Cooperative binding at lipid bilayer membrane surfaces

Article abstract of DOI:10.1021/ja021048a

The binding of copper(II) ions to membrane-bound synthetic receptors has been investigated. Complexation fitted a 4:1 receptor: copper(II) model, and the observed binding constants are significantly enhanced at the membrane relative to solution; these effects can be explained by the lower polarity of the membrane-water interface and the concentrating effect of the membrane, with no observed contribution from receptor preorganization. The stoichiometry of the complex formed is very sensitive to the concentration of the receptor in the membrane, and at low concentrations, binding is reduced relative to solution controls. This implies that by increasing or decreasing the number of receptors in their membranes, cells can finely tune biological responses such as chemotaxis that depend on the size of the receptor-ligand clusters formed.

Full text of DOI:10.1021/ja021048a

Published on Web 03/21/2003
Cooperative Binding at Lipid Bilayer Membrane Surfaces
†
Emma L. Doyle, Christopher A. Hunter,* Helen C. Phillips, Simon J. Webb, and
Nicholas H. Williams*
Contribution from the Centre for Chemical Biology, Krebs Institute for Biomolecular Science,
Department of Chemistry, The UniVersity of Sheffield, Sheffield, S3 7HF, U.K.
Received July 31, 2002; E-mail: N.H.Williams@Sheffield.ac.uk; C.Hunter@Sheffield.ac.uk
Abstract: The binding of copper(II) ions to membrane-bound synthetic receptors has been investigated.
Complexation fitted a 4:1 receptor:copper(II) model, and the observed binding constants are significantly
enhanced at the membrane relative to solution; these effects can be explained by the lower polarity of the
membrane-water interface and the concentrating effect of the membrane, with no observed contribution
from receptor preorganization. The stoichiometry of the complex formed is very sensitive to the concentration
of the receptor in the membrane, and at low concentrations, binding is reduced relative to solution controls.
This implies that by increasing or decreasing the number of receptors in their membranes, cells can finely
tune biological responses such as chemotaxis that depend on the size of the receptor-ligand clusters
formed.
Cooperative binding to receptors constrained to a membrane
surface is a fundamentally important process in biology. For
example, in chemotaxis bacteria can detect very small changes
in the concentration of ligands, such as sugars, over many orders
aggregates. Again, both antigen affinity and the size of the
aggregate is important.
5
Receptors that are anchored to a membrane surface can only
move in two-dimensions, and so binding interactions between
them are expected to be more thermodynamically favorable than
the corresponding interactions in solution, where the molecules
are free to move in three dimensions. Thus preorganization of
receptors on a membrane surface could account for the coopera-
tive intra-membrane binding interactions described above.
However, there have been few quantitative experimental studies
that shed light on the relative importance of the various factors
that might contribute to cooperative receptor aggregation in
membranes. There are two problems with such studies. If we
consider the aggregation of membrane-bound receptors around
a multivalent ligand, we cannot simply compare the association
constant for binding the ligand from solution (K1), with the
apparent association constants for subsequent interactions within
the membrane (K2), because the values of the latter will change
as a function of the concentration of the receptor in the
1
of magnitude. A recent mathematical model proposed to explain
this remarkable behavior suggested that signal transduction is
2
regulated by changes in lateral clustering of the chemoreceptors.
The importance of lateral clustering was confirmed when
multivalent ligands with pendant sugars were shown to induce
3
aggregation and give a chemotactic response. Furthermore, the
size of the cluster formed is also important; for example,
tetramers of the chemotaxic receptor Tar are significantly more
active during in vitro chemotaxic signaling than individual or
dimeric receptors.4
Another important biological process initiated by receptor
aggregation is the allergic response. This is mediated by
membrane-bound receptors that are formed when IgE immu-
noglobulins bind to membrane-embedded FCꢀRI stems on the
cell surface. During an allergic reaction a multivalent antigen
binds to the IgE-FCꢀRI receptors, inducing receptor aggregation.
The formation of these aggregates initiates a cascade of signaling
events, resulting in the release of chemical messengers that
trigger the immune response. The size of the aggregates is
unknown, but model studies with IgE oligomers have shown
that dimers do not trigger the response as effectively as larger
6
membrane. Second, the membrane interface is a quite different
environment from bulk solution, and this is likely to have a
dramatic effect on the intrinsic binding constant for any
interaction.
Here we report our results concerning the aggregation of
membrane-anchored receptors, using a model system that allows
us to quantify the membrane environment and therefore
investigate the relationship between receptor concentration and
the cooperativity of multicomponent assembly processes on the
membrane surface.
†
Current address: Department of Chemistry, UMIST, PO Box 88,
Manchester, M60 1QD, U.K.
(
1) (a) Mesibov, R.; Ordal, G. W.; Adler, J. J. Gen. Physiol. 1973, 62, 203-
2
23. (b) Jasuja, R.; Keyoung, J.; Reid, G. P.; Trentham, D. R.; Khan, S.
Biophys. J. 1999, 76, 1706-1719.
(5) (a) Receptors: models for binding, trafficking and signaling, Lauffenburger,
D. A.; Linderman J. J. Physical Aspects of Receptor/Ligand Binding and
Trafficking Processes; Oxford University Press: Oxford, UK, 1993; Chapter
4, pp 110-175. (b) Subramanian K.; Holowka D.; Baird B.; Goldstein B.
Biochemistry 1996, 35, 5518-5527. (c) Schweitzer-Stenner R.; Pecht, I.
Immunology Lett. 1999, 68, 59-69.
(
2) (a) Bray, D.; Levin, M. D.; Morton-Firth, C. J. Nature 1998, 393, 85-88
(
b) Levit, M. N.; Liu, Y.; Stock, J. B. Mol. Microbiol. 1998, 30, 459-466
c) Duke, T. A. J.; Bray, D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10104-
(
1
0108.
(
3) Gestwicki, J. E.; Strong, L. E.; Kiessling, L. L. Chem. Biol. 2000, 7, 583-
91.
4) Cochran, A. G.; Kim, P. S. Science 1996, 271, 1113-1116.
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(6) Mammen, M.; Choi, S.-K.; Whitesides, G. M. Angew. Chem., Int. Ed. Engl.
1998, 37, 2754-2794.
(
10.1021/ja021048a CCC: $25.00 © 2003 American Chemical Society
J. AM. CHEM. SOC. 2003, 125, 4593-4599
9
4593

A R T I C L E S
Doyle et al.
Table 1. Calculated Binding Constants to Copper(II) for Receptor 1 in Aqueous Buffer (pH 6, 50 mM MES), Receptor 1 in 4% Aqueous
a
Buffer in Methanol, and Receptor 2 in Vesicles
-1
membrane binding constants (M-1)
solution binding constants (M )
memb
memb
memb
receptor (medium)
(aq buffer)
(4% aq buffer in MeOH)
(vesicles)
K
1
K
2
K
3
K
4
K
2
K
3
K
4
2
4
1
1
2
1.2 ( 0.2 × 10
< 0.1
7 ( 1 × 10
nd
nd
nd
nd
3
3
8 ( 1 × 10
2
3
1.4 ( 0.2 × 10
1.0 ( 0.2 × 10
8 ( 4
1.5 ( 0.7 × 10
a
nd: too low to be determined accurately.
Scheme 1. Syntheses of Receptors 1 and 2
Results and Discussion
We chose a dansyl-ethylenediamine conjugate as our head-
7
group and cholesterol as the membrane anchor. The dansyl
group has a strongly environmentally sensitive fluorophore,
which provides information about the microenvironment sur-
8
rounding the probe, and the coordination of copper(II) to related
ligands quenches the fluorescence.9 Analysis of the degree
of quenching caused by titrating copper(II) into vesicular
suspensions of the membrane-anchored dansyl ethylenediamine
conjugate therefore allows direct monitoring of the binding
processes. Membrane receptor 2 was synthesized in two steps
from cholesterol (Scheme 1). Heating cholesterol with bromo-
acetyl chloride in THF under reflux afforded the bromoacetyl
ester. Subsequent displacement of the bromide with 1 in
acetonitrile, available through the condensation of dansyl
chloride with ethylenediamine, gave the receptor 2. Compound
,10
1
serves as a useful control system for quantifying the copper-
(II)-receptor interactions in solution.
Initial Binding Studies. The binding constants for complex-
ation of 1 by copper(II) in aqueous solution were measured to
provide reference binding constants for calibrating the analogous
interactions at vesicle surfaces. The affinity of 1 (0.2 mM) for
copper(II) was low in aqueous solution at pH 6: only the first
binding constant could be determined (K1, Table 1), and there
-
1
was no observable 2:1 (Cu12) binding (K2 < 0.1 M ). This
behavior strongly suggests a monodentate interaction rather than
a bidentate metal-ligand interaction; for glycine ethyl ester
-
1
binding to copper(II) K1 ≈ 200 M
ethylenediamine binding to copper(II) K1 ≈ 3 × 10 M at
pH 6.
at pH 6, but for
5
-1
1
1
We then prepared unilamellar vesicles (800 nm diameter)
containing 1 mol % 2, keeping the bulk concentration of receptor
at 0.2 mM as before. Aqueous copper(II) chloride was titrated
into this vesicular solution, and the decrease in the fluorescence
of the dansyl group monitored. Initial analysis of the titration
curve indicated that a 2:1 complex (Cu22) formed with observed
Thus the membrane-bound ligands have a much higher
affinity for copper(II) than the corresponding ligands in solution.
obs
obs
4
-1
obs
binding constants K1 and K2 both approximately 10 M .
The effect is particularly pronounced for K
which shows a
2
5
>
10 fold enhancement, apparently confirming that restricting
(
7) (a) Menger, F. M.; Caran, K. L.; Seredyuk, V. A. Angew. Chem., Intl. Ed.
Engl. 2001, 40, 3905-3907. (b) Barragan, V.; Menger, F. M.; Caran, K.
L.; Vidil, C.; Morere, A.; Montero, J.-L. Chem. Commun. 2001, 85-86.
8) Abel E.; Maguire G.; Murillo O.; Suzuki I.; De Wall S.; G o¨ kel G. W. J.
Am. Chem. Soc. 1999, 121, 9043.
9) (a) Corradini, R.; Dossena, A.; Galaverna, G.; Marchelli, R.; Panagia, A.;
Sartor, G. J. Org. Chem. 1997, 62, 6283-6289. (b) Dujols, V.; Ford, F.;
Czarnik, A. W. J. Am. Chem. Soc. 1997, 119, 7386-7387. (c) Grandini,
P.; Mancin, F.; Tecilla, P.; Scrimin, P.; Tonellato, U. Angew. Chem., Int.
Ed. Engl. 1999, 38, 3061-3064.
the ligands to a two-dimensional environment leads to strong
cooperative intra-membrane binding interactions. However, the
obs
(
(
observation of a 100-fold enhancement of K1 is important,
because anchoring the receptor in a membrane should have no
entropic advantage for this process. The increase suggests that
the environment at the membrane interface plays a significant
role in modulating binding interactions involving membrane-
anchored receptors.
The Polarity of the Membrane-Water Interface. To
quantify the effect of the membrane-water interface on binding,
we used the fluorescent headgroup to probe the polarity of the
environment experienced by the receptor. The fluorescence
(
10) Prodi, L.; Montalti, M.; Zaccheroni, N.; Dallavalle, F.; Folesani, G.;
Lanfranchi, M.; Corradini, R.; Pagliari, S.; Marchelli, R. HelV. Chim. Acta.
2
001, 84, 690-706.
(
11) (a) Martell, A. E., Ed. Stability Constants of Metal-Ion Complexes, Section
II: Organic Ligands; The Chemical Society: London, 1964; p 348.
(
Glycine ethyl ester.) (b) Perrin, D. D. Stability Constants of Metal-Ion
Complexes Part B Organic Ligands; Pergamon Press: Oxford, 1979; pp
7-58. (1,2-Diaminoethane.)
5
4
594 J. AM. CHEM. SOC. VOL. 125, NO. 15, 2003
9

Lipid Bilayer Membrane Surfaces
A R T I C L E S
in the vesicular system for 2. Thus the improvement in the
binding affinity due to constraining the receptors in vesicles
appears to be primarily due to solvation effects in the local
environment. Previous studies of interactions with membrane-
anchored receptors have not taken the effect of the membrane
environment into account, and the large cooperative effects
observed in these experiments could be caused by similar
1
5
effects.
The Effect on Binding of Vesicle Receptor Concentration.
The membrane concentration of the receptor should also be an
important factor in determining the magnitude of the observed
binding constants, and so far we have ignored this parameter.7
With an appropriate solution control established, i.e. K1 for
a,16a
obs
binding 1 in solution is the same as K1 for the membrane-
anchored receptor 2, we investigated how the membrane
obs
concentration of 2 affects the value of K2 relative to that
measured for 1 in solution.
We maintained the bulk concentration of 2 at 0.2 mM and
the vesicle size at 800 nm, but varied the concentration of
phospholipid. This has the effect of varying the number of
vesicles and hence the number of receptors per vesicle. Keeping
the overall concentration of the receptor constant ensures that
any differences observed in the binding behavior are purely due
to changes in the observed association constants. Five different
vesicular solutions were prepared containing 0.2, 1, 2.5, 5, and
Figure 1. Calibration curve showing the change in λmax of dansylamide
fluorescence with changing solvent polarity. The dansylamide fluorescence
was measured both in pure solvents (Blue dots: a, THF; b, CH2Cl2; c,
1-octanol; d, 1-butanol; e, ethanol; f, methanol; g, water) and water/methanol
mixtures. (Red dots, 10% increments. There is a linear relationship between
the volume fraction of a water-methanol mixture and its dielectric
12
constant. ) The dotted line shows the interpolation of λmax (538.4 nm) found
for receptor 2 in vesicles.
7
.5 mol % 2 in the lipid bilayer. Titration of copper(II) into
emission spectra of both 1 and 3 were measured in a series of
solvents. As solvent polarity increased, the dansyl fluorescence
intensity Imax decreased and wavelength maximum λmax in-
creased. The position of λmax and solvent polarity ꢀ were used
to construct a calibration curve (Figure 1). A good linear fit
was obtained for both 1 and dansylamide 3 with little difference
between the two compounds, showing that altering the polarity
of the surroundings is the major contributor to the changes in
λmax, and that changing the structure of the headgroup has a
negligible effect. The fluorescence emission spectrum of a
vesicular solution of 2 (1 mol % loading in 800 nm unilamellar
vesicles) had λmax ) 538.4 nm, and comparison with the
calibration curve indicates that the bilayer environment sur-
rounding the headgroups of the receptor 2 is akin to 4 ( 3%
water in methanol (ꢀ ∼ 35, Figure 1). This value is in good
agreement with other estimates of the polarity of the interface
these solutions revealed two effects as the membrane concentra-
tion of receptor increased: the overall binding affinity increased
dramatically and the stoichiometry changed from Cu22 to
Cu24 (Figure 2). There are clearly huge cooperative effects in
this system to the extent that the 4:1 complex, which is never
observed in solution, becomes the most stable species when the
receptors are anchored to the membrane.
We used an ML4 binding model and an iterative curve fitting
program to determine apparent bulk association constants at each
phospholipid concentration. The first binding constant K1 is
independent of the number of receptors per vesicle as expected,
but the observed values of K2 , K3 , and K4 increase with
increasing receptor to phospholipid ratio. The increase in K2
with increasing mole percent of receptor in the membrane is
shown in Figure 3. There is a clear linear correlation, and the
obs
obs
obs
obs
obs
between lipid bilayers and bulk aqueous solution,13 and shows
slope is related to the association constant in the membrane
Kn
membrane binding interaction (Figure 4).
memb
, which is the true measure of the strength of the intra-
that the receptor is positioned in a polar environment, although
clearly not in a fully aqueous environment. This clearly has to
be taken into account when interpreting the observed binding
constants. Theoretical studies by Sakurai et al.14 predict that
To understand this relationship, we define a binding constant
Knmemb in terms of the membrane concentration of the receptor:
binding interactions dependent either on hydrogen bonding or
between charged species are strongly affected by the presence
of the bilayer interface even when the local binding interaction
is positioned in the aqueous subphase.
To obtain more appropriate solution control association
constants for 1, we measured the binding constants in 4%
aqueous buffer in methanol. A 2:1 Cu12 complex was formed,
with both K1 and K2 significantly greater than in purely aqueous
buffer (Table 1). Both values are very close to those obtained
[
MR ]
memb
n memb
Kn
)
(1)
[MRn-1
]
memb[R]memb
The membrane concentration of each species X is related to its
bulk solution concentration by the fraction of the overall solvent
volume occupied by phospholipid:
[
X]solution ) [X]memb(V [PL])
(2)
m
where Vm is the molar volume of phospholipid17 and [PL] is
(
12) Jian-Zohong B.; Swicord M.; Davis C. J. Chem. Phys. 1996, 104, 4441.
13) (a) Epand, R. M.; Kraayenhof, R. Chem. Phys. Lipids 1999, 101, 57-64
(
(
b) Mazeres, S.; Schram, V.; Tocanne, J.-F.; Lopez, A. Biophys. J. 1996,
(15) (a) Try, A. C.; Sharman, G. J.; Dancer, R. J.; Bardsley, B.; Entress, R. M.
H.; Williams, D. H. J. Chem. Soc., Perkin Trans. 1 1997, 2911-2917. (b)
Sharman, G. J.; Try, A. C.; Dancer, R. J.; Cho, Y. R.; Staroske, T.; Bardsley,
B.; Maguire, A. J.; Cooper, M. A.; O’Brien, D. P.; Williams, D. H. J. Am.
Chem. Soc. 1997, 119, 12041-12047.
7
1, 327-335.
(
14) (a) Sakurai, M.; Tamagawa, H.; Inoue, Y.; Ariga, K.; Kunitake, T. J. Phys.
Chem. B 1997, 101, 4810-4816 (b) Tamagawa, H.; Sakurai, M.; Inoue,
Y.; Ariga, K.; Kunitake, T. J. Phys. Chem. B 1997, 101, 4817-4825.
J. AM. CHEM. SOC.
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A R T I C L E S
Doyle et al.
Figure 2. Titration curves showing the decrease in the fluorescence of
vesicle-bound receptor 2 upon the addition of copper(II). Receptor 2 (0.2
mM) was dissolved in vesicles with a receptor loading of 0.2 mol % (blue
dots), 1 mol % (red dots), 2.5 mol % (purple dots), 5 mol % (orange dots),
and 7.5 mol % (green dots). The solid lines represent curve fits calculated
using the binding constants in Table 1. The dotted lines represent curves
calculated at the tight binding limit for the Cu22 and Cu24 complexes.
Figure 4. Sequential binding events during the complexation of the
messenger (copper(II)) to membrane embedded receptor 2.
significantly less than K2 in solution. In other words, the intrinsic
binding strength for this interaction is lower in the membrane
than in solution. This presumably reflects difficulties in attaining
an appropriate geometric arrangement for binding. The potential
cooperativity associated with reducing the number of degrees
of freedom, i.e. constraining the receptors to move in two-
dimensions, does not appear to be significant in this system.
In this approach, we have assumed that the membrane-
anchored receptor is constrained to the volume of the lipid
membrane in order to define the membrane concentration, and
memb
so Kn
are three-dimensional binding constants. In reality,
it is unlikely that the polar headgroups can sample the full
volume of the membrane. They are constrained to the interfacial
regions, and so the association constants determined by this
method overestimate the true membrane binding affinities.
However, the advantage of this approach is that we can directly
compare membrane association constants with solution associa-
tion constants, even though membrane binding events are more
Figure 3. Change in the observed binding constant K2obs as a function of
the mole percentage of receptor in the vesicle membranes ø.
18
properly expressed as two-dimensional binding constants.
Using the data obtained at different phospholipid concentra-
tions, we used eq 4 to obtain three self-consistent membrane
the concentration of phospholipid. Combining eqs 1 and 2 gives
1
9
binding constants (Table 1) that fit all of the titration data
memb
[
MR ]
Kn
obs
n solution
(
16) (a) O’Brien, D. P.; Entress, R. M. N.; Cooper, M. A.; O’Brien, S. W.;
Kn
)
)
(3)
[
MR
]
[R]solution [PL]Vm
Hopkinson, A.; Williams, D. H. J. Am. Chem. Soc. 1999, 121, 5259-5265.
n-1 solution
(
b) Cooper M. A.; Williams D. H. Anal. Biochem. 1999, 276, 36-47. (c)
Cooper M. A.; Williams D. H. Chem. Biol. 1999, 6, 891-899.
3
-1
which can be expressed as
m
(17) We calculate V (0.84 dm mol ) from the dimensions of unilamellar
EYPC vesicles (thickness of the phospholipid leaflet ∼20 Å, cross sectional
2
area of the headgroup ∼70 Å . Balgavy, P.; Dubnickova, M.; Uhrikova,
memb
D.; Yaradaikin, S.; Kiselev, M.; Gordeliy, V. Acta Phys. SloV. 1998, 48,
Kn
obs
5
09-533) and so this factor relates to the relatively disordered bilayer phase,
Kn
)
ø
(4)
(
)
rather than the crystalline value.
1
00[R]TVm
(18) It is useful to also express the binding events at the surface of the membrane
as two-dimensional binding constants. These two-dimensional binding
obs
memb
constants can be related to the membrane binding constants K
n
and K
n
where [R]T is the total concentration of the receptor in the
solution (0.2 mM in our case) and ø is the mole percentage of
the receptor in the membrane. Using eq 4 and the slope of the
obs
2D
memb
2D
by K
n
) K
n
/A
m
[PL] and K
n
) dmemb
K
n
where dmemb is the
is the molar cross-
sectional area of the phospholipid headgroup (4.2 × 10 dm mol ). Data
from Balgavy, P.; Dubnickova, M.; Uhrikova, D.; Yaradaikin, S.; Kiselev,
M.; Gordeliy, V. Acta Phys. SloV. 1998, 48, 509-533.
thickness of a phospholipid leaflet (∼20 Å) and A
m
7
2
-1
-
1
memb
-1
plot in Figure 3 (6200 M ) we find that K2
is 100 M ,
4596 J. AM. CHEM. SOC.
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VOL. 125, NO. 15, 2003

Lipid Bilayer Membrane Surfaces
A R T I C L E S
Figure 6. Titration curves showing the decrease in the fluorescence of
receptor 2 in vesicles with a receptor-to-phospholipid ratio of (blue) 0.2
mol % and (purple) 2.5 mol % upon the addition of copper(II). The dashed
black line (- -) represents the titration curve constructed from the binding
constants of copper(II) to the headgroup 1 in 4% MES buffer pH 6 in
methanol.
Figure 5. Speciation plot showing the amounts of the Cu2 (green), Cu22
(blue), Cu23 (purple), Cu24 (red) complexes as fractions of all bound species,
as a function of the logarithm of the mole percent of receptor in the
membrane ø. The copper concentration is 0.1 mM and the concentration of
receptor 2 is 0.2 mM.
(
Figure 2). The stoichiometry of the complex is strongly
-
1
K2 (7000 M ), at which point inter-vesicular binding should
dependent on the membrane concentration of the receptor. The
speciation plot in Figure 5 shows that at a copper(II) concentra-
tion of 0.1 mM, a membrane concentration of 1 mol % gives
largely the Cu22 species, whereas 7.5 mol % gives the Cu24
species preferentially. The Cu23 species is never formed to any
significant extent, and in the absence of any structural informa-
tion, we prefer not to speculate as to the reasons.
At high mole percentages, species are formed that cannot be
observed in solution; this is simply because much higher local
concentrations of receptor are achieved in the membrane.
Conversely, at low mole percentages of receptor, aggregation
of the receptor is inhibited by localization in the membrane.
This is a straightforward implication of eq 4 and is demonstrated
by the experimental data in Figure 3. At membrane receptor
loadings above 1.1 mol %, positive cooperativity is observed
obs
maintain K2 ) K2. The fact that we observe inhibition of
obs
binding (K2 < K2) at low receptor loadings implies that there
are steric effects that prevent vesicle-vesicle interactions in this
system.
Therefore, both the binding strength and the type of complex
formed are highly dependent on the membrane concentration
of the receptor (Figure 7). This shows that by controlling the
concentration of receptor in its membrane a cell could control
both the strength of binding to a messenger, the type of cluster
formed, and interactions with other cells. Since both chemotaxis
and the immune response are sensitive to clusters of a particular
size, this effect gives the cell an extra degree of control over
the response to an external messenger molecule. For example,
overexpression of a receptor in a membrane could result in a
decrease in the cellular response by decreasing the number of
smaller active clusters in favor of larger inactive clusters.
obs
-1
since K2 is greater than 7000 M , the corresponding solution
value for 1 in 4% buffer in methanol. However, at loadings
less than 1.1 mol %, K2 is less than K2 and the system shows
obs
Conclusions
negative cooperativity. This negative cooperativity is highlighted
in Figure 6 which shows the titration data for 2 at membrane
loadings of 0.2 and 2.5 mol % compared with 1 in solution of
similar polarity.
Cooperativity during membrane binding events has only been
studied quantitatively in a few other systems. The effect of
changing the surface concentration of ligand on the formation
of the 1:2 complex between chloroeremomycin dimer and its
Thus at high receptor loadings, binding at the membrane is
more favorable than in solution, resulting in intra-vesicle
aggregation of the receptors. Conversely, at low receptor
loadings interactions within the membrane are less favorable
than interactions with species in solution, and so we might
expect inter-vesicle interactions to dominate. However, if inter-
vesicle interactions dominated at low loadings, then as the
vesicle-bound ligand, N-docosanoyl-Gly-Ala-D-γ-Glu-Lys(N-
obs
ꢀ-acetyl)-D-Ala-D-lactate, has been studied. The values of K2
were much larger than the equivalent process in solution, the
binding of chloroeremomycin to the N-acetyl-Gly-Ala-D-γ-Glu-
Lys(N-ꢀ-acetyl)-D-Ala-D-lactate, suggesting a large degree of
7(a)
obs
cooperativity in this system.
Although several values of K2
were obtained at different concentrations of phospholipid, the
obs
receptor loading decreased, K2 would decrease until it reached
memb
membrane binding constant K2
was not determined. A two-
(
19) Although this treatment ignores factors such as changes in surface charge,
this has been approximately calculated for 1 mol/mol % receptor and is a
minor effect. Data from Gennis, R. B. Biomembranes: Molecular Structure
and Function; Springer-Verlag: London, 1989.
dimensional binding constant was directly determined for the
bivalent binding of IgM to its hapten immobilized in EYPC
monolayers, but K2 in solution was not determined, so no
J. AM. CHEM. SOC.
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VOL. 125, NO. 15, 2003 4597

A R T I C L E S
Doyle et al.
The use of the dansyl fluorophore in our system has allowed
us to quantify the effect of the membrane environment on
binding, revealing that this is the dominant factor in determining
the strength of the receptor-ligand binding. Hence, we were
able to use a suitable control system to accurately quantify the
effect of membrane concentration on intra-membrane inter-
actions.
Receptor 2 bound in the membrane of phosphatidylcholine
vesicles formed Cu2, Cu22, and Cu24 complexes with copper-
(II). The affinity of 2 for copper(II) increased dramatically with
an increase in the membrane concentration of 2. In addition,
the nature of the complex formed is highly dependent on the
membrane concentration of 2. At membrane loadings below 2
mol % the Cu22 complex was the major species, as in the
solution binding experiments. However, at very low loadings
of receptor (<0.75 mol %), the intra-membrane binding
interactions were inhibited relative to interactions in solution,
and Cu2 was the dominant complex. In effect, under these
conditions the receptors are kept apart despite localization in
the membrane. The behavior of this system illustrates how cells
might control the production of a particular type of ligand-
receptor cluster by either increasing or decreasing the amount
of receptor expressed in the membrane. Receptor aggregation
gives a cell a more versatile mechanism to control its response
to external messengers compared to other signaling mechanisms.
Experimental Section
NMR spectra were recorded on Bruker AC 250 or AMX 400
spectrometers, UV-vis spectra on a Varian Cary 1 Bio spectropho-
tometer and fluorescence spectra on a Hitachi F-4500 fluorescence
spectrophotometer. ES , CI , and FAB (using an m-nitrobenzyl
alcohol matrix) mass spectra were obtained on Micromass Prospec and
Micromass Platform spectrometers, and MALDI-TOF mass spectra
were recorded on a Bruker Reflex III MALDI-TOF mass spectrometer.
Figure 7. Tuning the concentration of receptor in the membrane can be
used to control the identity of the major complex formed between copper-
+
+
+
(II) and receptor 2. As the membrane concentration of receptor increases,
the size of the complex increases. At low membrane concentrations (top),
intra-vesicular binding is inhibited and inter-vesicular binding should
predominate, though this was precluded in our system due to steric
constraints.
Column chromatography was carried out on 60 mesh silica gel. Water
was doubly distilled before use. Dansylamide 3 was used as purchased
from Aldrich.
comparison could be drawn between solution and membrane
Preparation of Dansyl Ethylenediamine, 1.10 A solution of dansyl
chloride (200 mg, 0.74 mmol) in dichloromethane (6 mL) was dropped
into 1,2-ethylenediamine (6.5 mL, 445 mg, and 7.42 mmol) while
stirring and cooling over ice. The mixture was stirred while warming
to room temperature for 1 h. The reaction mixture was acidified with
dilute HCl and then extracted with dichloromethane (2 × 20 mL). The
aqueous layer was made basic (pH 9) using 10 M NaOH and again
extracted with dichloromethane (2 × 20 mL). The organic layer was
7
b
binding events.
These and other such studies of the binding of multivalent
ligands at vesicular and cellular interfaces do not take into
account the difference in environment between the membrane
interface and bulk aqueous solution. Often K1 for binding to a
membrane-bound receptor is assumed to be the same as K1 for
binding to the receptor in aqueous solution.7
a,16
In fact, changes
4
dried over MgSO , filtered through a sinter and the solvent removed
in polarity and the hydration sphere around species embedded
in membranes are known to be important for many binding
processes, such as the binding of antibodies to surface-bound
haptens or the binding of antibiotics to bacterial cell wall
analogues.20 A large effect has been observed for the antibiotic
teichoplanin A3-1. This antibiotic, which does not insert into
vesicles, binds tightly to the vesicle-bound ligand (N-docosanoyl-
Lys(N-ꢀ-acetyl)-D-Ala-D-lactate) to form a 1:1 complex with
under reduced pressure to give (2-aminoethyl)-dansylamide as a yellow
1
solid (200 mg, 92%). H NMR: (CDCl
Hz, 2H; NHCH CH NH ), 2.80 (s, 6H; N(CH
CH NH ), 7.19 (d, J ) 7.6 Hz, 1H; 6-CH-dansyl), 7.52 (m, 2H; 7-CH-
dansyl and 3-CH-dansyl), 8.24 (m, 2H; 8-CH-dansyl and 4-CH-dansyl),
3
, 250 MHz); δ 2.63 (t, J ) 5.8
2
2
2
3
)
2
and m, 2H; NHCH
2
-
2
2
+
+
8
.47 (d, J ) 8.2 Hz, 1H; 2-CH-dansyl). MS (ES ): m/z 294 (MH );
1
Anal. Calcd. for C14
H
19
N
3
O S
2 1
2 2
‚ / H O: C, 55.61; H, 6.67; N, 13.90;
S, 10.60. Found: C, 55.72; H, 6.40; N, 13.77; S, 10.36.
4
-1
Preparation of Cholesteryl Bromoacetate.21 Cholesterol (383 mg,
1 mmol) was dissolved in dry THF (5 mL). Bromoacetyl chloride (250
µL, 3 mmol) was added dropwise and the reaction mixture was stirred
at reflux for 1.5 h. The solution was concentrated to an oil under reduced
pressure, and the residue recrystallized from ethyl acetate to give the
a binding constant of 6 × 10 M , whereas binding to the water
soluble derivative (N-acetyl-Lys(N-ꢀ-acetyl)-D-Ala-D-lactate)
-
1 7c
was too weak to be observed (<10 M ). Therefore large
increases in binding strength can result exclusively due to the
effect of the membrane environment on binding, which can lead
to overestimation of the degree of cooperativity.
product as a white powder (337 mg, 0.67 mmol, 67%). mp 160-162
1
°
C; H NMR (CDCl3, 250 MHz): δ 0.6-2.25 (m, 43H, cholesterol
(
20) Leckband, D. E.; Kuhl, T.; Wang, H. K.; Herron, J.; Muller, W.; Ringsdorf,
H. Biochemistry 1995, 34, 11467-11478.
(21) O’Connor, G. L.; Nace, H. R. J. Am. Chem. Soc. 1953, 75, 2118.
4598 J. AM. CHEM. SOC.
9
VOL. 125, NO. 15, 2003

Lipid Bilayer Membrane Surfaces
A R T I C L E S
protons), 3.8 (s, 2H, CH
H, 3.9 Hz 6-CH-cholesterol). MS (CI ) m/z 524 (M+NH
Preparation of 3-O-(2-(2-Aminoethyl)dansylamide)acetyl)cho-
lesteryl Ester, 2. Dansyl ethylenediamine 1 (21.9 mg, 74.6 µmol),
cholesteryl bromoacetate (34 mg, 67.8 µmol), and sodium carbonate
2
Br), 4.6 (m, 1H, 3-CH-cholesterol), 5.4 (d,
multilamellar vesicles. These were extruded through a single 800 nm
polycarbonate membrane using an Avestin Liposofast extrusion ap-
paratus to give unilamellar vesicles. Vesicle size was characterized by
static light scattering and membrane integrity confirmed through
carboxyfluorescein encapsulation.
+
+
4
).
1
(
18 mg, 169.5 µmol) were suspended in ca. 5 mL of acetonitrile. The
Calculation of Membrane Environment. (a) Calibration Curve.
reaction was stirred at reflux overnight. The resulting solution was
concentrated, dissolved in chloroform, filtered, and concentrated again.
The crude product was purified by column chromatography on silica
-8
Monomer probes 1 or 3 (2 × 10 mol) were transferred from stock
-4
solution (l00 µL from 1 × 10 M solution in THF) into each of eight
1
0 mL volumetric flasks. Solutions were made up to the 10 mL mark
(chloroform/ ethyl acetate, 4:1). The solvent was removed from the
using dichloromethane, THF, n-octanol, n-butanol, ethanol, methanol,
product containing fractions under reduced pressure and the residue
recrystallized by slow evaporation of the solvent from dichloromethane/
hexanes to give the product as a pale green solid (42 mg, 58 µmol,
-6
water, and MES buffer (pH 6) to give 2 × 10 M solutions. The
fluorescence emission spectrum of each solution was measured at 25
°
C. A calibration plot was constructed using λmax of emission versus
the dielectric constant of each of the solvents.
b) Measurements in Vesicles. Aliquots of the 1 × 10 M vesicular
stock solutions (200 µL) were diluted 100 fold using MES buffer (pH
). The fluorescence emission spectrum of each of these solutions was
1
8
6%). H NMR (CDCl
3
, 250 MHz): δ 0.6-2.25 (m, 43H, cholesterol
), 2.6 (s +t, 8H, N(CH
), 4.5 (m, 1H, 3-CH-cholesterol), 5.6
d, 1H, 6-CH-cholesterol), 7.1 (d, 1H, J ) 7.3 Hz, 1H, 6-CH-dansyl),
protons), 2.5 (t, 2H, ethylamine CH
ethylamine CH ), 3.0 (s, 2H, 2H
(
2
3
)
2
,
-4
(
2
c
6
7
8
2
7
.5 (m, 2H, 3- and 7-CH-dansyl), 8.2 (d, 1H, J ) 7.3 Hz, 8-CH-dansyl),
then recorded at 25 °C. The results were interpolated into the calibration
plot.
.3 (d, 1H, J ) 8.6 Hz, 1H, 4-CH-dansyl) 8.5 (d, 1H, J ) 8.2 Hz, 1H,
+
+
-CH-dansyl). MS (FAB ) m/z: found 720.475077; calcd for MH ,
20.477405. Anal. Calcd. for C43 S.C 14‚0.5H O: C, 72.20;
Titrations with Copper(II) Chloride. Aliquots of a 5 mM copper-
II) chloride solution dissolved in MES buffer (20 mM pH 6.0) were
added to the appropriate solutions. The fluorescence spectrum was
recorded after each addition. The stoichiometry and binding affinity
was determined by using an iterative curve-fitting program to fit the
decrease in emission intensity with increasing copper(II) concentration.
H N
64 3
O
4
6
H
2
(
H;9.90; N; 5.15; S, 3.95. Found: C, 72.0; H, 9.60; N, 4.95; S, 3.85.
Analysis by HPLC (silica, eluant: 9:1 PrOH:petrol with 0.1%
triethylamine, retention time 16.6 min) showed >98% purity.
Synthesis of Vesicles. We used egg yolk phosphatidylcholine
i
(EYPC, used as purchased from Sigma (type XVI from fresh egg yolk))
to make our vesicles as bilayers constructed from EYPC are in a fluid
2
2
Acknowledgment. This work was supported by the BBSRC
Grant Ref. B50/08373), EPSRC, and the Royal Society.
state at temperatures above -7°. Using phosphatidylcholines rather
than ethanolamines avoided complications arising from competitive
complexation to copper(II) by the phospholipid. The EYPC phospho-
lipid bilayers, though impermeable to fluorescein on the time scale of
the binding experiments, were permeable to copper(II) ions.23 This
eliminated the need to differentiate between binding to receptors on
the interior of the vesicle and receptors on the exterior of the vesicle.
Unilamellar vesicles were prepared by dissolving egg yolk phos-
phatidylcholine (64 mg, 320 mg for vesicles 0.2 mol % in the receptor)
and the required amount of 2 (0.2, 0.5, 1, 2.5, 5, and 7.5 mol %) in
spectroscopic grade ethanol-free chloroform (20 mL), followed by
removal of the solvent to give a thin film of phospholipid on the interior
of the flask. The buffer was added (MES 50 mM pH 6.0) to the flask,
and the thin film detached by vortex mixing to give a suspension of
(
Supporting Information Available: Details of experiments
to demonstrate membrane permeability to copper(II) and UV-
vis titration of recptors with copper(II). Tables detailing observed
binding constants for receptor 2 to copper(II) in different
concentrations of phospholipid, fluorescence emission λmax for
dansylamide and headgroup 1 in different solvents, fluorescence
emission λmax for dansylamide in mixtures of water and
methanol, fluorescence data for the titration of 1 in 4% MES
buffer in methanol with copper(II), fluorescence data for the
titration of vesicles containing 2 with copper(II), and values
used to calculate the curve fits. This material is available free
of charge via the Internet at http://pubs.acs.org.
(
22) Far ´ı as, R. N.; Chehin, R. N.; Rintoul, M. R.; Morero, R. D. J. Membr.
Biol. 1995, 143, 135-141.
(
23) Copper(II) ions rapidly diffused into vesicles containing the ion-sensitive
dye xylenol orange; see supplementary information.
JA021048A
J. AM. CHEM. SOC.
9
VOL. 125, NO. 15, 2003 4599