Received: 27 May 2010, Revised: 28 July 2010, Accepted: 28 July 2010, Published online in Wiley Online Librar y: 2011
Unraveling lipid/protein interaction in model
lipid bilayers by Atomic Force Microscopy
y
Andrea Alessandrini
a,b
and Paolo Facci
a
*
The current view of the biological membrane is that in which lipids and proteins mutually interact to accomplish
membrane functions. The lateral heterogeneity of the lipid bilayer can induce partitioning of membrane-associated
proteins, favoring protein–protein interaction and influence signaling and trafficking. The Atomic Force Microscope
allows to study the localization of membrane-associated proteins with respect to the lipid organization at the single
molecule level and without the need for fluorescence staining. These features make AFM a technique of choice to
study lipid/protein interactions in model systems or native membranes. Here we will review the technical aspects
inherent to and the main results obtained by AFM in the study of protein partitioning in lipid domains concentrating
in particular on GPI-anchored proteins, lipidated proteins, and transmembrane proteins. Whenever possible, we will
also discuss the functional consequences of what has been imaged by Atomic Force Microscopy. Copyright ß 2011
John Wiley & Sons, Ltd.
Keywords: protein–lipid interaction in model membranes; AFM; SLBs; transmembrane proteins
INTRODUCTION
In the Singer and Nicholson model (Singer and Nicholson, 1972)
of the biological membrane the lipids were considered mainly as
a solvent for the membrane proteins allowing them to diffuse in
order to interact with each other and perform their functional
activity. It has become clear now that lateral heterogeneity is the
main aspect of the lipid bilayer structure and that the lateral
organization of the membrane affects many membrane functions
such as signal transduction, protein and lipid sorting, and
endocytosis (Sackmann, 1984; Engelman, 2005). The first
evidences for the presence of laterally organized domains in
the lipid bilayer came from the study of model systems. Later on,
it was hypothesized that also biological membranes were
characterized by lateral heterogeneity as suggested by the
presence of detergent resistant domains (DRM). This experimen-
tal observation led to the introduction of the term ‘raft ’ to
designate globally the presence of specialized domains of lipids
and proteins in the lateral organization of lipid bilayers (Simons
and Ikonen, 1997; Jacobson et al., 2007). The identification of
these raft domains has remained elusive in biological mem-
branes, at variance with model systems, leading to the conclusion
that these functional domains, if they exist, must be small (in the
nanometer range) and transient (characterized by a short
aggregation lifetime for the constituent molecules) (Lingwood
and Simons, 2010). Hence, the biological structures associated
with the definition of ‘rafts’ are highly dynamic structures in the
nanometer range. It has recently become possible, with the
introduction of super-resolution microscopy, to obtain strong
evidences of the existence of transient domains also in the
membrane of living cells (Eggeling et al., 2009). Moreover, it has
been demonstrated that many membrane proteins require the
presence of specific lipids for proper function (Lee, 2009). For
example, anionic lipids are necessary for the functionality of
certain ion channels, probably through a binding to positively
charged residues (Schmidt et al., 2006; Marius et al., 2008). The
up-to-date view of the biological membrane is that in which lipids
and proteins strongly influence each other by fine mechanisms
involving chemical and physical interactions (Jensen and
Mouritsen, 2004; Lee, 2005).
In a reductionist approach it is possible to exploit model
systems of the complex biological membranes to retrieve
information about the physical rules, which regulate the phase
behavior of the membranes and the interplay between protein
and lipids. Three model systems are mainly used to accomplish
this task: Giant Unilamellar Vesicles (GUV) in which membrane
proteins have been reconstituted; Black Lipid Membranes (BLM),
which are useful for the studies of functional properties of
transport proteins; supported lipid bilayers (SLB) with recon-
stituted membrane proteins. It is clear that an approach based on
the exploitation of various model systems and different
investigation techniques offers a better understanding of the
complex lipid/protein interactions which might be relevant to
accomplish membrane functions. The SLB model system offers
the great advantage of being suitable to be studied with many
surface sensitive techniques. Among these techniques, of
particular relevance is the Atomic Force Microscopy (AFM)
(Mingeot-Leclercq et al., 2008), which provides high lateral and
vertical space resolution allowing the study of the presence of
different domains in the bilayer and the distribution of
(wileyonlinelibrary.com) DOI:10.1002/jmr.1083
Review
* Correspondence to: P. Facci, CNR-Istituto Nanoscienze, Via Campi 213/A, 41125
Modena, Italy.
E-mail: paolo.facci@unimore.it
a A. Alessandrini, P. Facci
CNR-Istituto Nanoscienze, Via Campi 213/A, 41125 Modena, Italy
b A. Alessandrini
Department of Physics, University of Modena and Reggio Emilia, Via Campi
213/A, 41125 Modena, Italy
y
This article is published in Journal of Molecular Recognition as a focus on AFM
on Life Sciences and Medicine, edited by Jean-Luc Pellequer and Pierre Parot
(CEA Marcoule, Life Science Division, Bagnols sur Ce
`
ze, France).
J. Mol. Recognit. 2011; 24: 387–396 Copyright ß 2011 John Wiley & Sons, Ltd.
387
membrane proteins relative to the domains. This capability is
connected to the fact that the coexistence of different lipid
phases can be detected by AFM due to the height difference
among the phases, which is related to the degree of chain order.
In this work we will review what the AFM technique can offer in
the study of lipid–protein interactions. The research field of
lipid–protein interactions includes the relations between trans-
membrane proteins and lipids, the interactions of a lipid bilayer
with surface bound proteins, lipoproteins, and peptides. In this
work we will mainly concentrate on lipoproteins and transmem-
brane proteins highlighting the information retrieved by AFM on
these systems in comparison with other techniques on different
model systems. The focus will be on the distribution of
membrane proteins relative to phase separation of the bilayer.
AFM has also provided a number of information on the structure
of the surface-exposed portion of transmembrane proteins and
on their oligomeric structure. Impressive results have been
obtained in this field establishing the AFM technique as the only
technique which can obtain subnanometer lateral resolution on
membrane proteins in physiologic-like environment. Moreover,
the possibility of observing the proteins at work in the real space
is another extremely useful advantage of AFM (Engel and Mu¨ ller,
2000). Several reviews about this topic can be found in the
literature and the interested reader is referred to these papers
(Mu¨ ller et al., 2002; Engel and Gaub, 2008). Moreover, AFM applied
in the force spectroscopy mode can provide a wealth of
information on protein/lipid interactions (Mu¨ ller, 2008). Exper-
iments in which single membrane proteins are extracted from the
lipid bilayer while measuring the opposing forces by AFM will not
be reviewed here. The interested readers can find excellent
reviews on this topic in the literature (Mu¨ ller and Engel, 2007).
This review is organized as follows. In Chapter 1, the model
system exploited in AFM studies will be critically described along
with techniques to prepare SLBs with reconstituted membrane
proteins on different surfaces. Chapter 2 will present results from
the literature on the study of membrane protein–lipid inter-
actions. In cases where information is available we will also relate
the data obtained from AFM studies to the functional behavior of
membrane proteins. In Chapter 3, we will describe the technical
approaches in which AFM is coupled to other biophysical
techniques, which can provide analytical information about
lipid–protein interactions. At the end, a summar y sec tion will
critically analyze the obtained results and will foresee the possible
future developments that AFM will enable in the field of
lipid–protein interactions.
CHAPTER 1: SUPPORTED LIPID BILAYERS
WITH RECONSTITUTED MEMBRANE
PROTEINS
Supported lipid bilayers consist of a lipid bilayer on a rigid
substrate such as glass, silicon oxide or mica. They were initially
developed by the McConnell’s group to study the interaction of
cells with lipid bilayers (McConnell et al., 1986; Castellana and
Cremer, 2006). They can be assembled by two different strategies:
the Langmuir Blodgett/Schaefer approach (Dufre
ˆ
ne et al., 1997)
and the vesicle fusion technique (Brian and McConnell, 1984). The
first technique is based on two consecutive transfers to a solid
substrate of lipid monolayers assembled at the liquid/air interface
in a Langmuir trough. The appealing feature of this approach is
connected with the possibility of forming lipid bilayers
characterized by a transbilayer lipid asymmetry, reproducing
the actual situation of biological membranes. The vesicle fusion
technique forms supported lipid bilayers from unilamellar
vesicles in solution. Upon contact with surfaces, under specific
conditions, unilamellar vesicles rupture, forming a planar bilayer.
In both preparation strategies, the presence of a thin water layer
between the bilayer leaflet nearer to the substrate (proximal
leaflet) and the substrate itself allows to preserve lipid diffusion
(Koenig et al., 1996). It is, however, to be stressed that the small
thickness of the water layer might somehow limit the lipid
diffusion with respect to the case in which a lipid leaflet is facing
bulk water (see discussion below). The thin water layer allows also
to host transmembrane proteins even in the case in which a small
portion protrudes from the bilayer towards the support side. The
vesicle fusion technique allows the incorporation of transmem-
brane proteins in the supported lipid bilayer more easily than the
Langmuir–Blodgett/Schaefer one. Even if strategies for the
incorporation of detergent solubilized transmembrane proteins
in already formed supported lipid bilayers have been developed
(Milhiet et al., 2006; Muller, 2006), the direct fusion of proteo-
liposomes on surfaces appears as a more practical approach.
Moreover, the drawback of the technique, which exploits the
addition of detergent to slightly destabilize the lipid bilayer in
order to favor the insertion of solubilized membrane proteins, lies
in the unknown amount of detergent which remains in the lipid
bilayer. The residual detergent could affect both the thermo-
dynamics of the lipid bilayer and the functionality of the proteins.
Vesicle fusion is usually performed starting from Small
Unilamellar Vesicles (SUV) or Small Unilamellar Proteoliposomes,
which means vesicles with a diameter of a few tens of
nanometers. According to the most probable pathway for
supported lipid bilayers formation from vesicles, the external face
of the liposomes will face the solid support in the final bilayer
configuration, while the internal layer will face the bulk of the
solution. However, contradictory results have been reported
about the formation of SLBs from vesicles containing transmem-
brane proteins (Contino et al., 1994; Salafsky et al., 1996). In some
cases it has been found that proteins which exposed the active
site to the bulk solution in vesicles also exposed the active site to
the bulk solution in the SLB. In other cases, it has been found that
a significant redistribution of protein orientation occurs during
the SLB formation. Probably, the scenario at work depends on the
specific case (Reimhult et al., 2009). Among the parameters to
consider there are the size of the vesicles, their lipid composition,
deposition temperature, and the nature of the support. The
orientation of the proteins in the lipid bilayer can be connected to
the orientation in the proteoliposomes, but the final structure on
the surface depends on the rupture pathway of the vesicles and
on possible reorientations of the proteins. The accurate
orientation of the proteins in the SLB can be established by
functional tests or by measuring the distribution of the height of
the inclusions (Liu et al., 2009).
When supported lipid bilayers with reconstituted membrane
proteins are studied by AFM, a homogeneous lipid to protein
ratio in the vesicles is highly desired. This situation would allow
more reproducible results and a uniform distribution of the
proteins in the supported lipid bilayer in the case of a
homogenous lipid phase (Vuong et al., 2010). In fac t, the
possibility that vesicles with different lipid to protein ratio have a
different affinity for the solid substrate could make the obtained
supported lipid bilayer with reconstituted membrane proteins
largely independent from the real vesicle composition in the
wileyonlinelibrary.com/journal/jmr Copyright ß 2011 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 387–396
A. ALESSANDRINI AND P. FACCI
388
solution. Even if the control over the lipid to protein ratio in the
vesicles is difficult, it is important to take into consideration this
parameter when interpreting the obtained AFM images. It is
usually found that the density of proteins in the supported lipid
bilayers is lower than the nominal concentration used to prepare
the proteoliposome sample (Seeger et al., 2009a). This behavior
could be the result of the presence of lipid vesicles without
proteins in solution, especially in the case of detergent removal
by dialysis. Removal of detergent by other techniques, such as the
use of Bio-Beads (Rigaud et al., 1997), offers the possibility of great
improvements in the results both for the reconstitution of
membrane proteins into vesicles and for the stripping of
detergent from destabilized supported lipid bilayers.
The main drawback in the SLB model system stems from the
small water gap between the lipid bilayer and the solid support
(0.5–2 nm) (Bayerl and Bloom, 1990; Johnson et al., 1991; Koenig
et al., 1996). This aspect could be a problem if membrane proteins
with a large extramembraneous portion have to be incorporated
in the bilayers. A contact between the proteins and the support
could affect the protein conformation and, consequently, the
function of the molecules. However, there are cases in which the
reconstitution of membrane proteins in-plane on a solid
substrate does not affect the protein functionality (Ataka et al.,
2004). The interaction is strictly dependent on the nature of the
substrate. On hydrophilic substrates such as mica, silicon oxide,
and glass, the interactions which are established between the
membrane and the substrate are sufficiently weak to prevent
alterations of normal molecular behavior.
Lateral diffusion is an aspect to be considered when
lipid–protein interactions are studied by exploiting supported
lipid bilayers. Diffusion firstly regards lipids and then also proteins
in the SLB. Recent experimental results have shown that the lipid
diffusion in free standing bilayers (GUV, Giant Unilamellar
Vesicles) is more than two times faster than in supported lipid
bilayers measured in the same conditions (the diffusion
coefficient is D ¼ 7.8 mm
2
s
1
for GUVs and D ¼ 3.1 mm
2
s
1
for
SLBs) (Przybylo et al., 2006). A different and more complicated
issue is the possible difference in diffusion coefficients between
the two leaflets of the bilayer. Asymmetric dynamic properties
involve the strength of the interleaflet coupling. In literature,
different results can be found. Hetzer et al. (1998) found that the
distal leaflet has a diffusion constant which is two times faster
than the proximal leaflet, pointing to an independent behavior of
the two leaflets. Recent results found the same translational
diffusion coefficient for both leaflets within a 10% experimental
uncertainty (Zhang and Granick, 2005). In the latter case a strong
coupling between the two leaflets could be the reason for the
same lateral mobility. We recently demonstrated that the
interleaflet coupling is strongly related to the experimental
details of the sample preparation, including preparation
temperature and the type of support (Seeger et al., 2009b;
Seeger et al., 2010). So, it is not always possible to compare the
obtained results even if related to the same system.
Many membrane proteins perform their tasks by forming
dynamic assemblies with other proteins in the membrane. The
single molecule level study of the molecular interactions would
increase our knowledge of biological processes. Dealing with the
diffusion of membrane proteins in supported lipid bilayers, it is
usually found that proteins are able to diffuse, but the diffusion
coefficient is orders of magnitude lower than expected from
proteins embedded in free standing bilayers (Mu¨ ller et al., 2003).
The reason for this behavior could be found in the thickness of
the water layer between the membrane and the support which
could increase frictional forces for membrane protein diffusion.
Another explanation for the low diffusion coefficient is the
presence of pinning points of the bilayer to the substrate which
could produce an obstructed diffusion. Different types of motion,
free diffusion, and obstructed diffusion, have indeed been
observed by AFM on SLBs (Mu¨ ller et al., 2003). It has been noted
that the diffusion of membrane proteins is related to the fluidity
of the lipid bilayer. Indeed, by increasing the temperature of the
sample it is possible to observe an increase of the displacements
of the proteins in the membrane, eventually reaching a situation
in which the proteins are no more visualized by AFM (personal
observations). A major limitation in the observation of protein
diffusion by AFM is the low time resolution of the technique. This
limit allows only the observation of slow dynamics. In particular,
depending on the area imaged and the time interval between
two consecutive images, limits in the determination of diffusion
coefficients are encountered (Hughes et al., 2004; Casuso et al.,
2009). For example, it has been hypothesized that the prevalent
observation of membrane proteins in association with the more
ordered regions of the lipid bilayer does not mean that the same
proteins are not interacting with the more liquid phases,
especially in the case of small proteins or peptides (Chiantia
et al., 2006c). In fact, highly mobile components in the lipid
bilayer might be not detected and a complementary technique
should be used to exclude the presence of small mobile proteins
in the fluid regions of the bilayer (see below). One of the most
exciting area of development for the AFM technique in biological
studies is the high-speed imaging which could allow the
acquisition of time-lapse images with a very short time interval
(see future trends) (Ando et al., 2007; Casuso et al., 2009). The
reduced protein diffusion constant does not however imply an
alteration of the functionality. For example, it has been
demonstrated in a recent fluorescence study on the cooperativity
of an ion-channel subunits performed on a solid SLB that, in spite
of an apparent absence of mobility for all the molecules, only a
small fraction showed no activity in response to a gating stimulus
(Blunck et al., 2008). A method which is worthwhile being
developed and exploited is that of assembling membranes on
supports in which holes have been produced (Gonc¸alves et al.,
2006). In this case it would be possible to image by AFM
membrane proteins separating two aqueous compartments (see
future trends), configuring thus a free standing membrane.
A further shortcoming of the use of supported lipid bilayers to
study lipid–protein interaction is connected to the planar
geometry of the model system. In many native membranes
the geometry is curved and the curvature is usually induced by
the presence of surface bound proteins or transmembrane
proteins. Two-dimensional projection of the native 3D structure
can be affected by artifacts in the apparent membrane
organization (Olsen et al., 2008). In particular, the role of the
membrane curvature in determining protein distribution or the
role of proteins in influencing the curvature may be under-
estimated.
CHAPTER 2: LIPID/PROTEIN INTERACTION
STUDIED BY AFM
The use of AFM on supported lipid bilayers containing proteins
has provided a wealth of information on the distribution of
membrane proteins relative to the lipid phase separation or on
J. Mol. Recognit. 2011; 24: 387–396 Copyright ß 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr
LIPID/PROTEIN INTERACTION IN MODEL LIPID BILAYERS
389
the effect of peptides and proteins on the struc ture of lipid
bilayers. Since the introduction of the idea that domains could
appear in biological membranes, it became evident that
understanding the distribution of peptides and proteins in lipid
bilayers would have been relevant for elucidating signaling
pathways. Both theoretical (Gil et al., 1998) and experimental
(Brown, 1994) efforts concentrated on the clustering and
partitioning of proteins in lipid membranes. From an exper-
imental point of view AFM represents one of the most suitable
approaches due to its high lateral and vertical spatial resolution.
The distribution of proteins and peptides along with the phase
behavior of the lipid bilayer can be studied by AFM in a
physiologic-like environment without the need of a labeling step.
One of the first studies in this context concentrated on the
distribution of peptides such as Gramicidin A, establishing the
possibility for those peptides to form clusters in the lipid bilayer
(Mou et al., 1996; Ivanova et al., 2003). In terms of membrane
protein/lipid interactions, the distribution of either GPI-anchored
proteins, lipidated proteins or transmembrane proteins in the
presence of phase separation in lipid bilayers is particularly
relevant for understanding trafficking processes of the mem-
brane and the possible influence of phase separation on
transmembrane protein function. This topic has been studied
by AFM (Milhiet et al., 2002; Seeger et al., 2009b). In the following,
we will concentrate on studies performed on these types of
membrane proteins. We forward the readers interested in AFM
studies on the interactions of small peptides, such as
antimicrobial peptides, fusogenic peptides and cell-penetrating
peptides with lipid bilayers to excellent reviews in the literature
(Brasseur et al., 2008; El Kirat et al., 2010). The reader can also
find interesting AFM works on the interaction of peripheral
membrane proteins such as cytochroms with supported lipid
bilayers (Choi and Dimitriadis, 2004; El Kirat and Morandat, 2009).
It should be stressed that the examples we will show do not
represent the general behavior of membrane proteins upon
phase separation in the bilayer and that the results obtained by
AFM should be as much as possible compared to the results
coming from other biophysical techniques. The last requirement
stems from the fact that the different model systems exploited
could give different results.
Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs)
are a class of proteins that are anchored to the membrane by
means of a posttranslational lipid chain insertion (Mayor and
Riezman, 2004). The lipid modification allows these proteins to be
somehow related to the membrane trafficking mechanisms and
to domain formation especially in the outer leaflet of the
biological membrane. Understanding the mechanisms under-
lying the sorting of GPI-APs in a lipid bilayer is important in the
context of the lateral heterogeneities in biological membranes
and its functional role. One of the GPI-anchored proteins which
has been studied by AFM is Placental Alkaline Phosphatase
(PLAP) (Saslowsky et al., 2002; Giocondi et al., 2008). The aim of
these studies was to establish whether PLAP was targeted to raft
domains in lipid bilayers. To that aim, proteoliposomes with
associated PLAP proteins were assembled and successively fused
on a mica support or were inserted into preformed SLBs. The
sample preparation technique assured the preservation of the
protein functionality in the supported planar lipid bilayer and the
proteins were mainly found associated with the most ordered
domains of the lipid bilayer composed by synthetic mixture of SM
and DOPC with and without cholesterol. The obtained results
constitute a paradigmatic example useful to understand the
relationship between different bilayer model systems. In fact,
experiments performed on a similar lipid system and the same
protein but on GUV, gave the strik ing result of the proteins mainly
concentrated in the liquid disordered regions instead of the
ordered ones (Kahya et al., 2005). The PLAP protein is considered
an example of Detergent Resistant Membrane (DRM) associated
protein. The finding that PLAP protein behaves differently in
DRMs and in GUV can be explained on the basis of the alteration
of the thermodynamics of the membrane when detergent is
inserted and temperature changed to identify DRM areas. The
behavior observed in AFM studies can also be affected by the
interaction of the proteins with the support. In conclusion, this
example highlights some of the problems connected with the
identification of lipid rafts in biological membranes. In the case of
GPI-anchored intestinal alkaline phosphatase (BIAP), the incorp-
oration of the protein in an already formed SLB, demonstrated
the preferential incorporation of the protein in the gel phase
domains of different lipid mixtures (Giocondi et al., 2007a, 2008)
(Figure 1). Also in this case, as in that of PLAP, the heterogeneity of
the lateral membrane organization is able to influence the
protein distribution. In the work on BIAP it was also possible to
observe a transfer of lipids from the fluid to the gel phase or vice
versa, upon protein insertion in the bilayer. This effect can be
deduced by the variation of the relative area of the fluid and gel
regions. The spontaneous insertion of these GPI-anchored
proteins into preformed lipid bilayers usually starts from the
interface between different phase domains. This phenomenon
can be related to the presence of both high line tension and
Figure 1. Sequence of AFM images showing the insertion of BIAP in DOPC/DPPC supported lipid bilayers. The insertion of the proteins was performed
after the lipid bilayer was formed on the substrate. (a) Lipid bilayer showing the coexistence of fluid and gel domains before the addition of BIAP. (b) AFM
image of the same area as in (a) 45’ after the addition of BIAP. Arrows point to the BIAP proteins which insert preferentially at the boundary between fluid
and gel domains. (c) After longer incubation time the proteins inserted also in the center of the gel domains (arrows). Scale bar ¼ 1 mm. Reproduced with
permission from (Giocondi et al., 2008).
wileyonlinelibrary.com/journal/jmr Copyright ß 2011 John Wiley & Sons, Ltd. J. Mol. Recognit. 2011; 24: 387–396
A. ALESSANDRINI AND P. FACCI
390
packing defects in this area. Considering the insertion and the
redistribution of the lipids it is probable that GPI-anchored
proteins, once in the lipid bilayer, recruit the most suitable lipid
environment most likely by exploiting the hydrophobic matching
interaction. The AFM can also be exploited to observe the
redistribution of GPI-anchored proteins after a temperature
variation (Giocondi et al., 2007b). The obtained information can
be fruitfully compared with results obtained from DRM extraction
at low temperature.
In the case of other lipidated proteins, N-Ras proteins, it has
been demonstrated by a combined fluorescence study on GUV
and AFM study on supported lipid bilayers that the proteins
behave in the same way in both model systems (Weise et al.,
2010). In particular, lipidated N-Ras proteins have a strong
preference for liquid disordered domains in the lipid bilayer
(Nicolini et al., 2006). In a time-lapse AFM study on lipidated N-Ras
proteins it has been demonstrated that the proteins preferentially
partition in the liquid disordered phase and then migrate towards
the domain boundaries where they decrease the domain line
tension. (Weise et al., 2009). Moreover, N-Ras proteins form
clusters which modify also the lipid distribution by creating lipid
domains which did not exist before the insertion of the proteins.
The Atomic Force Microscope can also be exploited to study
the partitioning of integral transmembrane proteins with respect
to the lateral phase heterogeneity of the lipid bilayer. When
integral membrane proteins are incorporated into lipid mem-
branes and domain formation occurs, two effects result. The
domain formation process appears to be modulated by the
presence of the proteins according to the lipid protein interaction
(Sperotto et al., 1989) and the domain structure influences the
function of the proteins by introducing tensions which can act on
the conformational changes of the proteins (Brown, 1994). The
hydrophobic matching principle is usually evoked to explain the
protein/lipid interactions and the distribution of the proteins in
the bilayer relative to different phases which might exist. At the
same time, if lipids of different hydrophobic thickness are present
in the lipid bilayer, the protein can perform a lipid sorting at its
interface on the basis of physical reasons, even in the absence of
any chemical specificity. The hydrophobic thickness of the lipids
strongly depends on the temperature of the system, especially
near a phase transition region. Thus, it is reasonable to expect
that the organization and the protein/lipid interactions change
with temperature. A further partitioning effect comes from the
existence of domain boundaries. It has been calculated that, for
particular lipid compositions, when the system is in the phase
transition region with a coexistence of gel and liquid domains,
the proteins tend to be adsorbed at the phase boundaries with a
region of fluid lipids around them (Dumas et al., 1997). This effect
represent a sort of interfacial adsorption which is generally
expected in a many-phase system with impurities that have no
particular preference for any given phase and are therefore
confined to the interface.
Seeger et al. (2009a) have studied the partitioning of the K
þ
channel KcsA (Doyle et al., 1998) in a POPE/POPG supported lipid
bilayer when a phase transition of the lipid bilayer was induced. In
that work the sample was prepared by the fusion of proteolipo-
somes on a mica surface and imaged by temperature controlled
AFM. The obtained sample most likely reflects the vectorial
incorporation of the KcsA molecules in the POPE/POPG lipo-
somes, where the channels are incorporated almost exclusively in
the outside-out configuration. This type of incorporation has
been confirmed by a proteolytic assay (Cuello et al., 1998).
The height distribution of the observed bumps points to a
monomodal distribution. Considering the strong difference
between the two extramembraneous portions of KcsA it is likely
that the vectorial reconstitution is preserved also in the
supported lipid bilayer. Figure 2 reports a sequence of AFM
images of the lipid bilayer with the reconstituted proteins
Figure 2. Series of AFM images (10 mm 10 mm) of the redistribution behavior of KcsA reconstituted in a SLB of POPE:POPG 3:1. (A) The SLB was
equilibrated in the liquid disordered phase. The KcsA proteins are randomly distributed in the SLB. The black line is the outline of the solid ordered
domain which formed in B. (B) A solid ordered domain (lighter area) was induced by cooling. The KcsA proteins were excluded from the solid ordered
region. (C–D) The solid ordered domain was allowed to equilibrate. (E–F) The phase transition and the protein redistribution proceeded upon further
temperature decrease.
J. Mol. Recognit. 2011; 24: 387–396 Copyright ß 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jmr
LIPID/PROTEIN INTERACTION IN MODEL LIPID BILAYERS
391
