ranging from bacteria to humans (Maiden et al., 1987).
In secondary transport, the energy of the (electro-)chemical gradient of one solute is converted into the
energy of the (electro-)chemical gradient of another solute (including ions). Secondary transport systems
can be subdivided in three categories. The transport is called uniport when it is solely driven by the
concentration gradient of the substrate and no coupling ions participate in the translocation process. An
example is the glucose transporter of Zymomonas mobilis (Weisser et al., 1995). The process is called
symport when more than one substrate move in the same direction. In this case the (electro-)chemical
gradient of one solute (usually proton or sodium-ion) is used to drive the uphill transport of another solute.
Since the direction of secondary transport is dependent on the direction of the gradients of transported
solutes, excretion of intracellularly formed metabolic end-products may in special cases lead to the
generation of a proton motive force (Lolkema et al., 1996). Finally, when substrates move in opposing
directions, transport is referred to as antiport. Depending on the charge and stoichiometry of the substrates,
the total driving force for the different transport reactions may differ considerably (Poolman and Konings,
1993).
General Introduction - 5 Since under normal physiological conditions the membrane potential (∆Ψ) is inside negative relative
to outside, and the pH gradient (∆pH) is inside alkaline relative to outside, a solute-H+ symport system will
catalyze the accumulation of the solute at the expense of the proton motive force (∆p):
∆p = ∆Ψ - (2.3RT/F)∆pH = ∆Ψ - Z∆pH
(mV)
where R is the gas constant, T the absolute temperature, and F the Faraday constant (2.3RT/F ~ 58.8
at 20 oC). The same is true for sodium motive force (∆S)-driven systems:
∆s = ∆Ψ + (2.3RT/F)∆pNa
As many primary sequences of secondary transport proteins have become available, it is clear that a
single family of homologous transporters can be composed of uniporters, symporters and antiporters. It
is thus impossible to infer from the primary sequences information regarding the coupling mechanisms of
the transporters. On the basis of structure predictions and various types of topological studies, the
secondary structure and folding of many of the proteins is thought to be similar, even among members of
different families. Although tertiary structures are not yet available, it is plausible that most of the
secondary transporters are composed of 10-14 hydrophobic segments in an α-helical configuration, which
each span the membrane (Marger and Saier, 1993).
SECONDARY LACTOSE TRANSPORT IN BACTERIA: LACY IN RETROSPECT
Already in 1956, Monod and co-workers discovered through genetic approaches the first sugar
transport system, i.e., the lactose carrier protein of Escherichia coli (the M protein, later called LacY)
(Rickenberg et al., 1956). Years later, Mitchell proposed that the lactose transport protein of E. coli was
a secondary transporter, which catalyzes lactose-H+ symport (Mitchell, 1963). It took, however, until the
1970's to show that lactose transport was really driven by lactose/H+ symport with a 1:1 stoichiometry
(West, 1970; West and Mitchell, 1973).
When lactose transport was measured from the distribution across the cytoplasmic membrane of
radiolabeled galactosides, it was observed that LacY is inactivated by N-ethylmaleimide (NEM) in a
substrate-protectable manner (Fox and Kennedy, 1965). Later it was shown that NEM is reacting with a
residue, Cys148, near the substrate binding-site (review Wu and Kaback, 1996). Labeling with radioactive
NEM made it possible to identify LacY as a membrane protein with an apparent Mr of 30,000 (Jones and
Kennedy, 1969).
Mechanistic studies in right-side-out (RSO) membrane vesicles led to the first models for the different
transport reactions catalyzed by LacY. In these membrane vesicles, different modes of lactose transport
have been studied, like: ∆p-driven uptake, efflux down the concentration gradient (vesicles loaded with
lactose are diluted into medium without), exchange transport (radio-labeled lactose on the inside is
exchanged with unlabeled lactose in the outer medium), and counterflow transport (unlabeled lactose on
the inside is exchanged for radio-labeled lactose in the medium) (Kaczorowski and Kaback, 1979;
Kaczorowski et al., 1979).
At the end of the seventies the gene encoding LacY was cloned and sequenced, and the protein was
overproduced (Teather et al., 1978; Büchel et al., 1980). Shortly thereafter, Newman and Wilson
solubilized the permease in octyl-β-D-glycopyranoside (octylglucoside) and successfully reconstituted
lactose transport activity in liposomes (Newman and Wilson, 1980). Reconstitution of the purified LacY
protein into proteoliposomes showed that the uptake, efflux, counterflow and exchange of the galactosides
are indeed all catalyzed by the LacY protein (Viitanen et al., 1986).
-6-
Chapter 1
Many transport proteins have now been discovered in bacteria, archaea, fungi, plants, animals, and
mammals, but still we do have little understanding about the molecular mechanisms by which these
secondary transporters operate (Maiden et al., 1987; Henderson, 1990; Marger and Saier, 1993; Poolman
and Konings, 1993; Reizer et al., 1993; Poolman et al., 1996; Pao et al., 1998). Because of the difficulties
in crystallizing membrane proteins, and the consequent lack of structures at atomic resolution, many efforts
have been made to study the structure/function relationships of these proteins by other methods. Examples
are given for LacY, but similar studies have been carried out for other membrane transport proteins.
Based on circular dichroism studies and hydropathy analysis, a secondary structure model for LacY
has been proposed, which represents the polypeptide as 12 transmembrane hydrophobic segments in αhelical configuration connected by more hydrophilic loops, and short hydrophilic amino- and carboxyterminal tails at the inner surface of the membrane (Foster et al., 1983). Evidence confirming some of these
features has been obtained from: laser Raman, and Fourier transform infra-red spectroscopy (Vogel et al.,
1985; Patzlaff et al., 1998), limited proteolysis and chemical labeling studies (Page and Rosenbusch,
1988), and from lacY-phoA fusion analysis (Calamia and Manoil, 1990).
Site-directed mutagenesis of every residue in LacY revealed that as few as six residues (out of 417)
are irreplaceable with respect to active lactose transport (Glu126, helix IV; Arg144, helix V; Glu269, helix
VIII; Arg302, helix IX; His322, helix X; Glu325, helix X). Mutation of any one of these residues
eliminates proton-driven uptake of lactose. Mutations of only one, Glu325, eliminates all partial transport
functions involving protons. There is strong evidence from site-directed excimer fluorescence and other
spectroscopic studies, isolation of second-site suppressor mutants, and disulfide-engineering that helix VIII
is close to helix X, helix VII is close to helices X and XI, helix IX is close to helix X, and that helix X is
indeed in an α-helical conformation (Wu et al., 1995; Frillingos and Kaback, 1996). The relationships
between helices V, VII, and VIII has been studied further by site-directed spin labeling and thiol
crosslinking experiments, which have also shown that helix I is close to helices V and VII, that helix II is
close to helices VII and XI, and that helix VI is close to helices V and VIII (Wu et al., 1996; Wu and
Kaback, 1996b; Wu and Kaback, 1996c). These results have led to a model for the helix-packing in LacY
of Escherichia coli. Furthermore, structural and functional data on the wild-type and numerous mutants
were merged into a mechanistic model that, to date, is the most detailed for any secondary transporter, and
that describes how conformational changes in the protein are related to the coupling of galactosides and
proton fluxes (Figure 1; for details see: Kaback, 1997; Lolkema et al., 1998).
Central to the mechanism of transport are six of the twelve transmembrane α-helices (V, VII, VIII,
IX, X, and XI), which interact with each other through charged or polar residues positioned at the
interfaces of the helices. The sugar binding site is situated between helices V and VIII and the proton
binding site is a glutamate residue in helix X (Glu325), at the interface with helix IX. These two binding
sites communicate through a redistribution of the interactions between the helices, which result in changes
in the tertiary structure of LacY, for instance, by rotation of the helices relative to one another or by
changes in the tilt of the helices.
In the unbound state, C- in the kinetic scheme of Fig. 1, helix X interacts electrostatically with helix
IX through the Arg302/Glu325 pair, the latter being the proton binding site. Helix X also interacts with
helix VIII through the Glu269/His322 pair. Binding of the substrate (C-:S) breaks the latter interaction and
Glu269 on helix VIII interferes with the interaction between helices IX and X. As a consequence, the
proton binding site is moved to a more apolar environment, which results in a pKa shift and binding of the
proton. In the ternary complex (C-:S:H+), the interactions between helices IX and X are lost, and helix X
interacts with helix VII. In this configuration translocation can take place. After release of the substrate,
deprotonation of Glu325 leads to re-establishment of the Arg302/Glu325 interaction, which allows the
protein to return to the “ground state”.
General Introduction - 7 -
IX
-
+
X
IX
XI
+
-
+
X
+
- -
VIII
VII
XI
+
-
- -
VIII
-
+
VII
V
V
S
C-
C-:S
H+
H+
C-:H+
C-:S:H+
S
IX
X
XI
+
+
+
-
- -
VIII
VII
V
IX
X
+
XI
+
+
-
- -
VIII
VII
V
Figure 1. Proposed mechanism of H+ and sugar binding by LacY of Escherichia coli. The center
shows a kinetic scheme for the random binding of the proton (H+) and a sugar (S) to the transporter.
The four models show the top view of the helix packing in the transporter protein. The top two and
lower, right models are simplifications of the ones presented in Kaback, 1997. The lower, left model
is hypothesized in Lolkema et al., 1998. Large circles indicate the transmembrane α-helices. Small
circles marked ‘+’ represent arginine or lysine residues, circles marked ‘-’ are glutamate or
aspartate residues, and unmarked circles correspond to histidine residues. The proton binding site
is the glutamate residue (Glu-325) in helix X and the square is the sugar binding site. Occupied
binding sites are shown in black. The arrows in the helices indicate the relative orientations in the
four models.
The model has recently been refined and in the new model Glu325 is protonated before the substrate
is bound (Frillingos et al., 1998). Glu269, Arg302, and His322 form a triad and ligand binding induces a
conformation change that disrupts this triad. With saturating substrate concentrations at both surfaces of
the membrane, the protonated form of Glu325 is stabilized and the permease can oscillate between
outward- and inward-facing conformations, thereby catalyzing exchange and counterflow without net
proton translocation. The model implies that the binding site moves across a permeability barrier upon the
binding and release of the proton, and that the substrate does not hop from site to site. Although much
work needs to be done to substantiate the model, it is clear that LacY, but also other secondary transport
proteins, are functioning as active enzymes, undergoing major conformational changes, rather than
channels that open and close in response to a specific signal.
-8-
Chapter 1
OUTLINE OF THIS THESIS
The protein that forms the topic of this study is the lactose transport protein (LacS) of S. thermophilus.
LacS is a member of a new family of transport systems that transport Galactosides, Pentosides or
Hexuronides (GPH). On the basis of the kinetic properties of mutants with altered cation and/or sugar
specificity, and the primary and secondary structure analysis, a cation binding pocket is proposed for these
transporters (Chapter 2 and 3).
To study LacS in further detail, it was essential that (mutant) proteins could be purified and
reconstituted into liposomes. Therefor the LacS protein was amplified in E. coli and S. thermophilus. The
protein was solubilized from membranes of S. thermophilus by commonly used detergents, and purified
in large quantities in a two step process involving nickel chelate affinity and anion exchange
chromatography. The process of membrane reconstitution of LacS was systematically studied. Importantly,
proteoliposomes have been obtained in which LacS was reconstituted with an inside-out orientation. The
orientation is a critical factor for the activity of the protein measured in proteoliposomes as the kinetics
of translocation are very different for the opposite directions of transport. Consistent with the in vivo
lactose/galactose exchange catalyzed by the protein, the maximal rate of lactose counterflow in the
proteoliposomes was much higher than that of the proton-lactose symport reaction (Chapter 4).
The activity of reconstituted LacS was markedly higher with Triton X-100 than with other detergents
(e.g., dodecylmaltoside) used to mediate the reconstitution. The macromolecular structures formed by the
lipids and nonionic detergents, that are intermediates in the reconstitution process, have been studied by
cryo-transmission electron microscopy. Whereas in case of Triton X-100 the bilayer structure was
maintained even with relatively high detergent concentrations, liposomes titrated with DDM were already
disrupted at the onset of solubilization. The membrane sheets were converted into long threadlike micelles
at higher DDM to lipid ratios. On the basis of the observed differences in macromolecular structures
formed, a model is proposed for the insertion of LacS mediated by Triton X-100 and DDM, which explains
the observed differences in orientation of the protein in the proteoliposomes (Chapter 5).
The reconstitution with DDM caused a significant loss of lipid during the reconstitution. The lipid
to protein ratio is of critical importance for the final transport activity and this partly explains the lower
activity when DDM was used for the membrane reconstitution of LacS. The reconstitution was studied in
more detail to confirm that the effect of DDM is primarily on the process of membrane insertion and not
on the protein itself (Chapter 6).
Finally, an overview of the topic of membrane protein reconstitution is given. The importance of
interactions between lipid, detergent and protein on the functional reconstitution of membrane proteins are
described (Chapter 7).