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Biochimica et Biophysica Acta 1778 (2008) 1601 – 1610

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Review
Formation of irreversibly bound annexin A1 protein domains on
POPC/POPS solid supported membranes
Simon Faiß a , Katja Kastl b,1 , Andreas Janshoff a , Claudia Steinem b,⁎
a
Institute of Physical Chemistry, University of Mainz, Welder-Weg 11, 55128 Mainz, Germany
b
Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstr. 2, 37077 Göttingen, Germany
Received 10 October 2007; received in revised form 9 December 2007; accepted 4 January 2008
Available online 12 January 2008

Abstract

The specific interaction of annexin A1 with phospholipid bilayers is scrutinized by means of scanning force and fluorescence microscopy,
quartz crystal microbalance, ellipsometry, and modeled by dynamic Monte Carlo simulations. It was found that POPC/POPS bilayers exhibit
phase separation in POPC- and POPS-enriched domains as a function of Ca2+ concentration. Annexin A1 interacts with POPC/POPS bilayers by
forming irreversibly bound protein domains with monolayer thickness on POPS-enriched nanodomains, while the attachment of proteins to the
POPC-enriched regions is fully reversible. A thorough kinetic analysis of the process reveals that both, the binding constant of annexin A1 at the
POPC-rich areas as well as the irreversible adsorption rate to the POPS-rich domains increases with calcium ion concentration. Based on the
thermodynamic and kinetic data, a possible mechanism of the annexin A1 membrane interaction can be proposed.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Ellipsometry; Fluorescence microscopy; Micropatterned membranes; Monte Carlo simulation; QCM; SFM

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1602
2. Binding of annexin A1 to solid supported lipid bilayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1602
2.1. Visualization of annexin A1 binding by fluorescence microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1602
2.2. Thickness determination of annexin A1 by means of ellipsometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1603
2.3. Scanning force microscopy of annexin A1 bound to POPC/POPS membranes . . . . . . . . . . . . . . . . . . . . . . . 1604
3. Adsorption and desorption kinetics of annexin A1 at different Ca2+ concentrations . . . . . . . . . . . . . . . . . . . . . . . . . 1605
3.1. Theoretical modeling of the adsorption/desorption data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1606
3.2. Results of DMC simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1608
3.2.1. Impact of the calcium ion concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1608
3.2.2. Influence of incubation time on the parameter space. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1608
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1609
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1609

Abbreviations: ASF, available surface function; β-BODIPY 500/510 C12-HPC, 2-(4,4-Difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-

hexadecanoyl-sn-glycero-3-phosphocholine; DMC, Dynamic Monte Carlo simulation; FRET, Fluorescence recovery after photobleaching; POPC, 1-Palmitoyl-2-
oleoyl-sn-glycero-3-phosphocholine; POPS, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; QCM, Quartz crystal microbalance; RSA, Random sequential
adsorption; SFM, Scanning force microscopy
⁎ Corresponding author. Tel.: +49 551 393294; fax: +49 551 393228.
E-mail address: Claudia.steinem@chemie.uni-goettingen.de (C. Steinem).
1
Current address: Toshiba Research Europe Ltd., Cambridge Research Laboratory, 260 Cambridge Science Park, Milton Road, Cambridge CB4 OWE, UK.

0005-2736/$ - see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamem.2008.01.003
1602 S. Faiß et al. / Biochimica et Biophysica Acta 1778 (2008) 1601–1610
1. Introduction
Annexins comprise a family of membrane proteins that bind
to negatively charged phospholipids in the presence of Ca2+ and
which are found in almost all organisms ranging from molds
to mammals [1–3]. In 1990, a common nomenclature was
proposed by Crumpton and Dedman based on the Greek term
“annex” (= to connect), which refers to the unifying property
of annexins to be able to connect opposing membranes [4].
Recently, this nomenclature has been fine tuned in such that
annexin genes expressed in vertebrates are termed “A”
annexins, those in invertebrates “B” annexins, while “C” an-
nexins are found in fungi and molds, “D” annexins in plants
and “E” annexins in protists. Mammals have 12 different an-
nexin genes (A1–A11 and A13) encoding for proteins with
molecular masses of typically 30–40 kDa. All proteins are
structurally characterized by a highly conserved C-terminal
domain (core domain) that harbors the Ca2+- and membrane
binding sites. Sequence analysis reveals that the core domain
is composed of four or eight (in case of annexin A6) annexin
repeats, which are highly homologous among each other and
among the annexin family. Within each annexin repeat a highly
conserved sequence of 17 amino acids is found, which is called
endonexin fold. In contrast to the core domain, the N-terminal
domains are rather variable. Their sizes range from a few amino
acids (annexin A5) to more than hundred amino acids in case
of annexin A7 and A11 and are thought to be responsible for
the different functions of the annexins within cells.
Assessing the function of an annexin is still a difficult task,
as some cell types express more than one annexin. This results
in a complex and overlapping expression pattern of a structu-
rally conserved protein family, which renders the elucidation
of their individual cellular functions very cumbersome.
In this mini-review, we focus on annexin A1, which has
been identified as the cytosolic factor that aggregates isolated
neutrophil specific granules and promotes exocytotic fusion
with the plasma membrane in a Ca2+ dependent fashion [5,6].
Since it has been localized at early endosomes, annexin A1 also
appears to be involved in endocytosis [7,8]. Moreover, it has
been shown that annexin A1 is expressed at higher levels in cells
of the innate immune system such as neutrophils, monocytes/
macrophages, and mast cells than in cells of the adaptive im-
mune response such as T and B lymphocytes [9]. For example,
blood neutrophils and monocytes have abundant levels of an-
nexin A1, representing about 3–4% of total cytosolic proteins.
Results of the last 20 years have demonstrated that annexin
A1 acts as glucocorticoid-regulated endogenous down-regu-
lator of inflammation: if it is externalized on the cell surface
of adherent neutrophils, it blocks their interaction with endo-
thelial cells [10]. Recently, the protein has also been suggested
to participate in the plasma membrane repair mechanism [11].
The crystal structure of annexin A1 was solved in the
presence [12] and absence [13] of Ca2+ ions showing the four
characteristic subdomains of the protein, each comprising five
α-helices. All eight Ca2+ binding sites are located at the convex
side of the slightly curved disk, while the N-terminus with an
intermediate length of 40 amino acids is presumably located at
the concave side and is buried within the core in the absence of
Ca2+. The Ca2+ affinities are different for the various binding
sites and increase by two orders of magnitude in the presence of
negatively charged phospholipids. Based on the high-resolution
structural studies of annexin A1, Rosengarth and Luecke have
proposed a two-step model for the annexin A1-membrane
interaction leading to membrane aggregation. Starting with the
protein in its inactive form, the first step would be the calcium
ion-mediated binding to anionic phospholipid headgroups of
one membrane. This process involves a change in conforma-
tion of the C-terminal core that results in the previously buried
N-terminal domain becoming solvent accessible. The second
step would be the binding of a second membrane via hydro-
phobic interactions with the now exposed amphipathic N-ter-
minal domain [12].
We were able to investigate both membrane binding pro-
cesses of annexin A1 separately by exploiting planar membra-
nes attached to a solid support. In a first step, the adsorption of
annexin A1 is monitored [14–17], while after removal of non-
bound protein from solution, the second membrane binding
can be followed by addition of unilamellar vesicles [18]. In this
mini-review, we discuss the results obtained from quartz cry-
stal microbalance (QCM), ellipsometry, and scanning force
microscopy measurements in conjunction with dynamic Monte
Carlo simulations to elucidate the binding properties of annexin
A1 and its ability to form protein domains as a function of
phosphatidylserine and Ca2+ concentration.
2. Binding of annexin A1 to solid supported lipid bilayers
The functionality of annexins, that is, the ability to bind to
acidic phospholipids in the presence of calcium ions, is often
investigated in vitro by vesicle copelletation assays [19,20].
In this method, however, annexin binding and vesicle aggre-
gation are intimately connected. In order to exclusively inves-
tigate annexin binding to one membrane interface, planar
solid supported lipid bilayers are advantageous. These are
available on various surfaces and can be investigated by fluo-
rescence microscopy, scanning force microscopy, and quartz
crystal microbalance.
2.1. Visualization of annexin A1 binding by fluorescence
microscopy
By means of fluorescence microscopy, it is possible to in-
vestigate the specificity of annexin A1 for acidic phospholipids.
Porcine annexin A1 was recombinantly expressed in Escheri-
chia coli and purified according to a procedure described by
Rosengarth et al. [21]. To allow for a direct comparison of
protein binding to membranes of different lipid composition
within one experiment, individually addressable micropatterned
lipid bilayers on glass substrates were employed. Micropat-
terned membranes can be prepared by forming polymeric ca-
pillaries on a hydrophilic glass substrate that are filled with
unilamellar vesicle suspensions [22–25]. Capillaries are created
by placing an elastomeric polydimethylsiloxane stamp on the
substrate forming a tight and sealed contact. Vesicle adsorption,
 S. Faiß et al. / Biochimica et Biophysica Acta 1778 (2008) 1601–1610 1603

Fig. 1. Fluorescence micrographs of micropatterned lipid membranes on a glass surface after addition of 0.2 μM Texas Red labeled annexin A1. The membranes were
labeled with 0.5 mol% β-BODIPY 500/510 C12-HPC A. The fluorescence image was taken using an excitation wavelength of 488 nm to monitor the BODIPY
fluorescence. B. The fluorescence image was taken with excitation at 543 nm to monitor the Texas Red fluorescence of the protein.

spreading and fusion to form planar lipid bilayers is restricted ter at higher POPS content. Ca2+ ions are expected to bridge the
to the capillaries, which results in the formation of parallel negatively charged SiO2 surface with the negatively charged
membrane stripes. Fig. 1 shows fluorescence micrographs POPS headgroups leading to an increased adhesion of the
of four membrane stripes with a width of 15 μm compo- adsorbed vesicles, which is required for vesicle rupture on the
sed of POPC/POPS (4:1) and neat POPC doped with 0.5 mol% surface. The overshoot of the parameter Psi as found for POPC/
β-BODIPY 500/510 C12-HPC. To visualize membrane bound POPS (95:5) vesicles is indicative of the deposition of intact
annexin A1 by fluorescence microscopy, the protein was labe- vesicles followed by rupture and fusion on the surface. This
led with a Texas Red fluorescent dye covalently linked to
the amino groups of the protein. The membrane stripes were
incubated with 0.2 μM Texas Red labeled annexin A1 for 15 min
in the presence of 1 mM CaCl2 and afterwards rinsed with
protein-free buffer. Fig. 1B shows the corresponding fluore-
scence image obtained upon excitation of the Texas Red dye
of the protein at 543 nm. It is obvious that the membrane
stripes containing 20 mol% POPS show a red fluorescence,
indicating the specific binding of annexin A1 to POPS lipids.
The POPC stripes are, however, black demonstrating that PC
lipids fully prevent non-specific binding of the protein. This is
not the case on the pure glass substrate, where a faint red
fluorescence is discernable as a result of non-specific binding
of annexin A1. In Fig. 1A, the same membrane stripes were
visualized by excitation of the β-BODIPY dye of the lipid at
488 nm. The pure POPC bilayers show the characteristic green
fluorescence of the lipid dye, while the green fluorescence of
the membranes, composed of POPC/POPS, on which anne-
xin A1 has been bound to, is greatly diminished as a result of a
fluorescence resonance energy transfer (FRET) between β-BO-
DIPY and Texas Red. This FRET indicates that the protein is in
close proximity to the membrane.

2.2. Thickness determination of annexin A1 by means of

ellipsometry

Ellipsometry offers the unique possibility to study the change

in thickness on a SiO2/Si surface upon adsorption of lipids
and proteins [26]. Fig. 2A displays the change of the two
ellipsometric angles (Delta and Psi) of a SiO2/Si wafer exposed
to a vesicle suspension (0.5 mg/mL) consisting of either POPC/
POPS (4:1) or POPC/POPS (95:5) in the presence of a buffer
containing 1 mM Ca2+ [26]. Consistent with the results of
Reimhult et al. [27], bilayer formation occurs considerably fas-
Fig. 2. A. Formation of a solid supported lipid bilayer on SiO2/Si followed by
ellipsometry. Addition of 0.5 mg/mL vesicle suspension composed of either
POPC/POPS (4:1) (△,▲) or (95:5) (□,■) to a cleaned silica surface leads to a
decrease in Delta (open symbols) and increase in Psi (closed symbols). B. Time-
resolved thickness change after addition of 0.4 μM annexin A1 to a bilayer
composed of POPC/POPS (4:1) in the presence of 1.0 mM Ca2+. After 20 min,
the sample was rinsed with a 1.0 mM CaCl2 containing buffer.
1604 S. Faiß et al. / Biochimica et Biophysica Acta 1778 (2008) 1601–1610

Fig. 3. Topographic scanning force microscopy images of POPC/POPS membranes with variable POPS content on silica in the presence of 0.05 mM CaCl2 after
addition of 0.4 μM annexin A1 and rinsing with protein-free buffer. A. 5 mol% POPS, B. 10 mol% POPS, C. 20 mol% POPS. The graph next to panel A shows the
corresponding height profile across two protein domains.

is reminiscent of comprehensive QCM studies put forward by
Kasemo and coworkers [28,29], Richter et al. [30–32] and
Janshoff and coworkers [17,33–36]. Eventually, a mean thickness
of the supported membranes of 4–5 nm is achieved as calculated
from the change in Delta [26], which demonstrates that lipid
bilayers with a low number of defects have been formed. The
adsorption of annexin A1 on these bilayers was also followed by
ellipsometry. Fig. 2B shows the binding of annexin A1 to a bilayer
composed of POPC/POPS (4:1) in the presence of 1 mM CaCl2.
An average thickness change of 2.5 nm is observed, which di-
minishes to about 2.2 nm if rinsed with buffer containing 1 mM
CaCl2 demonstrating that a substantial amount of annexin A1 is
bound irreversibly to the solid supported bilayer as long as cal-
cium ions are present. A similar thickness of about (2.2 ± 0.3) nm
was found for annexin A1 adsorbed on POPC/POPS membranes
imaged by scanning force microscopy (SFM) [16]. Compared to
the height of annexin A1 protein domains observed on DPPC/
DPPS bilayers, where a mean thickness of (3.2 ± 0.3) nm was
monitored by means of SFM [14], the thickness of the annexin A1
layer on POPC/POPS is considerably smaller. The reduced
thickness might be either attributed to a partial insertion of the
protein in the soft matrix of the POPC/POPS bilayer or the
thickness is reduced due to an incomplete coverage (ellipsometry)
and indentation of the protein layer into the soft POPC/POPS
bilayer (SFM), respectively.
2.3. Scanning force microscopy of annexin A1 bound to
POPC/POPS membranes
SFM is highly suitable to investigate the lateral organization
of membrane bound annexin A1 to POPC/POPS membranes.
Usually, membranes are prepared on silicon surfaces by spread-
ing and fusion of unilamellar vesicles [37]. In previous studies
investigating the interaction of annexin A1 and A2 with solid
supported bilayers, membranes were mainly immobilized on
mica surfaces [38,39]. However, it turned out that the mica
substrate influences the distribution of negatively charged PS
lipids so that the content of PS in the leaflet that faces the
aqueous solution is diminished, which does not occur if silica
substrates such as silicon wafers are used [40]. Fig. 3 shows
intermittent contact mode SFM images of POPC/POPS mem-
branes on silica substrates containing 5, 10 and 20 mol% POPS,
respectively, after adsorption of annexin A1 in the presence of
0.05 mM CaCl2 and subsequent rinsing with protein-free buffer.
 S. Faiß et al. / Biochimica et Biophysica Acta 1778 (2008) 1601–1610 1605

Fig. 4. Topographic scanning force microscopy images of POPC/POPS membranes with variable POPS content on silica in the presence of 1.0 mM CaCl2 after
addition of 0.4 μM annexin A1 and rinsing with protein-free buffer. A. 5 mol% POPS, B. 10 mol% POPS, C. 20 mol% POPS. The graph next to panel A shows the
corresponding height profile across two protein domains.

From the topographic images, it can be deduced that annexin A1
is organized in laterally confined, more or less round protein
domains. In the absence of annexin A1, the POPC/POPS bila-
yers show in rare cases POPS-enriched domains with an in-
creased height of 0.5–1 nm but mostly these domains were not
visible on silicon wafers. As shown in Fig. 3, the surface
coverage increases as a function of the POPS content of the
membrane. At 5 mol% POPS, individual protein domains with
an approximate height of about 2 nm can be identified, while
at 20 mol% POPS protein domains merge so that individual
domains can no longer be distinguished. The observed height of
around (2.2 ± 0.3) nm [16] is lower than that of membrane bound
annexin A1 on gel-phase lipids [14] as discussed above. The
mean domain radius decreases from (135 ± 70) nm at 5 mol%
POPS to (80 ± 45) nm at 10 mol% POPS, as inferred from grain
analysis. This might be discussed in terms of the presence of a
larger number of nucleation sites of protein binding at higher
phosphatidylserine content. Fig. 4 shows SFM images of mem-
brane bound annexin A1 at a 20 times larger CaCl2 concentration
(1 mM) as a function of the POPS content. Again, the protein
coverage increases with the amount of POPS present in the
membrane and individual annexin domains can only be obser-
ved at low phosphatidylserine concentration. At 5 mol% POPS,
the protein domains exhibit a mean radius of (145 ± 80) nm
and the coverage increases from 0.22 at 5 mol% POPS to one,
where individual domains can no longer be distinguished. A
comparison of the protein surface coverage at 5 mol% POPS
demonstrates that an increase in Ca2+ concentration results in
an increase in the surface coverage from 0.13 (0.05 mM CaCl2)
to 0.22 (1 mM CaCl2). From these experiments it was conclu-
ded that annexin A1 adsorbs irreversibly on POPC/POPS mem-
branes on silica and that the protein coverage increases with
both calcium ion concentration and POPS content. Lateral ag-
gregates of monomolecular height (domains) are formed and
the mean domain size decreases with increasing POPS content,
but is independent of the calcium ion concentration.
3. Adsorption and desorption kinetics of annexin A1 at
different Ca2+ concentrations
Adsorption and desorption of annexin A1 on and from POPC/
POPS membranes are well known to be a function of the Ca2+
concentration in solution. It is of great interest to elucidate to
what extend the amount of reversibly bound protein, the kinetic
parameters and the lateral organization of the membrane are
influenced by the Ca2+ concentration. To address these aspects,
1606 S. Faiß et al. / Biochimica et Biophysica Acta 1778 (2008) 1601–1610
Fig. 5. Time-resolved frequency changes of 5 MHz quartz resonators during
adsorption and desorption of annexin A1 on solid supported POPC/POPS (4:1)
membranes at different Ca2+ concentrations. QCM data (grey) and the results of
computer simulations (black lines) are shown [14]. The arrows indicate the time
of buffer flushing. Simulation parameters are compiled in Table 1.
the quartz crystal microbalance (QCM) technique has been
extensively used, which allows to follow the adsorption and
desorption of annexin A1 on and from membranes in a time-
resolved and label free manner [34]. For these experiments,
solid supported monolayers composed of POPC/POPS (4:1)
were deposited on a gold electrode of a quartz plate, which was
functionalized with a self-assembly monolayer of octanethiol.
The quality of the chemisorbed monolayer and the hybrid bila-
yer was monitored by impedance spectroscopy, which is very
sensitive to detection of small defects [41]. Protein binding to
the membrane was initiated by adding annexin A1 in the
presence of different Ca2+ concentrations. A protein concentra-
tion of 0.4 μM was used, which ensures that independent of
the Ca2+ concentration the frequency shift produced by annexin
Fig. 6. Illustration of the RSA computer simulations based on the model of heterogeneous surfaces. Annexin A1 binds irreversibly to circular POPS-rich domains in the
membrane, while protein adsorption to the POPC rich matrix is reversible.
A1 adsorption reaches saturation [15]. Annexin A1 binding to
the solid supported membrane is indicated by a decrease in
resonance frequency with maximum frequency shifts that are a
function of the Ca2+ concentration in solution (Fig. 5). Control
experiments, in which annexin A1 was either added to POPC/
POPS membranes in the absence of Ca2+-ions or added to POPC
membranes in the presence of 1 mM CaCl2, resulted in no
observable frequency shifts, which confirmed that the shifts in
resonance frequency can be attributed to a specific and Ca2+-
dependent binding of annexin A1 to acidic phospholipids.
Desorption of annexin from the POPC/POPS membranes was
initiated by rinsing the system with pure buffer solution con-
taining the corresponding CaCl2 concentration 10 min after
protein addition (Fig. 5). Independent of the Ca2+-concentration,
removal of annexin A1 from solution led to a small increase
in resonance frequency, which can be attributed to annexin A1
desorption from the membrane. This result implies that the
majority of the protein is irreversibly bound on the membrane,
while a minor part is reversibly bound at a given Ca2+ con-
centration. These findings are the basis for a kinetic analysis
based on computer simulations employing an off-lattice rever-
sible random sequential adsorption (RSA) algorithm on a he-
terogeneous surface.
3.1. Theoretical modeling of the adsorption/desorption data
The experimental evidence suggested that POPC/POPS mem-
branes display two kinds of binding sites for annexin A1. One
binding site is provided by POPS enriched domains, to which
annexin A1 binds in an irreversible manner, while the binding
to the POPC enriched matrix, with single POPS molecules or
small clusters, is reversible. Based on these findings a model
of annexin A1 binding to a heterogeneous surface has been
 S. Faiß et al. / Biochimica et Biophysica Acta 1778 (2008) 1601–1610 1607

transferred into dynamic Monte Carlo (DMC) computer simula-

tions of the adsorption and desorption process. Considering a
linear flow of particles to the surface, the time-dependent surface
coverage can be expressed by two different rate equations (Eqs.
(1) and (2)) for reversible and irreversible binding:

dH1 kon pa2 qbulk U1 ðH1 Þ  koff H1

¼ ð1Þ
dt 1 þ kkon U1 ðH1 Þ
tr

dH2 kirr pa2 qbulk U2 ðH2 Þ

¼ ð2Þ
dt 1 þ kkirr U2 ðH2 Þ
tr

Θ1 and Θ2 are the reversible and irreversible surface covera-

ges, kon, koff, kirr, and ktr are the kinetic rate constants as described
in Fig. 6, and Φ1 and Φ2 are the available surface functions
(ASFs). The ASF describes the available surface for deposition
of particles from bulk solution as a function of coverage. For
instance, the classical Langmuir kinetics are represented by
an ASF of Φ(Θ) = 1 − Θ. These rate equations are solved by an
RSA algorithm, in which the proteins are modeled as disks with
radius of a = 3 nm based on the crystal structure of annexin
A1 [13]. The particle density in bulk solution is denoted as ρbulk.
The particles are randomly placed on a square that contains
circular domains representing the POPS rich phase (Fig. 6).
An average radius of 50 nm of the POPS domains was assu-
med independent of the Ca2+ concentration, which was deduced
from SFM images [16,39]. All simulations were carried out on
a simulation area of (1.5 × 1.5) μm2.
In order to compare the data produced by the computer
simulations with the results of the QCM technique, the sur-
face coverage as obtained from the simulations has to be
converted into a frequency shift. This is achieved by multi-
plying the calculated coverage with a constant negative va-
lue given by a measured frequency shift that arises from a
known protein surface coverage. The multiplying factor P
has been calculated by taking the surface coverage of ir-
reversibly bound protein to the membrane (Θirr), as derived
from SFM images, into account and corresponds to the re- Fig. 7. Frequency changes (grey) of 5 MHz quartz resonators and corresponding
results of computer simulations (black) after addition of 0.4 μM annexin A1 with
sonance frequency shift Δfirr from QCM readouts that has been various incubation times. A. c2+ Ca = 0.05 mM, Θdom = 0.15, B. cCa = 0.5 mM,
2+

Θdom = 0.255, C. c2+

Ca = 1.0 mM, Θ dom = 0.335. For all other parameters see Table 2.

Table 1
Results of computer simulations as shown in Fig. 5 monitored upon irreversible binding of annexin A1 to the
c2+
Ca / mM kon⁎ / 103 koff / s− 1 K / 105 M− 1 kirr⁎ / 105 Θdom Θsim
irr membrane (Eq. (3)):
(M s)− 1 (M s)− 1
Dfirr
0.01 0.51 0.007 0.73 0.34 0.065 0.035 P¼ ð3Þ
0.05 0.89 0.007 1.27 0.43 0.150 0.083 Hirr d Hjam
0.10 1.19 0.005 2.38 0.85 0.240 0.137
0.50 2.86 0.010 2.86 10.2 0.255 0.158 For the adsorption of 0.4 μM annexin A1 on a POPC/POPS
1.00 2.04 0.007 2.91 11.9 0.335 0.208 (4:1) membrane at 1 mM CaCl2 the resonance frequency shift
The parameters correspond to the binding of 0.4 μM annexin A1 to POPC/POPS is − 19.5 Hz and the corresponding surface coverage was
(4:1) membranes and an incubation time of 10 min. kon⁎ and koff are the rate determined to be 0.38 as deduced from SFM images. With a
constants of reversible adsorption and desorption on and from the POPC- theoretical maximal coverage of the surface referred to as the
enriched phase. kirr⁎ is the rate constant of the irreversible adsorption on the jamming limit of Θjam = 0.547, the absolute surface coverage is
POPS-enriched domains. K = kon⁎ / koff is the reversible binding constant, Θdom is
the fraction of the simulation area covered by POPS-enriched domains, and Θsim
0.21 and the multiplying factor is P = − 94 Hz. The conversion
irr
is the surface coverage of irreversibly adsorbed protein. The average domain factor P is slightly larger than that reported in an earlier study
radius was set to 50 nm. [16], since the SFM images allow for a more detailed estimation
1608 S. Faiß et al. / Biochimica et Biophysica Acta 1778 (2008) 1601–1610

Table 2 range from 0.005–0.010 s− 1 and show no obvious dependency

A. Kinetic parameters obtained from adapting dynamic Monte Carlo simulations on the calcium ion concentration.
to QCM measurements carried out in buffer containing different CaCl2
concentrations (see Fig. 7)
3.2.2. Influence of incubation time on the parameter space
0.05 mM CaCl2
tInc / s kon⁎ / 103 (M s)− 1 −1
koff / s 5
K / 10 M −1
kirr⁎ / 10 (M s)
5 −1
Coverage
Since it is well conceivable that the lateral organization of the
35 1.36 0.010 1.36 0.43 0.016 protein clusters as well as the lipid domains change over time, it
108 1.36 0.010 1.36 0.43 0.057 remained to be elucidated whether the kinetic parameters also
720 0.95 0.007 1.36 0.43 0.083 change as a function of incubation time. The incubation time is
1800 0.54 0.004 1.35 0.43 0.088 defined as the time between protein addition and flushing with
0.50 mM CaCl2
buffer. By terminating the adsorption process at a very early
tInc / s kon⁎ / 103 (M s)− 1 koff / s− 1 K / 105 M− 1 kirr⁎ / 105 (M s)− 1 Coverage time point, it should become possible to find evidence whether
34 2.8 0.010 2.9 10.2 0.06 lipid domains were present in full scale before adsorption takes
90 2.8 0.010 2.9 10.2 0.144 place or increase in size during adsorption [16]. Although it was
600 2.8 0.007 2.9 10.2 0.158

1.0 mM CaCl2
tInc / s kon⁎ / 103 (M s)− 1 koff / s− 1 K / 105 M− 1 kirr⁎ / 105 (M s)− 1 Coverage
35 2.04 0.007 2.91 11.9 0.066
75 2.04 0.007 2.91 11.9 0.181
600 2.04 0.007 2.91 11.9 0.208
1800 1.45 0.005 2.90 13.6 0.211
Adsorption kinetics of annexin A1 to POPC/POPS (4:1) membranes was
interrupted at time tInc by rinsing with buffer containing the given Ca2+
concentration.

of the protein surface coverage, which is slightly below the

theoretical jamming limit.

3.2. Results of DMC simulations

Prior to the kinetic analysis, the transport rate to the surface

ktr had to be quantified. Experimentally and theoretically a rate
of ktr = 10− 6 m/s was found and used throughout the simulations
[34,42].

3.2.1. Impact of the calcium ion concentration

Fig. 5 shows the simulated frequency changes for different
Ca2+ concentrations together with the QCM measurements.
Rate constants kon, koff, and kirr were stepwise adapted to ac-
hieve the best accordance between the QCM data and the
simulation. Noteworthy, the rate constants are defined in dif-
ferent parts of the curve, thus rendering the manual fitting
procedure very robust. The obtained values for the rate con-
stants are summarized in Table 1. The most important outcome
of the simulations and fitting procedure is that an increase in
Ca2+ concentration is accompanied by a substantial increase
of the overall domain area, on which irreversible adsorption
takes place. At 1 mM Ca2+, Θdom, the surface covered by the
POPS-enriched phase, was set to 0.335 to achieve a surface
coverage of 0.21 corresponding to the value obtained from the
analysis of SFM images, while Θdom decreases to 0.065 at
10 μM Ca2+ resulting in protein coverage of only 3.5%. Both
the reversible binding constant K ¼ kkonoff⁎ (k ⁎ ¼ kon pa2 NA with
the Avogadro number NA) and the rate constant for irreversi-
⁎
ble binding kirr ¼ kirr pa2 NA increase with the calcium ion con- Fig. 8. A. Course of the resonance frequency of adsorption Δfads (●) and
desorption Δfdes (▲) of annexin A1 binding to POPC/POPS (4:1) membranes as
centration. At 0.5 mM and 1 mM Ca2+, however, they are a function of Ca2+ concentration. B. Course of the binding constant K and C. the
almost constant and the change of the frequency course is rate constant of irreversible binding kirr⁎ as a function of Ca2+ concentration. The
mainly caused by the difference in domain area. The koff values time of protein incubation was 10 min for all measurements.
 S. Faiß et al. / Biochimica et Biophysica Acta 1778 (2008) 1601–1610
shown by SFM on pure bilayers that POPS-enriched domains
exist, it is unclear what the impact of protein binding on the
domain organization might be. Therefore, the time resolved
protein adsorption was monitored by QCM at different calcium
ion concentrations with incubation times ranging from about
30 s to 30 min. Dynamic Monte Carlo simulations were per-
formed and the simulation parameters (kon⁎, koff, and kirr⁎) ad-
justed to match the experimental data.
Fig. 7 shows the simulated frequency courses for different
incubation times at three different Ca2+ concentrations together
with the QCM results. The domain area was kept constant for a
fixed calcium ion concentration. The particle density for short
incubation times was corrected for the inevitably existing
protein concentration gradient at the beginning of the measure-
ment [37]. Table 2 summarizes the results obtained for three
different calcium ion concentrations and various incubation
times.
The simulated frequency courses agree well with the ex-
perimental QCM data. While K and kirr remained constant, the
rate constants of reversible binding, kon⁎ and koff, had to be
adjusted to lower values for longer incubation times [16,37]. At
high Ca2+ concentrations (0.5 and 1.0 mM) protein binding
is virtually irreversible within the first 35 s and only a small
amount of reversibly bound annexin can be released after 75
and 90 s incubation time. The measurements at 0.05 mM Ca2+,
however, show a larger impact of the incubation time on the
reversible binding dynamics. Already after 108 s, the same
amount of reversibly bound protein (3 Hz) is detectable as after
12 min (3 Hz) or even as at a later time point. This finding
indicates that within the time scale of the experiment, a slight
rearrangement of proteins and lipids might occur at low cal-
cium ion concentration, which changes the nature of binding
dynamics. A possible explanation of the apparently reduced
adsorption dynamics at low Ca2+ concentrations might be a
limited accessibility of free POPS molecules at later times,
which is responsible for the reduction of kon⁎. At later times,
reversibly bound annexin A1 might recruit more POPS mo-
lecules, which eventually leads to a reduced desorption rate.
Notably, a dynamic lateral rearrangement during the adsorp-
tion experiment is not captured by the RSA simulation and is
inferred only indirectly from the change in the parameter space
necessary to describe the experimental data. Interestingly, the
binding constant K is preserved giving rise to an apparently
consistent adsorption isotherm, which misses to display subtle
shifts from reversible to irreversible binding in the case of low
Ca2+ concentration over time.
Dynamic Monte Carlo simulations in conjunction with ki-
netic QCM measurements show that the reversible and ir-
reversible binding as well as the lateral organization of the
membrane is only slightly dependent on the incubation time
suggesting a rather stationary picture of the adsorption pro-
cess. POPS aggregates are preformed due to the presence of
calcium ions and are not affected by protein addition. This
is confirmed by SFM images of POPC/POPS bilayers immo-
bilized on mica surfaces (no protein present) revealing small
domains of POPS in the presence of calcium ions [16,39].
However, at low Ca2+ concentration a subtle tendency towards
1609
a lower binding and unbinding dynamics is found by compari-
son of the adsorption and desorption rate constants as a function
of incubation time.
Generally, the reversible binding constant K and the irre-
versible rate constant kirr⁎ increase both with the calcium ion
concentration (Fig. 8 B,C). Plotting the parameters on a half
logarithmic scale shows in both cases a sigmoidal curve with an
inflection point of 7·10− 5 M Ca2+ (K) and about 2.5·10− 4 M Ca2+
(kirr⁎). The considerable change of these two parameters at Ca2+
concentrations in the 10− 4 M range is consistent with the ob-
served change in resonance frequency, which shows for both,
Δfads and Δfdes a point of inflection at 7·10− 5 M Ca2+ (Fig. 8A).
Even though the results summarized in this article are all
obtained from in vitro experiments with well defined lipid com-
positions that mimic the inner leaflet of the plasma membrane,
one might ask the question of how such observed POPS ag-
gregation and annexin A1 protein domain formation affects the
protein's function within the cell. A conceivable scenario can be
envisioned as follows: A defined threshold of calcium ions is
needed to induce the formation of POPS clusters within a matrix
of POPC, which renders adsorption of annexin A1 irreversible as
long as the calcium ion concentration is constant. If calcium ions
are removed, demixing is reversed and annexin A1 is released.
In fact, the presence of POPS domains entirely abolishes de-
sorption of annexin A1, thus creating small protein islands on
the surface, which might serve as new adsorption platforms
for other newly arriving proteins and membrane structures. The
binding properties of annexin A1 to POPS are thus modulated by
PS-clustering as a result of the Ca2+-concentration. This way, the
cell may avoid a high binding constant of annexin A1 per se,
which would interfere with fast dynamics but rather allows
controlling the binding affinity of annexin A1 by a modification
of membrane organization as a function of calcium ions.
Acknowledgements
A.J. gratefully acknowledges the DFG (SFB 625) for fi-
nancial support.
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