522 Biochemical Society Transactions (2007) Volume 35, part 3
Self-assembling layers created by membrane
proteins on gold
D.S. Shah*, M.B. Thomas†, S. Phillips*, D.A. Cisneros‡, A.P. Le Brun†, S.A. Holt§ and J.H. Lakey†1
*Orla Protein Technologies Ltd, Nanotechnology Centre, Newcastle upon Tyne NE1 7RU, U.K., †Institute for Cell and Molecular Biosciences, University of
Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K., ‡Biotechnology Center, Dresden University of Technology, Tatzberg 49,
01307 Dresden, Germany, and §ISIS, Rutherford Appleton Laboratory, Chilton, Didcot OX11 0QX, U.K.
Membrane systems are based on several types of organization. First, amphiphilic lipids are able to
create monolayer and bilayer structures which may be flat, vesicular or micellar. Into these structures
membrane proteins can be inserted which use the membrane to provide signals for lateral and orientational
organization. Furthermore, the proteins are the product of highly specific self-assembly otherwise known
as folding, which mostly places individual atoms at precise places in three dimensions. These structures
all have dimensions in the nanoscale, except for the size of membrane planes which may extend for
millimetres in large liposomes or centimetres on planar surfaces such as monolayers at the air/water
interface. Membrane systems can be assembled on to surfaces to create supported bilayers and these have
uses in biosensors and in electrical measurements using modified ion channels. The supported systems also
allow for measurements using spectroscopy, surface plasmon resonance and atomic force microscopy. By
combining the roles of lipids and proteins, highly ordered and specific structures can be self-assembled in
aqueous solution at the nanoscale.
Biochemistry is by its very nature a nanoscale science. The
major players proteins, nucleic acids and membranes all
operate on length scales of nanometres and above. Thus
measure at the nanoscale. The benefits of nanotechnology,
other than benefits of scale, result largely from the novel
behaviour of materials at these dimensions.
To benefit from the combination of biochemistry and
nanotechnology there is a need to create a link that is either
physical or virtual. By virtual we mean in this context an
ability to communicate between the biological and physical
elements without a tangible link. In this respect the addition
of a latex bead to a biomolecule for manipulation by optical
tweezers is a physical link but the laser trapping of the bead
is a virtual link. In most cases where we wish to exploit
biomolecules we will require some form of physical link,
e.g. labelling of quantum dots, protein arrays, cell culture
scaffolds. In this respect the more precise the nanoscale ap-
plication, the more precise the linkage should be and the
exact placing of a defined number of molecules is to be
expected to be a normal requirement.
The electronics revolution, which has been driven by
microtechnology since the 1950s, kept its momentum by an
ever decreasing size of the processors that could be manu-
Key words: atomic force microscopy (AFM), Fourier-transform infrared (FTIR), impedance
spectroscopy, neutron reflection, outer membrane protein F (OmpF), thiolipid.
Abbreviations used: AFM, atomic force microscopy; FTIR, Fourier-transform infrared; OmpF,
outer membrane protein F; SLD, scattering length densities.
1To whom correspondence should be addressed (email firstname.lastname@example.org).
factured. Thus Moore’s law was obeyed by reducing the
size of the smallest features that could be manufactured .
Manufacture was by externally applied guidance, such as
photolithography, applied to large substrates and was termed
‘top-down’. Beyond the feature-size limits imposed by the
minimum wavelength of light we can now use electron-beam
methods to go to angstrom-sized structures but these are
increasingly expensive approaches. Bottom-up methods,
where we move from small molecules to increasingly larger
and more complicated structures, are required if we wish to
reduce costs, increase productivity and ensure robustness.
The heart of the bottom-up revolution is the concept of
self-assembly, which implies that the individual small-
molecule components possess the ability to create a structure
of greatly reduced entropy with levels of organization much
above the individual molecule level .
Amphipathic molecules, those that have both water soluble
and non-polar regions, are the stars of the self-assembly
world, most of us has carried out nanotechnology within
our very early childhood years by playing with foam in
the bath or by blowing bubbles using detergent solutions.
Here the free energy change that accompanies the hiding
of polar headgroups and water away from air in the soap
film is enough to stabilize structures of great complexity .
In aqueous solution, several groups have shown how this
simple structural device can create liposome-like structures
whose architecture can be fine-tuned by modifications such
C ?2007 Biochemical Society
Bionanotechnology: From Self-Assembly to Cell Biology523
of self-assembling monolayers originally  uses covalent
attachment to surfaces and hydrophobic self-assembly to
make highly ordered interfaces from randomly dissolved
single molecules. This has been coupled with a top-down
approach called microcontact printing to create patterned
self-assembling structures .
the self-organizational properties of DNA, the usefulness
of which relies on the specific recognition and binding of
complementary sequences. The four-base code means that
the number of unique combinations of binding partner for
a sequence n bases long is n4, although with sequences
longer than 20 residues the ability to discriminate a single
base change becomes more problematic especially at low
temperatures. This approach has been used widely to easily
create complex complementary nanostructures based on
synthesized oligonucleotides .
Proteins as self-assembling structures in
technology: they are capable of the most complex self-
assembly known, they can precisely recognize and respond
to a huge range of targets and, finally, they have a diverse
range of intrinsic catalytic activities.
The self-assembly of proteins provides a palette of
nanoscale molecules in large amounts, with good quality
the nucleic acids direct their own part of the assembly
process. The self-assembly continues after translation with
protein folding, a true and, probably, the most complex
self-assembly step known . First demonstrated in the
laboratory by Anfinsen et al. , the self-assembly involved
in protein folding requires only the information contained
within the protein sequence. In the cell, assistant protein
molecules called chaperonins guide proteins through the
as misfolding or aggregation. This is achieved by protecting
hydrophobic areas of the cell until folding hides them or
by increasing the rate of protein folding by catalysing the
isomerization of prolyl bonds or the formation of disulfide
links between cysteine residues. These are usually found
in some of the proteins destined to leave the cell and be
secreted into the surrounding medium. Anfinsen et al. 
andmanyotherssincehave shownthat theprocessof protein
folding can also be achieved in vitro. Normally this consists
of holding the protein in a denatured state by addition of
high concentrations of urea or guanidine chloride before
dilution (∼10-fold for most proteins) allows the folding to
begin. After this step a folded protein, usually monomeric,
is achieved in which the individual atoms and amino acid
residues occupy unique positions. Admittedly some regions
of proteins are flexible and some entire proteins even display
all the properties of being unfolded, but this flexibility is
often closely linked to function and often these regions bind
specific target molecules [9,10].
Thus the proteins provide a way to create reproducible
nanoscale structures of high complexity and with recombin-
ant methods of gene expression these can be made in large
amounts. However, in general, it is not simple to create your
problem’. This is due to the fact that we do not, apart from a
small number of exceptions, know the relationship between
amino acid sequence and complex tertiary structure. Thus
we can exploit simple rules to make small domain proteins
 but in general the larger structures and the enzymes
we use are those that have evolved naturally. These proteins
discovered in the natural world possess the properties that
we seek and by transferring the genes to a suitable host we
can start to use them for nanotechnology [12–15]. Here
the subject is membrane proteins and this is interesting to
nanotechnology because we have yet one more level of self-
assembly available to us if we use this group of molecules.
As shown in Figure 1 we can add two-dimensional oriented
assembly to the list if we use membrane proteins. This in fact
exploits the properties of amphiphiles to create the large self-
assembled and ordered structures discussed above but this
time to control and order whole protein molecules. Another
advantage of using a membrane system is that the proteins
reside in an electrically insulating layer which can be used
to separate two aqueous compartments [16–18]. The unique
relationship between the protein and the lipid layer means
that it is always precisely positioned and in some cases the
protein can be 100% oriented as well.
Self-assembly of membrane systems
Membrane systems can be assembled on to surfaces in a
systems to be applied to solid devices . The simplest
method is to add liposomes to surfaces such as silicon oxide
where they form bilayers. The surface layer of water on the
oxide layer ensures that the bilayer is stable. Any proteins
reconstituted into the vesicles can thus be arranged in a
lipidic medium. A larger water reservoir is made possible
by tethered lipid bilayers in which a lipid is attached to the
surface via a hydrophilic linker such as poly(ethylene glycol).
These lipids do not fill the lower leaflet of the bilayer but as
their name implies tethers a freely fluid bilayer to the surface
. We used this method to make an ion-channel-based
biosensor which included the Escherichia coli OmpF (outer
membrane protein F) porin . This was used to measure
the binding of the colicin toxin by surface plasmon resonance
and impedance spectroscopy, which measured the increase in
bilayer resistance when the ion channel in the OmpF protein
was blocked by the toxin. Subsequently we investigated
whether it is possible to adhere the OmpF protein directly to
the gold surface via the thiol group on a cysteine residue in
a periplasmic turn . This enabled the protein to bind at
densities close to half that of a two-dimensional crystal and
C ?2007 Biochemical Society
524 Biochemical Society Transactions (2007) Volume 35, part 3
Routes to self-assembly of membrane protein systems
After translation on the ribosome (A), we can obtain two main groups of membrane proteins for use in nanotechnology.
(B) Integral membrane proteins (here OmpA from the outer membrane of E. coli bacteria ) can be purified from the host
membrane or refolded from insoluble inclusion bodies [28,29]; in either case the purified form is stabilized by detergent
micelles as shown here. The protein can then be reconstituted into lipid bilayers (C) by removal of the detergent in the
presence of free lipid. Membrane-active toxins are secreted mainly by bacterial hosts into the surrounding medium as
water-soluble precursors, which later insert into the membrane without detergent. In this case the monomer protein (LukF;
PDB: 1LKF)  (D) heptamerizes (E) to form a transmembrane ion channel (F) (α-haemolysin; PDB: 7AHL)  which has
been engineered in many ways to show the usefulness of protein-based nanopores.
the rest of the surface to be completed with a monolayer of
thiolipid lacking a long tether, followed by free phospholipid
which adhered to the hydrophobic surface. The protein
retained activity by binding the colicin toxin and grazing
incidence FTIR (Fourier-transform infrared) measurements
confirmed the retention of β-structure.
This approach has been extended to the monomeric porin
OmpA, which due to its β-structure is a very useful protein
is easily refolded from bacterial inclusion bodies and the
to larger protein domains. It can be assembled as the
OmpF was previously (Figure 2)  by use of an inserted
cysteine mutation. Insertion of peptide sequences that code
for cell attachment shows that layers can be made for cell
culture applications (Figure 2), while inserted epitopes can
make surfaces useful for antibody detection. Larger domains
assembled in this way can functionalize surfaces for enzyme
The structure of the immobilized porin on gold was
recently confirmed as native by the use of AFM (atomic
force microscopy) . Two clear results were achieved;
for the first time high resolution was achieved on proteins
not packed in a two-dimensional crystal and this was made
possible by the compensatory rigid packed thiolipid without
which it was not possible to image the proteins at high
resolution. Providing proteins can be fixed to the gold
this appears to be a useful method for imaging membrane
proteins. It is very interesting that it could be achieved with
only the lower thiolipid monolayer present, leaving a bare
hydrophobic surface . The height of the monolayer is
sufficient because outer membrane proteins are shorter than
their stability. The hydrophobic surface may attract mobile
material, such as residual detergents, that we do not see by
AFM but in principle there appears to be no reason why
it should not allow the high quality of imaging that we
observe. The water/methylene group interface may simply
consist of ordered water as in the classical clathrate models
of water structure at such surfaces or in view of recent
neutron reflection data it may simply consist of decreased
water density . The protein appears not to be affected
since it resembles closely the structure of OmpF from two-
dimensional crystals and X-ray crystallography.
The immobilization of the proteins on to gold enables
them to be stable enough for study by a range of biophysical
techniques (Figure 3). In addition to AFM, FTIR and
impedance spectroscopy mentioned above, we have recently
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Bionanotechnology: From Self-Assembly to Cell Biology525
Stages in the assembly of an OmpA self-assembling layer
The OmpA gene is engineered to include the new protein sequence, expressed as bacterial inclusion bodies then refolded in
a detergent mixture, and it contains a cysteine mutation which provides the thiol or -SH group which anchors the protein to
the gold. After assembly of the protein from detergent solution, thiolipid in detergent is added, which forms a dense layer
wherever gold is exposed between the proteins. Finally, a top layer of free lipid is added to complete the surface. In the
picture, fibroblast cells are shown to adhere preferentially to an area (A) containing an OmpA protein which has an inserted
RDGS sequence compared with the area of lipid filler (B).
A summary of the methods used to investigate the structure of immobilized protein layers on gold
IS (impedance spectroscopy) [14,32,33], CD [25,34], ATR (attenuated total internal reflectance)-FTIR spectroscopy  might
be used from the lower side (as opposed to grazing incidence FTIR which must be used from the top and needs a dry
sample) but a very thin gold layer is needed. Other methods include AFM  and SPR (surface plasmon resonance) .
Neutron reflectivity is achieved by guiding the neutron beam through the silicon substrate [26,36].
employed CD . The advantage of this method is that
a full liquid layer is retained, whereas the grazing incidence
However, the method is at the limits of sensitivity of current
machines and our published results use an α-helical protein
. Nevertheless, we were able to see an unfolding and
folding cycle in the presence of the urea denaturant. We have
also used neutron reflection to measure the distribution of
protein, lipid and water in these layers in the z-axis . This
method uses the different neutron ‘refractive indices’ called
SLD (scattering length densities) of the components to build
up a layer-by-layer model of the substrate (silicon), gold,
protein, lipid and water. By exchanging the water phase for a
C ?2007 Biochemical Society
526 Biochemical Society Transactions (2007) Volume 35, part 3 Download full-text
of the sample can be tuned to enhance the differentiation of
the layers. Recently we have employed the two polarization
states of neutrons to enable two independent sets to be
obtained from one sample; this will significantly increase the
ability to effectively model multilayer systems.
In summary, membrane protein monolayers on gold
provide easily assembled, precisely arranged structures that
can be used for both biotechnology and fundamental
We thank the BBSRC (Biotechnology and Biological Sciences
Research Council) and the Wellcome Trust (grants 056232, 040422,
055979, 066850 and 049735) for support.
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Received 18 December 2006
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