biomimetic approaches for studying membrane …razj/mol-bio-sys.pdfbiological processes occurring on...

Biomimetic approaches for studying membrane processes

Raz Jelinek* and Liron Silbert

Received 8th April 2009, Accepted 13th May 2009

First published as an Advance Article on the web 25th June 2009

DOI: 10.1039/b907223n

This short review focuses on recent innovative systems and experimental approaches designed to investigate

membrane processes and biomolecular interactions associated with membranes. Our emphasis is on

‘‘biomimetics’’ which reflects the significance and contributions of the chemistry/biology interface in

addressing complex biological questions. We have not limited this review to discussion of new ‘‘sensors’’ or

‘‘assays’’ per se, but rather we tried to review new concepts employed for analysis of membrane processes.

1. Introduction

The plasma membrane constitutes a critical platform for diverse

biological processes such as ligand recognition,1 drug action,

vesicle fusion2 and endocytosis3 pore-formation by membrane-

active peptides,4 and others. Numerous studies have been

conducted to decipher structural, functional, and mechanistic

aspects pertaining to membrane processes. Due to the complexity

of physiological membranes, a wide variety of model membrane

systems has been developed over the years to provide insight into

biological processes occurring on membrane surfaces or within

membrane lipid bilayers. Several reviews on varied aspects of

model membrane assemblies and their applications for studying

membrane processes were recently published.5,6

2. Model membranes and reconstituted membrane

assemblies

Model membrane assemblies have been used in biochemical

studies for several decades already. Small and large

multilamellar and unilamellar vesicles have been extensively

used in membrane studies,7 as well as Langmuir phospholipid

monolayers.8 Giant vesicles (GVs) have attracted interest due

to their larger size, which is close to cell dimensions.9 Indeed,

the lower curvature of GVs as compared to conventional small

or large vesicles has been proposed as an important parameter

that distinguishes membrane events occurring on GV

surface.10 Beside conventional application of GVs as

biomimetic membranes, these assemblies have been also used

in innovative biosensing approaches, such as templates for

conducting polymer biosensor assemblies.11 These researchers

used GVs as templates for assembly of polypyrrole, further

forming elongated tubules and wires of the polymer.

Artificial membrane assemblies have been essential tools for

pharmaceutical screening and development. The parallel

artificial membrane permeability assay (PAMPA), tradition-

ally prepared by impregnating a porous filter with

lipid mixtures, has been a well-known ‘‘work-horse’’ in

pharmaceutical research and development.12 This assay is

generally used for predicting drug permeability through

membranes, but often its simplicity and generic nature some-

what mask shortcomings in term of stability, predictability,Department of Chemistry, Ben Gurion University of the Negev,Beer Sheva, 84105, Israel. E-mail: [email protected]

Raz Jelinek

Currently at the department ofchemistry at Ben GurionUniversity, Israel, Raz Jelinekobtained his BSc in chemistryfrom the Hebrew University ofJerusalem, Israel, and hisPhD from the University ofCalifornia, Berkeley. He wasa Cancer Research Institutepost-doctoral fellow at theUniversity of Pennsylvania,and is currently a VisitingProfessor at the Departmentof Chemical and BiomolecularEngineering at Johns HopkinsUniversity. Professor Jelinek

has over 80 scientific publications and 10 patents to his name,and has received several scientific awards, including theRoger–Siegel–Brown Award of the Israeli Academy of Scienceand the Ruth L. Kirschstein National Research Service Award ofthe National Institutes of Health, USA.

Liron Silbert

Liron Silbert was born in BeerSheva (Israel) in 1978. Shereceived her MSc in chemistryfrom Ben Gurion University ofthe Negev (Israel) in 2006.She currently pursues herPhD studies in biophysicalchemistry in the laboratory ofProf. Raz Jelinek at the BenGurion University. Her re-search focuses on the use ofbiomimetic chromatic vesiclesas membrane biosensors.

This journal is �c The Royal Society of Chemistry 2009 Mol. BioSyst., 2009, 5, 811–818 | 811

REVIEW www.rsc.org/molecularbiosystems | Molecular BioSystems

and overall performance. Varied modifications of the basic

artificial membrane design have been reported, aiming to

improve the assay properties and its preparation.12

Chen et al., for example, constructed a lipid/oil/lipid tri-layer

assembly in a porous filter setting, which displayed better

predictability for highly permeable compounds and good

correlation to cell assays. The trilayer system also proved

compatible with different solvent systems and exhibited high

stability in storage, both contributing to the practicality of the

new assay.

Solid-supported membranes have been widely used, mostly

for studying transport properties of ions by membrane-

embedded ion channels and transporters.13 This family of

techniques is based upon reconstitution of natural membranes

or lipid bilayer assemblies on the solid substrate that generally

constitutes a part of an electrical circuit. Generation of

solid-supported lipid bilayers have particularly contributed

to applications of sophisticated bioanalytical techniques, such

as surface plasmon resonance (SPR) and quartz crystal

microbalance (QCM). Utilization of solid-supported bio-

membranes has faced considerable technical challenges, primary

due to maintaining the structural and functional properties of

the membrane bilayers upon the solid surface. Merz et al.

describe several systems in which negatively-charged lipid

films mimicking bacterial membranes were successfully

immobilized on common biosensing solid supports through

careful optimization of Ca2+ concentrations needed to

promote vesicle fusion with the solid surfaces.14 That work

exemplifies the technical advances that are essential for

introduction of solid-supported membrane biosensors employ-

ing more complex lipid assemblies. Kasuya et al. demonstrates

a technique for immobilization of intact vesicles on a QCM

chip surface through anchoring to short amphiphilic

peptides.15 The method did not adversely affect the integrity

of the vesicles and the structural and functional properties of

lipid receptor components within the bilayers were retained,

allowing highly sensitive detection of glycolipid/protein

binding at the membrane surface.

Variations of solid-supported membrane construction

approaches have been described. Tethered membranes

deposited on solid substrates have been useful functional

platforms for electrochemical analysis of ion transport by

bilayer-embedded peptides and proteins and studying redox

processes in membranes.16 Tethered membranes have been

employed not only in electrochemistry-type experiments, but

also as a vehicle for studying other membrane processes, such

as cell adhesion.

A key challenge for potential utilization of tethered

membrane systems has been to retain the functionality of the

membrane bilayer through immobilization onto the solid

surface. Purrucker et al. have demonstrated the construction

of tethered membranes using lipopolymers that were chemi-

cally coupled to solid substrates.17 The use of lipopolymers—

essentially synthetic entities—facilitated fine control of the

stability and fluidity of the produced lipid bilayers. In a later

study it has been shown that the use of lipopolymer tethers

allowed incorporation of integrin, a large transmembrane

protein receptor.18 The researchers examined their tethered

membrane models using giant vesicles displaying the cell

recognition RGD peptide determinant; adhesion of the

vesicles was found to be more than an order-of-magnitude

stronger than integrin incorporated within lipid vesicles

conventionally immobilized on solid substrates.

Recent advances in materials sciences and nanotechnology

have expanded the ‘‘substrate tool-box’’ employed for

immobilization of membrane assemblies and investigation of

membrane properties and processes. In particular, newly-

developed micro- and nano-porous materials were shown to

constitute highly effective substrates for such purposes.19 In

their review, Reimhult et al. emphasized the significant

contribution of manufactures ordered arrays of small surface

apertures to electrochemistry-based techniques and micro-

fluidic cells designed to serve as membrane biosensors and

devices for rapid analysis of the functions and modulations of

membrane-embedded receptors and other biomolecules.19

Indeed, progress in nanolithography and microfabrication is

expected to continue to play an important role in the

development of new sensing approaches for membrane

processes and membrane-associated molecules.

Microarray technology is a particularly promising albeit

challenging frontier for screening membrane interactions, due

in large part to the difficulty for maintaining the functionality

of model membranes in microarray settings. Yamazaki et al.

have recently demonstrated a technology for generating cell

membrane microarrays that was successfully used for high

throughput functional screening.20 The researchers succeeded

to fabricate a microarray of membranes that were physio-

logically fluid, a critical property which is essential for

achieving real functional screening of membrane-embedded

molecules. The researchers have demonstrated the use of their

microarray technology for analysis of ligand–receptor inter-

actions and drug effects involving membrane-associated

receptors such as gangliosides and lipopolysaccharides. Such

microarray technologies are expected to play an increasingly

important role in industrial research-and-development focused

on membranes and membrane processes.

3. In vivo systems

Varied techniques have been developed for studying molecular

recognition on membrane surfaces in vivo. The majority of

such approaches rely upon actual cellular systems biologically

manipulated to enhance signals arising from the desired

membrane process. Schwarzenbacher et al. recently described

a method in which micropatterned cells were functionalized

with antibodies designed to dock membrane protein ‘‘baits’’

onto the membrane surface.21 The displayed membrane

protein receptors were consequently employed for capturing

fluorescently-labeled soluble protein ligands, and the extent of

affinity between the proteins was evaluated through

fluorophore distribution in the cell membrane.

Manipulating cellular membrane with foreign antibodies

has been exploited in other applications. Moschopoulou

et al. presented an interesting biosensing approach in which

virus-specific antibodies were loaded into the membranes of

fibroblast cells.22 These engineered cells were capable of

capturing and detection of specific viral strains through

changes in membrane potential induced through viral binding.

812 | Mol. BioSyst., 2009, 5, 811–818 This journal is �c The Royal Society of Chemistry 2009

The authors have shown that highly specific detection of viral

homologues was feasible electrochemically. This elegant work

exemplifies an approach in which a well-known membrane

event (change in hyperpolarization) can be induced through

incorporation of foreign recognition elements into the cell

membrane (antibodies) and used for high specificity biosensing

(viral detection).

The use of fluorescent probes has greatly aided our under-

standing of processes occurring within membranes of living

cells. Fluorescent membrane-associated dyes have been

particularly useful for studying ligand–receptor interactions

and molecular recognition at the cell membrane. Varied

synthetic dyes have been designed to respond to changes in

the viscosity of the membrane, allowing analysis of the

numerous membrane-active substances which affect the

fluidity of the lipid bilayer. Beside well known fluidity-sensitive

fluorescent dyes such as diphenylhexatriene (DPH) and its

derivatives,23 new probes are being synthesized. Yasuhara

et al. reported the synthesis of a dye that is capable of

distinguishing between different phases of lipid bilayers.24

Potential-sensitive dyes have been employed for screening

agonists and antagonists of the nicotinic receptor channel.25

Such fluorescent probes have been similarly useful for studying

peptide–membrane interactions, since peptide insertion

into the membrane (and specifically peptide-induced pore-

formation) generally results in significant modulation of the

membrane potential.26

4. New biomimetic systems for studying membrane

processes

A successful biomimetic platform for studying membrane

processes has to adhere to two primary conditions: the

molecular system produced should retain, as much as possible,

the physico-chemical properties of the actual cellular membrane

(such as lipid and protein organization and fluidity), and the

availability of measurement schemes that will reliably report

upon the molecular processes occurring within the biomimetic

membrane. Varied elegant approaches have been designed to

address these two often formidable challenges.

Placement of lipid bilayers on solid supports has been a

popular approach for mimicking membrane environments.27

Recent approaches have expanded this concept to new organic

and inorganic entities. Baksh et al. coated conventional silica

beads with a lipid membrane having controlled lipid

contents.28 These authors discovered that colloidal phase

transitions of the membrane-coated silica beads provided a

simple and label-free method for analysis of membrane

interactions. Specifically, the detection method relied on

evaluation of the pair interaction potential between adjacent

coated beads; through careful modulation of the lipid

composition the researchers could place the system close to

the phase transition so that small perturbations to the lipid

bilayers (induced for example through docking of

biomolecules on the membrane surface) resulted in

pronounced changes in the macroscopic organization of the

colloids.

Other intriguing systems in which membranes have been

coupled to advanced materials have been described.

Bauer et al. presented an elegant method for construction of

vesicle/nanotube networks.29 The technique makes possible

reconstruction of membrane environments on a dense network

of carbon nanotubes. The resultant biomimetic assembly

could allow investigation of membrane proteins, molecular

recognition, and transport across lipid bilayers. The

noteworthy feature of the system is the conceptual similarity

of the nanotube framework to the cytoskeleton network

connected to the lipid membranes in a cell.

Varied artificial membranes and model systems have been

developed to study membrane events. The biochemical

questions and systems studied are often critically dependent

upon the molecular composition and organization of the

membranes (both in its physiological state as well as in model

assemblies). Accordingly, the success of new experimental

setups and technical concepts depend on large part upon

satisfying the above requirements.

Spatial organization of molecules that constitute part of the

membrane is a critical factor affecting diverse biological

processes. Lipid rafts, for example, are believed to be a

primary determinant for docking of membrane-associated

molecules. In particular, different biochemical experiments

and specifically designed assays have shown that these

sphingomyelin and cholesterol-rich domains within the cell

membranes closely affect binding and insertion of amylo-

idogenic peptides and proteins into membrane bilayers.6,30–32

Several investigations have suggested that these specialized

membrane microdomains promote assembly of signaling

molecules, modulate membrane fluidity and affect trafficking

of membrane proteins.33 Thus disruption of rafts by inter-

acting peptides and proteins would have the significant bio-

logical consequences associated with different diseases.

Biomimetic vesicles comprising polydiacetylene (PDA) as a

vehicle for biological sensing have been used as useful

platforms for analysis and rapid screening of membrane

processes and biomolecular recognition events. Conjugated

polydiacetylene (PDA) is a remarkable polymeric system

which exhibits unique organization and chromatic

properties.34–36 PDA is formed through 1,4-addition of

aligned diacetylenic monomers, initiated by ultraviolet (UV)

irradiation (Fig. 1).37 The resulting polymer is intensely blue to

the eye, due to electronic delocalization within the conjugated

framework, giving rise to an absorption at around 650 nm in

the visible region of the electromagnetic spectrum.

Importantly, PDA can undergo both rapid blue–red

colourimetric transitions38–41 and in parallel fluorescence

transformations,42 induced by external stimuli which perturb

the conjugated framework of the polymer.37,40,43,44

Diverse avenues have been reported for utilization of

PDA-based vesicle systems for biological and chemical

applications. Several laboratories have explored immobilization

and patterning of derivatized PDA on solid substrates and

utilization of such assemblies for high throughput screening of

analytes.45,46 These studies take advantage in particular of the

intense fluorescence of PDA after it undergoes structural

transformations induced by the analytes to be detected. These

researchers have also coupled PDA vesicle immobilization to

advanced nanolithography techniques, creating biosensor

chips for proteins, bacteria and viruses. Several reports


depicted additional schemes for immobilization of PDA-

based vesicles, while retaining the chromatic properties of

the polymer.47 These authors have incorporated the vesicles

within polyelectrolyte multilayers, particularly important for

construction of possible biosensing devices.

Mixed lipid/PDA vesicles comprising the polymer as well as

phospholipids and/or other constituents of the cell membrane

have been shown to mimic cellular environments. The advan-

tages of such mixed assemblies stem from the observation that

the PDA framework can act both as a ‘‘scaffolding’’ for

stabilization of lipid bilayers or entire membrane domains,

while it also serves as a transduction vehicle for generation of

color/fluorescence signals that are induced through membrane-

associated events.

A particularly important feature of lipid/PDA vesicle

systems is the feasibility for incorporating a significant

concentration of lipid constituents within the PDA matrix—

up to 50% (mole ratio). This architecture is designed to

better mimic the cell surface and is radically different from

PDA systems containing smaller quantities of lipid ‘‘dopants’’,

both in structure as well as functionality. Essentially,

such mixed vesicles comprise distinct lipid domains embedded

within the polymer framework that still retains its structural

and chromatic properties39,48–50 Fig. 2 depicts a schematic

description of biomolecular sensing with lipid/PDA

vesicles. Previous studies indicated that the lipids and

polydiacteylene most likely form interspersed microscopic

phases within the vesicles.49 The phospholipids incorporated

within the PDA matrix adopt a bilayer structure, the

dominant lipid organization within cellular membranes.

Published data further point to the contribution of changes

in fluidity within the lipid domains in inducing the blue–red

transitions.48,49

Early examples of lipid/PDA vesicle membrane sensing

applications include the incorporation of gangliosides into

the PDA vesicle framework, exploiting the affinity between

the ganglioside headgroup and cholera toxin for viral

detection.44,51–53 Another example of the implementation of

novel sensing schemes using lipid/PDA vesicles is the

incorporation of cholesterol moieties as a ‘‘bait’’ for colori-

metric sensing of pore-forming toxins produced by bacteria.54

That scheme has been further expanded, employing

fluorescently-labeled lipid insertion into the vesicles for

bacterial sensing.55

The observation that the lipid components in the mixed

vesicles form distinct bilayer domains is significant in the

context of biosensing applications; such vesicles could then

closely mimic the membrane surface of a cell. Enhancing the

utilization of lipid/PDA vesicles for biological applications has

been the capability of incorporating within the vesicles

varied synthetic and natural phospholipids, glycolipids, lipo-

polysaccharides, cholesterol, or total membrane extracts,

essentially mimicking the lipid compositions of different

membranes and cellular systems.56

The mechanisms accounting for the induction of colouri-

metric transformations in lipid/PDA vesicle systems by lipid-

bond biomolecules have not been fully elucidated, however

several studies have shed light on the factors contributing to

the blue–red changes.39,49 Specifically, previous studies

determined that the PDA framework in the mixed lipid/PDA

vesicles retains its conjugated backbone structure, accounting

to the initial blue colour of the vesicles. The externally-

induced colourimetric transformations are a consequence of

structural and dynamical perturbations within the lipid

domains which affect the PDA through the lipid/polymer

interfaces.39,48–50

Fig. 1 Structural features of polydiacetylene. (A) Creation of the polydiacetylene backbone from the diynoic acid monomers and induction of the

blue–red colour transitions. (B) Structural transition within the PDA backbone leading to the blue–red change.

Fig. 2 Colourimetric sensing with lipid/PDA vesicles. Structural/

colourimetric transformations of PDA (blue) induced by molecules

(grey ovals) interacting with the lipid bilayer domains (black).

Interaction mechanisms that have been distinguished include surface

binding (top right) and vertical insertion (bottom right).


Lipid/PDA vesicle systems have been used for studying

membrane interactions of short antimicrobial peptides

(AMPs).39,57–60 Such applications have been highly

informative, particularly in light of the widely-recognized

effects of AMPs upon the cell membrane, including pore

formation, lipid micellization, and bilayer disruption.61

All these processes are expected to induce significant

chromatic transitions within the lipid/PDA systems.

Moreover, studies have shown that important biophysical

parameters, such as the degree of penetration of the peptides

into lipid bilayers and mechanisms of peptide–lipid binding

affect the extent and dynamics of the colourimetric

transitions.57,59

Investigations employing the lipid/PDA vesicle assay for

screening and analysis of antimicrobial peptides have been

reported, including evaluation of the contribution of specific

lipid molecules in the bilayer to peptide adsorption and

penetration,57–59,62,63 comparative study of the contributions

of specific residues within antimicrobial peptide sequences to

their membrane interactions,50,55,60 and membrane binding of

pre-fibrillar assemblies and its significance to amyloid

toxicity.64 The observation of rapid colourimetric transitions

induced by antimicrobial peptides opens the way for using

lipid/PDA vesicles as a useful bioanalytical tool. The assay

could be applied as a vehicle for rapid colourimetric screening

of bilayer interactions and membrane binding of antimicrobial

compounds, or the absence of such interactions.

Lipid/PDA vesicles have been shown to constitute a useful

platform for evaluation of membrane interactions of

pharmaceutical materials.65 The unique chromatic properties

of PDA could prove particularly advantageous from a

practical point of view, since the PDA-based system could

easily facilitate colorimetric screening of a large number of

membrane-active compounds. An important observation

reported recently has been the apparent relationship between

the degree of bilayer penetration by pharmaceutical

compounds and the extent of colourimetric response elicited

following their addition to DMPC/PDA vesicles.65

Incorporation of natural and artificial receptors within

lipid/PDA assemblies has been a particularly important

development in the utilization of the chromatic vesicles for

colourimetric detection of biological analytes. The design of

new systems for rapid detection of interfacial biomolecular

interactions has to fulfill two main objectives. First, the

chemical construct should allow physical access and binding

between the receptor and the ligand in an aqueous solution.

The second requirement is that the ligand/receptor inter-

actions could be reported through easily detected chemical

or physical transformations within the system. Lipid/PDA

vesicles embedding recognition elements adhere to the above

requirements. In such vesicles the phospholipid framework is

exploited as an anchoring platform for receptors containing

hydrophobic moieties, overall facilitating display of the

recognition elements at the vesicle surface.

Colourimetric detection of ligand/receptor interactions

through physical incorporation of receptors within lipid/PDA

vesicles presents important advantages over chemical

attachment of recognition units to the PDA itself, discussed

above. First, chemical derivatization of PDA can be

technically demanding, and the organic synthesis procedures

limit the scope of this approach. Furthermore, attaching

additional chemical units onto the diacetylene monomers

often disrupts the organization and self-assembly of the

monomers and adversely affects polymerization. Con-

sequently, the abundance of recognition modules in previously

reported derivatized PDA vesicles is low.51,66 Such limitations

are generally not encountered in lipid/PDA vesicles

incorporating recognition elements. No chemical modification

of the diacetylene monomers is needed because the lipid

moieties constitute the scaffolding modules for anchoring the

receptor modules. In addition, a higher number of receptors

can be incorporated in the vesicles because of the high mole

ratio—almost 50% of the lipids in the mixed lipid/PDA

vesicles.57,67 Another noteworthy feature of the lipid/PDA

system as a vehicle for receptor display is the generic nature

of this approach—in principle, attachment of appropriate

lipophilic residues is the only precondition for displaying any

receptor unit at the vesicle surface.

Incorporation of biological receptor modules in PDA-based

vesicles can be combined with other scaffolding systems for

creation of versatile sensing modules. For example, sol–gel

assemblies comprising phospholipid/polydiacetylene vesicles

that further contained immunoglobulins were shown to

respond to the presence of specific antigens through visible

blue–red changes.67 Below we describe several sensor systems

utilizing receptor/lipid/PDA vesicles for specific molecular

recognition.

5. Advanced bioanalytical techniques for studying

membrane events

Methodologies designed to couple reconstituted and artificial

membrane systems to advanced bioanalytical technologies

have been reported. Such approaches generally aim to exploit

powerful detection and measurement methods in conjunction

with samples that are designed to mimic the environment of

the cell membrane. Atomic force microscopy (AFM) is a

powerful surface characterization tool which is increasingly

applied for membrane characterization. AFM offers a spatial

resolution (approaching E1 nm) that allows visualization of

supramolecular assemblies of membrane protein and other

molecules, structural transformations of membrane-embedded

species, and also local changes in fluidity and elasticity within

the membrane68 In recent years, AFM has evolved from a

somewhat strict imaging technique, into a multi-prong sensor

for analysis and manipulation of cellular membranes and

membrane processes.

Surface acoustic wave sensing is an effective technique for

high sensitivity measurements for biomolecule (particularly

peptide and protein) interactions with membranes. The

method relies on the intrinsic dependence of acoustic waves

upon the mass and viscoelastic properties of a monolayer

deposited on the sensor surface.69 While challenges still exist

as to achieving effective lipid immobilization and overcoming

non-specific interactions, surface acoustic wave biosensing has

been shown to be a powerful technology for analysis of

peptide–membrane interactions.69 The authors succeeded to

reconstruct highly uniform and rigid bilayers of phospholipids


and lipopolysaccharides. These assemblies have revealed

significantly enhanced rigidity of the lipid bilayers upon

surface binding by a cationic antimicrobial peptide.

Several bioanalytical techniques have been applied for

studying membrane processes using unique artificial organiza-

tions of lipid molecules. Lipid environments that confer

alignment of embedded molecules have been shown to be

particularly useful in solid-state NMR studies of membrane

proteins.70 Such systems allow application of simplified

structural analysis due to orientation of the studied molecules

in the magnetic field of the NMR spectrometer. Lipid bicelles,

in particular, have become in recent years a useful biomimetic

membrane platform for NMR experiments.71,72 The rapid

anisotropic tumbling of bicelles makes possible application

of a plethora of both solution and solid-state NMR

experiments. Employing small bicelles, high-resolution NMR

experiments have illuminated interactions of a membrane

protein with its bilayer environment.73 The cylindrical

symmetry of lipid bicelles, in particular, have been shown to

be particularly advantageous to NMR structural analyses,

since the symmetry axis can be uniformly oriented with respect

of the external magnetic field.74

A variation of lipidic ‘‘nano-discs’’ was used in an elegant

mass spectrometry (MS) experiment for carrying out

functional assays of membrane-associated membrane

proteins.75 In that study, lipid-based nanodiscs were used for

capturing the membrane proteins rhodopsin and transducin,

which were subsequently immobilized onto a bio-chip surface.

Using a variant of conventional matrix-assisted laser

desorption ionization time-of-flight (MALDI-TOF) termed

self-assembled MALDI-TOF (SAMDI), the researchers were

capable of observing the membrane-associated light-activated

interactions between rhodopsin and transducin.

A related surface plasmon resonance (SPR) application

employing immobilized lipid nanodiscs has been described.76

Similar to the MS experiment depicted above, the nanodiscs

were chemically immobilized onto a solid substrate (a SPR

biochip, Fig. 3). Specifically, immobilization of discoidal

assemblies was done through a histidine-tag moiety which

was conveniently bound onto a Ni2+-coated sensor surface.

Ligand–receptor binding involving membrane-associated

molecules could thus be monitored via the plasmon signal,

allowing kinetic and structural analysis.

Micelles have been another biomimetic membrane system

employed as a useful model in both NMR and MS studies.

The rapid tumbling rate of micelles or micelle–protein

complexes in water allows application of advanced

NMR spectroscopy for studying membrane-associated

biomolecules.77 A recent MS experimental technique in which

micelle-embedded membrane protein complexes were

‘‘nanoelectrosprayed’’ was shown to be highly useful in

preventing micelle aggregation in the gas phase, thus

facilitating investigation of protein–protein interactions.78

Progress in microscopy technologies and fluorescence

imaging methods have opened the way for high-resolution

analyses membrane-associated species in living cells.

A methodology developed by van de Linde et al. was applied

for probing lateral organization of proteins in cellular

membranes, specifically the inner leaflet of the mitochondrial

membrane.79 The researchers have termed their technique

‘‘direct stochastic optical reconstruction microscopy’’

(dSTORM), and it is based upon the photoswitchable

properties of several carbocyanine dyes. Essentially, the

excitation-induced transformation of the dye between a

fluorescent and non-fluorescent state allows localization of

the molecule through stochastic analysis.80 Through

application of the dSTORM technique, the authors have

achieved sub-diffraction spatial resolution of down to 20 nm

of antibodies labeled with fluorescently labeled dye, which

were targeted to the mitochondrial membrane.79

Membrane properties were studied by imaging ellipsometry,

an imaging technique based upon solid-supported monolayer

thickness measurement.81 The researchers analyzed inter-

digitation of lipid molecules composing a lipid film and the

effect of lipid mixing upon the monolayer phase transitions.

The technique was further employed to determine the effect of

cholesterol, a prominent modulator of membrane organization

and fluidity, on the extent of acyl-chain mixing.

6. Conclusions and future directions

The convergence of chemistry and biology in recent years has

led to considerable conceptual and technical advances for

better understanding membrane processes. This trend will

most likely continue, with the increasing sophistication of

nano-technological methodologies expected to continue to

contribute to membrane studies. In particular, biomimetic

chemistry and biomimetic systems will continue to play

significant roles in the development of innovative approaches

for elucidation of important biological events occurring at, or

associated with the cellular membrane.

Manipulation of lipid assemblies, either bilayers or

monolayers, has been the mainstay for experimental

approaches designed to elucidate membrane processes. The

introduction of lipid bicelles, nanodiscs, lipid/polymer

assemblies, and similar systems was aimed to both mimic

cellular membranes, as well as allow studying specific structural

and functional parameters. Furthermore, the introduction

of new chemical platforms for studying membrane processes

generally goes hand-in-hand with the dramatic progress in the

capabilities of advanced bioanalytical techniques, such as mass

spectrometry, microscopy, NMR spectroscopy, and others.

Fig. 3 Ligand–receptor interactions studied via immobilized lipid

nanodiscs. Discoidal assemblies attached to a SPR chip allows detec-

tion of specific ligand binding, see text.


Sophisticated measurement techniques will remain pivotal in

determining future experimental directions, involving both

living cell systems, as well as new artificial models.

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