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The effect of nanoscale surface curvature on the
oligomerization of surface-bound proteins
M. Kurylowicz, H. Paulin, J. Mogyoros, M. Giuliani and J. R. Dutcher
J. R. Soc. Interface 2014 11, 20130818, published 26 February 2014
Supplementary data
"Data Supplement"
http://rsif.royalsocietypublishing.org/content/suppl/2014/02/21/rsif.2013.0818.DC1.htm
l
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The effect of nanoscale surface
curvature on the oligomerization of
surface-bound proteins
rsif.royalsocietypublishing.org
M. Kurylowicz, H. Paulin, J. Mogyoros, M. Giuliani and J. R. Dutcher
Department of Physics, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Review
Cite this article: Kurylowicz M, Paulin H,
Mogyoros J, Giuliani M, Dutcher JR. 2014
The effect of nanoscale surface curvature on
the oligomerization of surface-bound proteins.
J. R. Soc. Interface 11: 20130818.
http://dx.doi.org/10.1098/rsif.2013.0818
Received: 4 September 2013
Accepted: 3 February 2014
Subject Areas:
biophysics
Keywords:
protein oligomerization, surface curvature,
nanoparticles, single-molecule force
spectroscopy
Author for correspondence:
J. R. Dutcher
e-mail: [email protected]
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsif.2013.0818 or
via http://rsif.royalsocietypublishing.org.
The influence of surface topography on protein conformation and association
is used routinely in biological cells to orchestrate and coordinate biomolecular
events. In the laboratory, controlling the surface curvature at the nanoscale
offers new possibilities for manipulating protein–protein interactions and
protein function at surfaces. We have studied the effect of surface curvature
on the association of two proteins, a-lactalbumin (a-LA) and b-lactoglobulin
(b-LG), which perform their function at the oil–water interface in milk emulsions. To control the surface curvature at the nanoscale, we have used a
combination of polystyrene (PS) nanoparticles (NPs) and ultrathin PS films
to fabricate chemically pure, hydrophobic surfaces that are highly curved
and are stable in aqueous buffer. We have used single-molecule force
spectroscopy to measure the contour lengths Lc for a-LA and b-LG adsorbed
on highly curved PS surfaces (NP diameters of 27 and 50 nm, capped with a
10 nm thick PS film), and we have compared these values in situ with those
measured for the same proteins adsorbed onto flat PS surfaces in the same
samples. The Lc distributions for b-LG adsorbed onto a flat PS surface contain
monomer and dimer peaks at 60 and 120 nm, respectively, while a-LA contains a large monomer peak near 50 nm and a dimer peak at 100 nm, with a
tail extending out to 200 nm, corresponding to higher order oligomers, e.g. trimers and tetramers. When b-LG or a-LA is adsorbed onto the most highly
curved surfaces, both monomer peaks are shifted to much smaller values
of Lc. Furthermore, for b-LG, the dimer peak is strongly suppressed on the
highly curved surface, whereas for a-LA the trimer and tetramer tail is suppressed with no significant change in the dimer peak. For both proteins, the
number of higher order oligomers is significantly reduced as the curvature of
the underlying surface is increased. These results suggest that the surface curvature provides a new method of manipulating protein–protein interactions
and controlling the association of adsorbed proteins, with applications to the
development of novel biosensors.
1. Introduction
The delicate balance between different interactions within and between biological molecules gives rise to intricate structures and dynamics. In many cases,
subtle changes to their environment can give rise to dramatic rearrangements
of the molecules. For example, changes in pH can alter the oligomerization
state and binding of ions to proteins that changes their biological function.
This sensitivity to environmental conditions is very common in biology, and
cells have evolved elaborate, nanostructured machinery that allows them to
respond to and even exploit environmental changes and stresses, such as
changes in pH, temperature and pressure, to ensure their viability. In addition,
the curvature of surfaces within cells is exploited to manipulate biomolecular
events and this has the advantage of targeting specific locations within the cell.
Cells also respond to the topography of surfaces on which they grow. Micropatterning of surfaces has been used to geometrically control the growth and
apoptosis of epithelial cells [1] and to direct the migration of various mammalian cells [2] by controlling cell shape. Curvature has been specifically identified
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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J. R. Soc. Interface 11: 20130818
NPs of diameter less than approximately 100 nm. Most of
these investigations have used optical spectroscopy techniques
to measure colloidal suspensions of protein-coated NPs,
including tryptophan fluorescence [26,27], circular dichroism
(CD) [28–31], ultraviolet–visible [29,32,33], infrared (IR)
[27,29,34] and surface plasmon resonance [32,35,36] spectroscopies. Silica NPs have been used to induce structural
changes in lysozyme [28,31,37–39], BSA [34,39–41], fibrinogen
[34], human carbonic anhydrase I [30], ribonuclease A [42],
haemoglobin [40] and b-lactoglobulin (b-LG) [27]. Changes
in secondary structure from a-helix to b-sheet were detected
in a model peptide covalently attached to thiolated Au NPs
[43]. Other surfaces with nanoscale curvature, such as liposomes [44] and single-walled carbon nanotubes [45,46], have
been investigated for their effect on the catalytic activity of
enzymes. These measurements suggested that proteins could
be stabilized on surfaces with nanoscale curvature more
readily than on flat surfaces by suppressing unfavourable lateral
protein–protein interactions. Recently, we have found that the
surface curvature can also suppress favourable protein–protein
interactions that hold together dimers and higher order
oligomers [47].
In this study, we compare the effect of nanoscale surface
curvature on two surface-binding proteins, a-lactalbumin
(a-LA) and b-LG, using single-molecule force spectroscopy
(SMFS). SMFS is an atomic force microscopy (AFM)-based
technique that has been developed to study protein unfolding
at the single-molecule level [48,49]. Protein molecules will
attach spontaneously to an AFM tip that is brought into contact
with a layer of adsorbed protein molecules, and the proteins
can be unfolded by translating the tip away from the substrate
while measuring the deflection of the AFM cantilever. This
mechanical denaturation of proteins can be reversible for
large proteins [49,50] and irreversible for small proteins [51].
As the retraction distance leading to a force peak is a measure
of the length of an unfolded protein complex and the chain
length of a monomer is known from the amino acid primary sequence, SMFS can be used to reliably measure the
oligomerization state of a surface-bound protein.
b-LG and a-LA are ideal proteins for investigating interfacial phenomena. Although they are water soluble, they
adsorb readily onto both hydrophilic and hydrophobic surfaces
[52–55]. Their native environment is at the oil–water interface
in bovine milk, where they act as emulsifiers. Bovine a-LA also
serves as the regulatory component of lactose synthetase [56].
The structure of both proteins has been studied in detail (Protein
Data Bank (PDB) codes 3BLG for b-LG and 1A4 V for a-LA).
Under physiological conditions, the ruminant variety of b-LG
forms a dimer in solution, but dissociates into monomers
below pH 3 [57]. X-ray crystallography has revealed a hexameric
quaternary structure for a-LA both with and without Ca2þ at
physiological pH (PDB codes 1F6S and 1F6R, respectively
[58]). Recently, a-LA has attracted renewed attention as a complex with oleic acid, in a technique called HAMLET/BAMLET
(human/bovine a-LA made lethal to tumours), which selectively causes apoptosis in tumour cells as a very high-order
oligomer [59–61].
Although the structures of b-LG and a-LA are known in
solution, much less is known about the conformation of the
proteins when they are adsorbed onto surfaces. Nuclear magnetic resonance (NMR) has been used to investigate a-LA
when adsorbed onto the surface of polystyrene (PS) NPs
[62] and the structure has been identified as a molten globule
rsif.royalsocietypublishing.org
as a causal factor in the differentiation of mesenchymal stem
cells, with cells that were trapped inside a convex shape differentiating preferentially into adipocytes while the concave
curvature promoted osteoblast generation [3]. Nanoscale topography has been used to influence cell adhesion [4] and even to
induce morphological, genomic and proteomic changes in
bacteria [5]. These studies have controlled the tissue microenvironment of cells to regulate their development, but an
important biological question remains to be elucidated: do
cells detect the geometry of the underlying surface by an
internal mechanism (for example, signalling through cytoskeletal strain [3]), or do they detect biochemical or geometric
changes in the membrane or protein film to which they are
attached? Both effects are probably relevant and it is important
to study each in detail. This means that it is important to understand the impact of substrate structure on cells as well as
individual proteins that are many orders of magnitude smaller.
An excellent example of the effect of the underlying substrate structure is the localization of the peripheral membrane
protein SpoVM at the outer forespore membrane as a result of
recognition of convex curvature during sporulation of Bacillus
subtilis [6]. Moreover, it has been shown in vitro that native
SpoVM selectively targets membrane vesicles smaller than
5 mm, while a non-functional mutant is much less size selective
and localizes in vesicles up to 20 mm in diameter [7]. Concave
membrane curvature has also been shown to influence the
localization of the peripheral membrane protein DivIVA [8].
In these cases, the mechanism by which the micrometre-scale
curvature influences a nanometre-scale object has yet to be elucidated, though it may be that biological membranes act as
curvature amplifiers by generating packing defects that can
be detected by individual proteins [9]. Importantly, it remains
to be shown whether surface-binding proteins respond to geometric curvature itself ( perhaps by altering surface-induced
folding landscapes) or whether phospholipid membranes
must act as biochemical assistants or possibly transducers.
The availability of a wide variety of synthetic nanostructures [10] has made it possible to directly probe the effect of
nanoscale surface geometry on the structure and function
of adsorbed proteins. Recent studies have highlighted the
importance of characterizing the complex and dynamic
‘corona’ of adsorbed proteins on nanoparticles (NPs) in vivo
[11–13], emphasizing that cellular responses to nanomaterials
in a biological medium are likely to stem from the adsorbed
biomolecular layer rather than the material itself [14,15]. For
example, it has been demonstrated that the adsorption of
fibrinogen on tantalum can be affected by nanoscale surface
roughness, whereas the adsorption of bovine serum albumin
(BSA) was not affected [16]. On titanium surfaces with similar
nanoscale roughness, no changes in fibrinogen adsorption
were observed with increasing roughness [17], whereas BSA
adsorption was significantly increased on platinum surfaces
with nanoscale roughness [18]. These results illustrate the
subtle interaction of surface chemistry and geometry on the
adsorption of proteins, with different substrate materials yielding different trends. Understanding the influence of nanoscale
geometry on molecular events will not only further our understanding in vivo but also lead to technological developments,
such as biomolecular NP conjugates for use in biosensing
[19,20], drug delivery [21,22] and the creation of new classes
of advanced biomaterials [23–25].
A number of studies have demonstrated changes in protein
structure and function due to the adsorption of proteins onto
Downloaded from rsif.royalsocietypublishing.org on February 26, 2014
PS cap
5
H2O + methanol
floating
PS film
PS NPs
2
NP monolayer
4
H2O
Figure 1. Schematic of the sample preparation procedure. Starting with a
thin film of PS spin-coated onto Si (1), a surfactant-stabilized aqueous suspension of PS NPs was mixed with dioleoyl-phosphatidylcholine (DOPC) and
methanol and then spin-coated onto a thin film of pure PS on Si (2), followed by a methanol rinse to remove the DOPC. This created a textured
surface with curved regions of NP monolayers and bare flat regions (3). To
create a well-defined surface chemistry for this textured surface, we floated
a 10 nm thick film of pure PS film on mica onto a clean water surface and
captured this film onto the NP-coated substrates (4), capping the highly
curved substrate with pure PS for use in the protein adsorption studies (5).
unfolding intermediate [63]. CD and fluorescence have also
been used to detect a loss of tertiary structure of a-LA
upon binding to PS NPs [64]. For b-LG at an oil –water interface, CD measurements indicated an increase in alpha helical
content for the adsorbed protein as compared with that in
solution [65], with similar results obtained using Fourier
transform infrared (FT-IR) spectroscopy [66]. Although it is
clear that the function of a-LA is to promote the production
of lactose in milk, no clear function has been identified for
b-LG. Other lipocalins have previously been classified as
transport proteins and have also been demonstrated to play
roles in coloration, olfaction and prostaglandin synthesis
[67]. There is some consensus that b-LG may be a convenient
nutritional protein in milk, which may be ‘left over’ from
another function in the evolutionary past of its host [68].
Recently, we used SMFS to determine that nanoscale curvature suppressed the oligomerization of b-LG on highly curved
PS surfaces [47]. The propensity of a-LA to form hexameric
oligomers in solution suggested to us that the effect of nanoscale surface curvature on oligomerization could be even
more dramatic for a-LA than for b-LG. In this paper, we
show that the nanoscale surface curvature shifts the equilibrium of surface-bound oligomers to smaller sizes for a-LA,
as it did for b-LG, with an interesting difference: although trimers and tetramers of a-LA are suppressed on curved surfaces,
the population of dimers is not significantly affected.
2. Results
The fabrication of highly curved, chemically pure hydrophobic surfaces that are stable in buffer allowed us to
perform SMFS experiments on proteins immobilized on surfaces under biologically relevant conditions. A schematic of
our sample preparation technique is shown in figure 1 (for
details, see the Material and methods section). First, we
spin-coated a thin (approx. 80 nm) film of pure, monodisperse PS onto a Si wafer to form a flat hydrophobic PS
substrate. Next, we spin-coated a dilute aqueous suspension
J. R. Soc. Interface 11: 20130818
3
3
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PS film
SiOx
1
of surfactant-stabilized PS NPs onto the PS substrate, resulting in close-packed monolayers of the NPs separated by
bare regions of the underlying substrate. As the NP suspensions used in this study are stabilized by surfactants, the
surface chemistry of the NPs is necessarily different from
that of pure PS. To produce chemically pure, highly curved
surfaces, we used a water transfer procedure [69] to cap our
PS NP surfaces with an ultrathin (10 nm thick) film of the
same PS that was used to create the flat PS substrates
(figure 1; also see Material and methods). The transfer of
the ultrathin PS film onto the top of the PS NP surface
ensured that the surfaces were chemically pure PS that was
identical on the flat and curved regions of the samples, eliminating the effect of the ill-defined surface chemistry of the
NPs. By choosing the thickness of the PS capping layer to be
sufficiently small (10 nm thick) and by performing the water
transfer procedure at an elevated temperature, we ensured
that the capping film would conform to the shape of the
underlying NPs for even the smallest NP diameters (27 nm).
The morphology of the PS-capped PS NP substrates
was measured using both optical microscopy and AFM, and
representative images are shown in figure 2. By carefully selecting the spin-coating conditions, it was possible to obtain NPcovered regions that contained well-ordered, close-packed
layers of NPs perforated by large regions several tens of micrometres to hundreds of micrometres in size that exposed the
flat PS substrate, caused by dewetting of the water-based suspension during the spin-coating process. We emphasize that,
although it may be possible to create NP films with more uniform coverage, the presence of highly curved and flat regions in
the same microscopic domains allowed us to perform in situ
comparisons using SMFS of proteins adsorbed to both
curved and flat surface topographies. The 10 nm thick PS capping film was clearly visible in the optical micrographs as a
result of optical interference with the underlying film or NPs,
which allowed us to target only the PS-capped surfaces for
curved and flat regions in the AFM experiments. Figure 2 also
shows an AFM image of the capping layer and its edge, demonstrating that the capping film conforms around the NPs and
maintains the desired nanoscale curvature of the surface.
We alternately collected AFM images and SMFS data,
which allowed us to perform SMFS measurements on the
top of the NPs where the surface curvature is very well
defined by the radius of the NP and the thickness of the
capping layer. For each sample, we collected hundreds of
force –distance curves and used robust selection criteria (see
Material and methods) to select those curves that contained
a well-defined worm-like chain (WLC) detachment peak
(the last and longest peak in the force –distance curve). We
then fitted each of these selected curves to equation (5.1)
(see Material and methods) to obtain the best-fit contour
length for the detachment peak, which we refer to as the
detachment length Lc. A representative force –distance curve
with a WLC-like detachment peak is shown in the electronic
supplementary material, figure S1, together with a schematic
of the SMFS measurement on a protein dimer.
In figure 3, we show distributions of the detachment
lengths Lc for the curved and flat regions of 27 and 50 nm
diameter NP-coated samples for both a-LA and b-LG. To
facilitate comparisons between the different histograms, we
have divided the number of counts in a given histogram by
the total number N (shown in the legends of each plot)
of the force –distance curves included in that histogram. As
Downloaded from rsif.royalsocietypublishing.org on February 26, 2014
200 nm
100 µm
100 nm
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1 µm
capped
uncapped
curved
70 nm
20
10
0
–10
–20
100 200 300 400 500
nm
Figure 2. Optical microscopy image (left) and AFM topography image (right) of a 50 nm diameter NP surface, with part of the surface covered with a 10 nm PS
capping film, indicating both the capped/uncapped and curved/flat regions. The AFM line scan in the lower right corner corresponds to the white line indicated in
the AFM image directly above the line scan.
(a) 0.25
0.20
frequency
(b)
a-LA
flat (1262)
50 nm (926)
0.15
b-LG
flat (1372)
50 nm (438)
0.10
0.05
(c)
0
a-LA
flat (1262)
27 nm (310)
frequency
0.20
0.15
(d)
b-LG
flat (1372)
27 nm (397)
0.10
0.05
0
50
100 150 200
contour length (nm)
250
0
50
100
150 200
contour length (nm)
250
300
Figure 3. Comparison of histograms of detachment lengths Lc measured for a-LA and b-LG on flat PS surfaces with those measured on (a,b) capped 50 nm
diameter NP-coated PS surfaces and (c,d ) capped 27 nm diameter NP-coated curved PS surfaces. The number of force– distance curves included in each histogram
is shown in the inset to each plot.
each Lc distribution measured for the flat surfaces exhibits a
well-defined peak that occurs at a value close to the contour
length of a single protein (approx. 50 nm for a-LA and
approx. 60 nm for b-LG), we refer to this peak as the monomer peak, as in Kurylowicz et al. [47]. Smaller peaks observed
at larger values of Lc are identified as higher order oligomers,
e.g. dimers, trimers, etc., indicating that the AFM tip has
pulled on two or more proteins that are associated with the
surface [47]. For the 50 nm diameter NP surfaces (figure
3a,b), the numbers of measured Lc values that would correspond to the dimer peak for b-LG and the trimer peak for
a-LA are reduced with respect to those for the flat surface,
with the distributions shifted to smaller Lc values while
preserving their overall shape. For the 27 nm diameter NP
surfaces (figure 3c,d ), the distributions are shifted to even
smaller Lc values, with the numbers of measured Lc values
that would correspond to the dimer peak for b-LG and the
higher order peaks, e.g. trimers and tetramers, for a-LA
strongly suppressed or not present at all. Therefore, for
both proteins, the number of higher order oligomers is significantly reduced as the curvature of the underlying
surface is increased. It is interesting to note that the peaks
in the Lc histograms for the 27 nm diameter NP surfaces
occur at Lc values (approx. 30 nm for b-LG and approx.
12 nm for a-LA) that are considerably less than the contour
lengths of the a-LA and b-LG molecules. It is possible that
J. R. Soc. Interface 11: 20130818
flat
4
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(a)
0.25
(b)
frequency
0.15
flat
a-LA (1262)
27 nm
a-LA (310)
b-LG (1372)
b-LG (397)
rsif.royalsocietypublishing.org
0.20
5
0.10
0.05
50
100 150 200
contour length (nm)
250
0
50
100 150 200
contour length (nm)
250
300
Figure 4. Comparison of histograms of detachment lengths Lc measured for a-LA and b-LG on (a) flat PS surfaces and (b) capped 27 nm diameter NP-coated
curved PS surfaces. The number of force – distance curves included in each histogram is shown in the inset to each plot.
these values correspond to the distances between distinct,
adjacent anchoring points of the protein on the underlying
PS surface, and that these distances are observed more clearly
on the most highly curved surfaces. In the electronic supplementary material, figure S2, we have compared the
cumulative distribution function for each pair of Lc histograms
shown in figure 3, and we have used the two-sample Kolmogorov–Smirnov (KS) test [70] to determine the corresponding
p-value for each case, where p , 0.05 is generally accepted as
a stringent threshold indicating a significant difference
between distributions (e.g. [71]). The two-sample KS test is a
general non-parametric method for comparing two distributions of data that does not rely on any assumptions about
the distributions. Further details about the procedure used to
implement the two-sample KS test are provided in the electronic supplementary material. The largest p-value obtained
for our data is 0.001 for the comparison of the Lc histograms
for the flat and 50 nm diameter NP surfaces for b-LG, and all
of the other p-values are much smaller. Therefore, all of the
distributions can be considered to be statistically different
and this has allowed us to draw meaningful conclusions from
comparisons of the different histograms shown in figure 3.
In figure 4, we have replotted the data shown in figure 3 to
highlight the comparison between the data collected for the
two proteins, for both the flat PS and 27 nm diameter NP surfaces. It can be seen from these plots that the highly curved
surfaces produce a decrease in the relative number of higher
order oligomers for both proteins, with some significant differences. In particular, we note that for a-LA the relative number
of dimers is preserved, whereas for b-LG it is not.
In the electronic supplementary material, figure S3, we
compare the detachment lengths Lc and detachment forces Fd
measured for an AFM tip retraction rate of 3.0 Hz (used in all
of the above measurements) with those measured for a lower
retraction rate of 0.5 Hz for a-LA on flat PS surfaces. For the
lower retraction rate, there is a decrease in the longest
measured detachment lengths and a correspondingly small
but measurable shift in the detachment forces to smaller values.
3. Discussion
One of the key aspects of this study is the development of a
unique sample geometry that provides several advantages
for studies of proteins on highly curved surfaces. Through
the use of arrays of polymer NPs capped by ultrathin
polymer films, we can directly compare the results for proteins adsorbed onto flat surfaces with those obtained for
proteins adsorbed onto highly curved surfaces that are chemically pure. This is a major advance in the study of the properties
of proteins adsorbed onto NP surfaces, e.g. protein–NP conjugates. Although NMR [62,63] and optical spectroscopy [26–36]
have been used to characterize the ensemble average behaviour
of suspensions of protein–NP conjugates, our use of SMFS provides the first investigation of the properties of proteins
adsorbed to NP surfaces at the single-molecule level. Moreover, previous studies of bulk suspensions of protein–NP
conjugates have suffered from the presence of impurities
because suspensions of NPs are typically stabilized by charge
or surfactants before the introduction of proteins. In the case
of polymeric NPs, there are additional impurities arising
from the emulsion polymerization process. Our ability to
ensure the chemical purity of the nanoscale curved surfaces
by capping the surface-bound NPs with a pure, ultrathin PS
film solves a major problem in controlled experimental investigations of protein–NP conjugates. The capping of highly
curved surfaces with ultrathin polymer films also offers the
possibility for surface curvature investigations using different
polymeric materials.
Using this sample geometry, we have compared the
association of two different surface-bound proteins, a-LA
and b-LG, on flat and highly curved surfaces. It can be
seen from the histograms shown in figures 3 and 4 that the
effect of increasing the curvature of the underlying surface
disrupts the oligomerization of the proteins because the relative size of the monomer peak increased for both proteins at
the expense of the higher order peaks for the most highly
curved surfaces (27 nm diameter NP surfaces). This effect
can be understood in simple terms: each adsorbed protein
molecule has more available space and therefore fewer tendencies to interact with neighbouring molecules relative to
its local environment on a flat surface. This results in fewer
higher order peaks observed in the detachment length Lc histograms for the surfaces with the highest curvature. As the size of
the protein molecules is small compared with the diameter of
even the smallest NPs, it is likely that the surface coverage of
b-LG molecules is comparable on NPs of different diameters
and that the dominant effect with decreasing NP diameter is
weakened lateral interactions between adsorbed proteins [47].
Our SMFS data also show the expected increase in the
detachment force Fd with increasing retraction rate (electronic
supplementary material, figure S3). The corresponding
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In this study, we have used SMFS to study changes in the oligomerization of surface-bound a-LA and b-LG adsorbed
onto hydrophobic PS substrates, as the curvature of the surface
5. Material and methods
A 1.5% (by mass) solution of PS (Mw/Mn ¼ 1.12, Mw ¼ 675 000)
in toluene was spin-coated at 1500 r.p.m. onto a 1 1 cm oxidized Si(100) wafer and annealed for 18 h at 1058C under an
oil-free vacuum. The thickness of the resulting films was between
70 and 80 nm, as measured by ellipsometry and AFM. The resulting hydrophobic surface was then used as a substrate to spin-coat
PS NPs of two different diameters: 27 and 50 nm.
PS NPs (Bangs Labs) were purchased with a concentration 10%
by mass. The 27 nm diameter NPs were spin-coated directly from
this stock suspension. The 50 nm diameter NP suspensions were
diluted to 1% by mass with Milli-Q water, then mixed in a 1 : 1
ratio with a solution of 400 parts methanol to 1 part dioleoyl-phosphatidylcholine (DOPC) by mass (Avanti; lyophilized powder). The
DOPC acted as a surfactant to allow 50 nm diameter NP suspensions to wet the hydrophobic PS film substrate with a much lower
contact angle than the pure aqueous suspensions, allowing for convective self-assembly of NPs at the evaporative front during spincoating. The stock 27 nm diameter NP suspension had a much
lower contact angle than the others, and we hypothesize that the
27 nm diameter NPs are small enough to act as their own surfactant,
as in Pickering emulsions. After spin-coating, all samples were
flushed thoroughly with methanol to dissolve the DOPC and then
dried in clean nitrogen gas, including the 27 nm diameter NP
samples to which DOPC was not added.
For the capping film, a 0.25% (by mass) solution of PS
(Mw/Mn ¼ 1.12, Mw ¼ 675 000) in toluene was spin-coated at
2000 r.p.m. onto a freshly cleaved 2 10 cm piece of muscovite
mica and annealed for 18 h at 1058C under an oil-free vacuum.
The resulting film was 10 nm thick as measured using AFM (see
figure 2). A razor blade was used to score this film into a grid of
twenty 1 cm2 squares. The film-coated mica was then lowered at
a 458 angle into a pool of Milli-Q water at 708C. As mica is hydrophilic and PS is hydrophobic, the PS film floats onto the water
surface. After 1 min of thermal equilibration under water, the
1 cm2 Si-PS-NP-PS substrates were lifted at an angle of approximately 308 under a piece of floating PS film to transfer the PS
film onto the solid substrate. Samples were put under vacuum
for 1 h to remove water and then stored in air. Although the
6
J. R. Soc. Interface 11: 20130818
4. Conclusion
was varied at the nanoscale. A key aspect of this study was the
preparation of monolayers of monodisperse PS NPs that
allowed us to achieve a well-defined, controllable surface curvature. Capping of the PS NP monolayers with an ultrathin
PS film allowed us to obtain chemically pure, highly curved,
hydrophobic PS surfaces that were stable in buffer. The
samples contained regions of close-packed NP monolayers as
well as bare flat regions, allowing for in situ comparisons of
the effect of surface curvature on the oligomerization of a-LA
and b-LG. By pulling on individual proteins using SMFS, we
obtained clear evidence that the association of neighbouring
protein molecules was significantly reduced for both a-LA
and b-LG molecules on the highly curved surface compared
with the flat surface. These results are consistent with the curvature of the surface providing more space for adsorbed
proteins, reducing their interactions with neighbouring proteins. In addition, the most highly curved surfaces reveal the
dominance of Lc values that are considerably smaller than
the contour lengths of the protein molecules, which may correspond to the distances between well-defined anchoring points
of the proteins with the underlying surface. The ability to alter
protein oligomerization with surface curvature provides
promising applications to biosensing applications, modifying
biological function by tailoring surface topography.
rsif.royalsocietypublishing.org
detachment length Lc results (electronic supplementary
material, figure S3) show that the longest detachment lengths
are measured for the fastest retraction rates, suggesting that
the dissociation time of an oligomer is comparable to the tip
retraction time. It is for this reason that we performed the
SMFS experiments using the fastest retraction rate.
Proteins can bind to different vertical locations on the
AFM tip because of the vertical compression of the protein
layer due to the pressing of the AFM tip onto the sample
surface. This compression can allow neighbouring proteins to bind to the AFM tip at locations that are displaced
vertically from the AFM tip apex. As the maximum compression of the protein layer is equal to the protein
diameter (only several nanometres), this limits the attachment
of neighbouring proteins to points on the AFM tip that are
displaced vertically from the tip apex by the maximum
value of several nanometres. In our histograms of the data
presented in this paper, the bin size is 10 nm (considerably
larger than the maximum difference in vertical binding distance on the AFM tip) and the changes in Lc values due to
changes in the surface curvature are approximately 25 nm
(much larger than the maximum difference in the vertical
binding distance on the AFM tip). Therefore, the possible
variation in the vertical binding location of the proteins is
considerably smaller than measured shifts in the Lc values
as a result of the surface curvature, with the uncertainty in
the Lc values limited to several nanometres.
As the oligomerization state of a protein often modulates
its function [72], the manipulation of protein –protein interactions is of great interest for medical and biotechnological
applications. Many possibilities are suggested by the ways
in which dimerization is used in biological cells. For example,
dimerization can regulate signal transduction in cell surface
receptors, where ligands induce dimerization in the extracellular domain of the protein that triggers kinase activity
within the cytoplasm [73,74]. Dimerization enables such
remarkable effects by catalysing reactions by bringing substrates and active sites together in favourable orientations
and enhancing specificity by increasing the effective surface
area of interaction [75]. Transient changes from the monomeric to the dimeric state of the protein can also provide
dynamic triggers for conformational changes in the local
environment of the protein, can facilitate chemical modifications or exchanges and can provide temporary storage or
stability for the monomeric state [72].
Protein–protein interactions can also be altered by external
changes. In biomedical products, for example pharmaceutical
formulations, chemicals or co-solvents can be added to either
stabilize or compete with contacts between proteins [76]. Oligomerization of proteins can also be modified by changing the
primary sequence of the protein at specific protein-coupling
interfaces [76,77]. These changes will, of course, affect entire
protein populations. The advantage of using substrate curvature to control protein oligomerization is that native protein
structures can be targeted at specific locations within cells
and on biosensing surfaces.
Downloaded from rsif.royalsocietypublishing.org on February 26, 2014
FLp 1
x 2 1 x
þ ,
¼ 1
Lc
4 Lc
kB T 4
(5:1)
where F is the measured force and x is the tip – sample separation,
T is temperature, kB is Boltzmann’s constant and Lp is the persistence length of the chain (fixed at a value of 0.4 nm for amino
acids). The best-fit value of the detachment length Lc was
obtained by fitting the detachment peak data to equation (5.1).
Funding statement. We gratefully acknowledge financial support from
the Natural Sciences and Engineering Research Council of Canada,
the Canadian Foundation for Innovation and the Ontario Research
Fund. J.R.D. is the recipient of a Canada Research Chair in Soft
Matter and Biological Physics.
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neighbours. Typically approximately 100 force–distance curves
were collected per NP, with approximately 10% success rate for
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