Báo cáo khoa học: Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Ab1)40 peptide in solid-supported lipid bilayers ppt

Effects of sphingomyelin, cholesterol and zinc ions on the
binding, insertion and aggregation of the amyloid Ab
1)40
peptide in solid-supported lipid bilayers
Savitha Devanathan
1
, Zdzislaw Salamon
1
,Go
¨
ran Lindblom
1
, Gerhard Gro
¨
bner
2
and Gordon Tollin
1
1 Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ, USA
2 Department of Biophysical Chemistry, Umea
˚
University, Sweden
The 39–42 amino acid residue amyloid b peptide (Ab)
is a seminal etiologic factor in Alzheimer’s disease
(AD), a member of the large family of neurodegenera-
tive disorders with a common pathology in the form
of aberrant protein folding [1–4]. The unifying theme
for all of these amyloidogenic diseases is the pathologi-
cal conversion of specific proteins into toxic assem-
blies. In the case of AD, its key substance, Ab peptide,
is released as a soluble monomer, but seems to require

a minimal level of aggregation to exert its neurotoxic
action [3–9]. In particular, soluble oligomeric and pro-
tofibrillar Ab structures are the primary toxic agents
Keywords
Alzheimer’s disease; amyloid toxicity;
microdomains; plasmon-waveguide
resonance spectroscopy; rafts
Correspondence
G. Tollin, Department of Biochemistry and
Molecular Biophysics, University of Arizona,
Tucson, AZ 85721, USA
Fax: +1 520 621 9288
Tel: +1 520 621 3447
E-mail:
(Received 8 December 2005, revised 25
January 2006, accepted 2 February 2006)
doi:10.1111/j.1742-4658.2006.05162.x
We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow
the effects of sphingomyelin, cholesterol and zinc ions on the binding and
aggregation of the amyloid b peptide
1)40
in lipid bilayers. With a dioleoyl-
phosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no
aggregation occurred over a period of 15 h. In contrast, similar binding
was found with a brain sphingomyelin (SM) bilayer, but in this case an
exponential aggregation process was observed during the same time inter-
val. When the SM bilayer included 35% cholesterol, an increase of
 2.5-fold occurred in the amount of peptide bound, with a similar increase
in the extent of aggregation, the latter resulting in decreases in the bilayer
packing density and displacement of lipid. Peptide association with a bilay-

er formed from equimolar amounts of DOPC, SM and cholesterol was fol-
lowed using a high-resolution PWR sensor that allowed microdomains to
be observed. Biphasic binding to both domains occurred, but predomin-
antly to the SM-rich domain, initially to the surface and at higher peptide
concentrations within the interior of the bilayer. Again, aggregation was
observed and occurred within both microdomains, resulting in lipid dis-
placement. We attribute the aggregation in the DOPC-enriched domain to
be a consequence of lipid mixing within these microdomains, resulting in
the presence of small amounts of SM and cholesterol in the DOPC micro-
domain. When 1 mm zinc was present, an increase of approximately three-
fold in the amount of peptide association was observed, as well as large
changes in mass and bilayer structure as a consequence of peptide aggrega-
tion, occurring without loss of bilayer integrity. A structural interpretation
of peptide interaction with the bilayer is presented based on the results of
simulation analysis of the PWR spectra.
Abbreviations
Ab, amyloid b
1)40
peptide; AD, Alzheimer’s disease; AFM, atomic force microscopy; DOPC, dioleoylphosphatidylcholine; POPC,
palmitoyloleoylphosphatidylcholine; PWR, plasmon-waveguide resonance; SM, brain sphingomyelin; TFA, trifluoroacetic acid; TFE,
trifluoroethanol.
FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1389
currently associated with the neuropathological events
occurring in patients with AD [3,7–9]. Nevertheless,
globular and nonfibrillar Ab peptides are continuously
released during normal metabolism in healthy people,
with no problems observed, and therefore fundamental
questions behind the toxic mechanism in AD are
unsolved [10–12]. Recently, the discovery of various
soluble amyloid oligomers having a common structure,

independent of their location, has brought new insight
into possible mechanisms of toxicity [10]. The inhibi-
tion of their toxicity by a common oligomer-specific
antibody, connected to cell parts that are accessible by
extra- and intracellular regions, has pointed strongly
to cell membranes as a potential prime target [9,10,13–
15]. This is not surprising because Ab has inherited a
transmembrane domain from its precursor protein (a
highly conserved integral membrane protein with a
single transmembrane domain), providing it with an
amphipathic nature, which makes it an ideal target for
toxic events associated with neuronal membranes
[5,13,16–22]. The effects of Ab on membranes and
lipid systems, and their possible roles in neurotoxicity,
include changes in membrane fluidity, leading to
membrane depolarization and disorder [23], mem-
brane-mediated aggregation of Ab triggering neuronal
apoptotic cell death [24], lipid peroxidation via H
2
O
2
produced by Cu
2+
reduction by Ab [22,25], and even
the formation of calcium-permeable membrane ion
channels [26].
Owing to the amphipatic nature of Ab, a second
process plays a potential key role in AD, namely the
enhancing effects of specific neuronal membranes on
Ab peptide conversion into toxic-acting oligomers.

Various lipid membranes have been shown to induce
an electrostatically driven surface accumulation, fol-
lowed by dramatically increased misfolding of Ab,at
rates much higher than in a membrane-free environ-
ment [13–23,25–28]. Membrane components, such as
anionic lipids, gangliosides or cholesterol, were shown
to be involved in various stages of Ab aggregation,
and raft-like neuronal membranes seem to play a signi-
ficant role in the regulation of Ab-production and its
cytotoxic products [20–23,29,30]. Interestingly, brain
lipid composition in patients with AD is significantly
altered, suggesting a link between lipid composition
and increased susceptibility to neuronal cell death
[16,31,32]. Because of its amphipathic nature and the
fact that patients with AD have altered neuronal lipid
compositions [16,17], in the present study we investi-
gated the role of raft-mimicking model neuronal
membranes on the behavior of Ab peptide. Using plas-
mon-waveguide resonance (PWR) spectroscopy [33,34]
we elucidated features of the peptide–membrane inter-
action, which might be important for raft membrane-
dependent aggregation and neurotoxic action, in par-
ticular the presence of sphingomyelin, cholesterol and
zinc ions.
Results and Discussion
In order to characterize the interaction of Ab with lipid
membranes, we used PWR spectroscopy to study the
association with bilayers composed of single lipids, of
binary lipid mixtures, or of a ternary mixture composed
of dioleoylphosphatidylcholine (DOPC) ⁄ sphingomyelin

(SM) ⁄ cholesterol (1 : 1 : 1 mole ratio), the latter in the
presence and absence of added zinc ions. This methodo-
logy has previously been used in our laboratory to
characterize the composition of, the formation of, and
insertion into, microdomains in bilayer membranes
formed from binary mixtures of DOPC ⁄ SM and palm-
itoyloleoylphosphatidylcholine (POPC) ⁄ SM, both in
1 : 1 mole ratios [35,36].
Interaction of Ab with single lipid bilayers
Figure 1A,B shows that for a DOPC bilayer, interac-
tion with increasing Ab concentrations produced small
shifts to higher-incident angles in p-polarized and
s-polarized spectra (11 and 5 mdeg shifts, respectively,
at a peptide concentration of 5 lm in the aqueous cell
compartment; s-polarized data not shown). The spec-
tral shifts can be ascribed to an overall mass increase
in the membrane as a result of peptide association with
the bilayer. These shifts followed a single hyperbolic
curve with an apparent dissociation constant (K
D
)of
0.16 ± 0.02 lm (Fig. 1B; Table 1). No further spectral
changes were observed with time (over a time period
of  15 h), indicating that no peptide aggregation
occurred during this interval.
The addition of Ab to a lipid bilayer containing only
SM again resulted in increasing spectral shifts to higher
incident angles at peptide concentrations up to 5 lm
(Fig. 2A,B) for both p- and s-polarized spectra (17 ver-
sus 14 mdeg, respectively; s-polarized data not shown).

This is similar to what was observed for the DOPC
bilayer, and occurred with a similar binding affinity
(Fig. 2B; Table 1). However, in the SM bilayer, after
peptide binding and upon further equilibration with
time (up to  15 h), a slow progressive increase in spec-
tral position to higher-incident angles was observed.
These changes, which we attribute to peptide aggrega-
tion, are plotted in Fig. 2C; they occurred exponentially
with a half-time of  4.6 h (Table 2). We have obtained
similar results to these with bilayers composed of
DOPC and SM in a 1 : 1 mole ratio (data not shown).
Factors affecting Ab binding in lipid bilayers S. Devanathan et al.
1390 FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS
Interaction of Ab with binary lipid bilayers
As shown by the data in Fig. 3, and by the K
D
values
in Table 1, Ab was bound fivefold more tightly to an
SM bilayer containing cholesterol (1 : 0.35 mole ratio)
and resulted in an approximately twofold larger mag-
nitude in the spectral shifts obtained at 5 lm peptide
concentration (40 mdeg for p-polarization versus 27
mdeg for s-polarization; s-polarized data not shown).
Again, time-dependent aggregation of the peptide
occurred, with a half-time of  4.2 h (Fig. 3C;
Table 2). However, in this case the spectral changes
involved shifts towards lower-incident angles (for
p-polarization, )23 mdeg and for s-polarization,
)9 mdeg; s-polarized data not shown), contrary to that
observed with the SM bilayer in the absence of choles-

terol. A spectral shift to smaller angles is caused by a
decrease in refractive index. As this parameter is pro-
portional to mass density, we attribute this shift to a
net loss in mass resulting from the removal of lipid
molecules from the bilayer and transfer to the Gibbs
border, which occurs upon peptide insertion into the
bilayer and aggregation leading to lipid displacement
(see below for further discussion). This contrasts with
the process of peptide interaction and aggregation with
the SM bilayer in the absence of cholesterol, where a
net mass increase was observed as a result of peptide
accumulation and possibly also lipid recruitment from
the Gibbs border.
Interaction of Ab with ternary lipid bilayers
For a ternary mixture composed of DOPC ⁄ SM ⁄ choles-
terol (1 : 1 : 1 mole ratio), and using a PWR resonator
design with a higher resolution that enabled the obser-
vation of membrane microdomains [35], two resonances
were obtained corresponding to less-ordered thinner
domains at lower-incident angles (DOPC enriched),
and more ordered and more densely packed thicker
microdomains (SM enriched) occurring at higher-inci-
dent angles (Fig. 4A; cf. ref. 35 for evidence supporting
this assertion). In this case, the Ab-binding process was
biphasic (the initial phase is shown in the inset to
Fig. 4B), with an initial positive shift followed by a neg-
ative shift as the peptide concentration was increased,
and occurred preferentially into the SM-rich microdo-
main. The latter is shown by the larger shift observed
for the resonance corresponding to this domain than

for the DOPC-enriched domain (compare Fig. 4B,C).
Table 1. Amyloid b
1)40
peptide (Ab) binding affinities (p-polariza-
tion). DOPC, dioleoylphosphatidylcholine; SM, sphingomyelin.
Bilayer K
D
(lM)
Resonance
shifts (mdeg)
a
DOPC 0.160 ± 0.02 11 ± 1
SM 0.220 ± 0.03 17 ± 1
SM ⁄ cholesterol 0.043 ± 0.01 40 ± 2
DOPC ⁄ SM ⁄ cholesterol
DOPC-rich domain 0.370 ± 0.02 )6±2
SM-rich domain 0.004 ± 0.001 (K
D1
)3±1
0.110 ± 0.01 (K
D2
) )15 ± 2
DOPC ⁄ SM ⁄ cholesterol (+ 1 m
M Zn)
DOPC-rich domain 0.450 ± 0.09 )20 ± 2
SM-rich domain 0.003 ± 0.001 (K
D1
)20±2
0.022 ± 0.001 (K
D2

) )32 ± 1
a
Extrapolated to infinite peptide concentration.
63.5 63.6 63.7 63.8
0.0
0.2
0.4
0.6
2
1
Reflectance
Incident angle (deg)
A
012345
0
5
10
15
B
Resonance shifts (mdeg)
Aβ (µM)
Fig. 1. Binding of amyloid b
1)40
peptide (Ab) to a dioleoylphosphat-
idylcholine (DOPC) bilayer. (A) Plasmon-waveguide resonance
(PWR) spectra for p-polarization are shown for a solid-supported
DOPC bilayer before (curve 1) and after (curve 2; squares) the addi-
tion of Ab (5 l
M bulk concentration in the aqueous cell compart-
ment). The buffer used in the sample compartment was 10 m

M
Tris (pH 7.4). Experiments were performed at 25 ± 0.1 °C. (B) Plot
of p-polarized PWR resonance minimum spectral shifts as a func-
tion of the concentration of Ab added to the PWR sample compart-
ment. The binding data were fit by a single hyperbola (solid line),
and the binding affinity values and the magnitude of the spectral
shift are given in Table 1.
S. Devanathan et al. Factors affecting Ab binding in lipid bilayers
FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1391
It is worth noting that accumulation of peptide at the
microdomain surface, rather than insertion into its
interior, is also possible. We attribute the initial positive
shift to a mass increase resulting from peptide binding
to the bilayer surface, and the subsequent negative shift
to peptide insertion into the bilayer and lipid displace-
ment. The higher spectral resolution allowed us to
observe both of these processes in this experiment. Con-
sistent with the larger spectral shifts for the SM-rich
microdomain, and thus a higher peptide concentration,
the binding affinity of the peptide for this domain was
approximately fourfold larger than for the DOPC-
enriched domain (Table 1). Note that a shift to lower-
incident angles occurred for both microdomains, again
indicating insertion of peptide within the bilayer, pro-
ducing a less densely packed bilayer as a result of
expulsion of lipid molecules. That this occurred in both
microdomains is probably a consequence of the fact
that during microdomain formation a small amount of
SM and cholesterol is mixed into the DOPC portion of
the bilayer, and a small amount of DOPC and choles-

terol is mixed into the SM-enriched microdomain [35].
Peptide aggregation also occurred in this system, and
within the first 3 h the spectral changes occurred pre-
dominantly in the SM-enriched region (Fig. 4A, curve
3), as evidenced by the smaller spectral shift that
occurred in the DOPC-enriched domain. Figure 4D
shows the time course of resonance minimum shifts
upon peptide aggregation. At 15 h, the magnitude of
the spectral changes was approximately twice as large
for the SM-rich microdomain. In the DOPC-rich
microdomain, the half-time was 4.5 h, whereas it was
slightly shorter in the SM-rich domain (Table 2).
0246
0
10
20
Resonance shifts (mdeg)
Aβ (µM)
B
63.6 64.0 64.4
0.0
0.2
0.4
0.6
0.8
3
2
1
Reflectance
Incident angle (deg)

A
0 5 10 15
15
30
45
C
Resonance shifts (mdeg)
Time (h)
Fig. 2. Binding and aggregation of Ab in a
sphingomyelin (SM) bilayer. (A) p-Polarized
plasmon-waveguide resonance (PWR) spec-
tra are shown for an SM bilayer before
(curve 1) and after (curve 2; circles) the addi-
tion of Ab (5 l
M bulk concentration in the
aqueous cell compartment). Curve 3 (trian-
gles) shows the spectrum after equilibration
for 15 h. Other conditions were as in Fig. 1.
(B) Plot of PWR spectral shifts induced by
Ab binding to the bilayer with an increasing
concentration of added peptide. The data
were fit by a single hyperbola, with the
binding constant and total spectral shift
given in Table 1. (C) Plot of the time course
of spectral changes for p-polarization associ-
ated with peptide aggregation. A single
exponential fit to the data is shown (solid
line) with a half-time and total spectral shift
as presented in Table 2.
Table 2. Aggregation kinetics (p-polarization). DOPC, dioleoylphos-

phatidylcholine; SM, sphingomyelin.
Bilayer
Half-times
(h)
Resonance
shifts (mdeg)
a
DOPC – –
SM 4.6 ± 0.4 46 ± 1
SM ⁄ cholesterol 4.2 ± 0.3 65 ± 3
DOPC ⁄ SM ⁄ cholesterol
DOPC-rich domain 4.5 ± 0.5 )30 ± 5
SM-rich domain 3.7 ± 0.2 )58 ± 1
DOPC ⁄ SM ⁄ cholesterol
(+ 1 m
M Zn)
t
1
¼ 0.53 ± 0.06 )15 ± 3
t
2
¼ 2.96 ± 0.12 194 ± 5
t
3
¼ 17.9 ± 0.51 58 ± 3
a
Extrapolated to infinite time.
Factors affecting Ab binding in lipid bilayers S. Devanathan et al.
1392 FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS
Effect of Zn

2+
on Ab interaction with a ternary
lipid bilayer
The binding of metal ions, such as Zn
2+
,toAb pep-
tides has been shown to facilitate peptide penetration
of the hydrocarbon core of the membrane and subse-
quent aggregation [22,37]. In order to test the effect of
zinc addition in the present experiments on peptide
binding and bilayer structural changes caused by
aggregation, we used a DOPC ⁄ SM ⁄ cholesterol (1 : 1 : 1
mole ratio) bilayer in contact with a buffer containing
1mm Zn
2+
, present on both sides of the bilayer. The
spectral changes produced as a result of peptide bind-
ing and aggregation in the presence of zinc are shown
in Fig. 5A,B. Control experiments showed that no
PWR spectral changes occurred as a result of Zn
2+
interaction with the lipid bilayer in the absence of pep-
tide. Figure 5A shows the spectral changes for p-polar-
ization at various early time-points up to 200 min after
the addition of 5 lm peptide. The binding and aggre-
gation process was observed to follow biphasic kinet-
ics, with an initial shift to lower-incident angles that
occurred in  30 min, followed at later time-points by
a shift to higher angles accompanied by a decrease in
spectral amplitude for the lower-incident angle reson-

ance (owing to the DOPC-rich domain), and an
increase in amplitude and a large shift to higher angles
for the resonance associated with the SM-rich domain
(Fig. 5A,B). These results suggest that a major change
in bilayer structure occurred as a result of peptide
insertion into the bilayer and aggregation. The spectral
changes are consistent with a high degree of mass
accumulation occurring predominantly within the
SM-rich microdomain. However, it is important to
note that the bilayer remained intact (i.e. no large
holes were formed that exposed resonator surface to
the aqueous phase). This is evidenced by the fact that
no resonances were observed corresponding to the bare
prism surface in direct contact with the aqueous
medium.
The binding isotherms for peptide association with
the bilayer are shown in Fig. 5C,D, with the binding
constants given in Table 1. Again, the initial binding
process occurred with high affinity (inset to Fig. 5C),
similar to that observed in the absence of zinc. How-
ever, in this case the second phase of binding had a
fivefold higher affinity than in the absence of zinc
(Fig. 5C; Table 1). The peptide interacted with the
68.0 68.5
0.4
0.6
0.8
A
3
2

1
Reflectance
Incident angle (deg)
B
012345
0
10
20
30
40
Resonance shifts (mdeg)
Aβ (µM)
0481216
-20
0
20
40
C
Resonance shifts (mdeg)
Time (h)
Fig. 3. Binding and aggregation of Ab in a
SM ⁄ cholesterol (1 : 0.35) bilayer. (A) p-Polar-
ized PWR spectra are shown for a bilayer
before (curve 1) and after (curve 2; squares)
Ab was added to the sample cell (5 l
M bulk
concentration in the aqueous compartment).
Curve 3 (triangles) shows the spectrum
after equilibration for 15 h. Other conditions
were as in Fig. 1. (B) Plot of the spectral

shifts associated with binding of the peptide
to the bilayer as a function of added Ab con-
centration. The binding constant was
obtained by a hyperbolic fit to the data (solid
line) with a K
D
value and total spectral shift
as given in Table 1. (C) Plot of the time
course for p-polarization spectral changes
associated with peptide aggregation. The
data were fit with a single exponential (solid
line), and the half-time and total spectral
shift are given in Table 2.
S. Devanathan et al. Factors affecting Ab binding in lipid bilayers
FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1393
DOPC-rich domain with a single K
D
value 20-fold
weaker than with the SM-rich domain, and with a
smaller spectral shift (Fig. 5D).
The kinetics of the spectral changes are shown in
Fig. 6, and the half-times obtained by curve fitting are
given in Table 2. These reveal at least three kinetic
phases: two at early time-points with half-time values
of 0.53 ± 0.06 h and 2.96 ± 0.12 h; and a third,
slower, phase corresponding to a half-time of
17.9 ± 0.5 h. Thus, the mechanism by which aggrega-
tion occurs is quite complex in this system, and further
studies will be necessary to obtain mechanistic insights.
These results are consistent with the reported effect of

zinc on the formation of a helical peptide structure that
facilitates insertion and pore formation as a result of
aggregation [22,37,38]. Furthermore, the atomic force
microscopy (AFM) studies of Lin et al. [39], on solid-
supported bilayers, have shown that the interaction of
Ab results in the formation of conducting ion channels
that have rectangular or hexagonal shapes with four or
six subunits, are 80–120 A
˚
in diameter and protrude
 10 A
˚
from the bilayer surface. The large PWR spec-
tral changes shown in Fig. 5 are consistent with struc-
tures of this magnitude inserted into the bilayer.
Fig. 4. Binding and aggregation of Ab in a DOPC ⁄ SM ⁄ cholesterol (1 : 1 : 1) bilayer. Spectra were obtained using a high-resolution sensor.
Other experimental conditions are as given in the legend to Fig. 1. (A) p-Polarized PWR spectra are shown for the membrane before (curve
1) and after (curve 2; squares) addition of the peptide (5 l
M bulk concentration in the aqueous cell compartment). Based on previous results
[35], the resonance at smaller-incident angles is ascribed to a DOPC-enriched microdomain, and the resonance at larger incident angles to
an SM-enriched microdomain. The initial addition of peptide resulted in binding to both microdomains, but to a larger extent in the SM-rich
domain. Spectra are also shown after the sample had equilibrated for 3 h (curve 3; circles) and 15 h (curve 4; triangles). These changes are
ascribed to peptide aggregation. (B,C) Plots of initial peptide binding, resulting in shifts to larger angles (inset to B) and binding at higher con-
centrations resulting in shifts to smaller angles. Binding to the SM-rich domain is shown in (B) and to the DOPC-rich domain in (C). Data
were fit with three hyperbolic curves (solid lines), yielding the affinity constants and spectral shifts in Table 1. (D) Plots of the time course of
spectral changes for the DOPC-rich microdomain (open triangles) and the SM-rich microdomain (closed triangles) associated with peptide
aggregation. Solid lines correspond to single exponential fits, with half-times and total shifts given in Table 2.
Factors affecting Ab binding in lipid bilayers S. Devanathan et al.
1394 FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS
Spectral simulation and structural modeling

The purpose of PWR spectral simulation is to quantify
the changes in the optical properties of the membrane
that occur during the processes of lipid bilayer–peptide
binding and subsequent time-dependent peptide aggre-
gation. This can provide insights into the changes in
mass density and structure of the system caused by
these events. The simulation procedure is described in
detail in our previous publication [35]. Briefly, it is
based on Maxwell’s equations that provide an analyt-
ical relationship between experimental spectral parame-
ters and the optical properties of the bilayer, the latter
defined by refractive index, n, extinction coefficient, k,
and thickness, t. These three parameters can provide a
unique fit to the experimental spectra. (In these experi-
ments, the k-value is caused by light-scattering effects
and will be ignored.) In the present analysis, based on
the structure of the peptide and the literature data, we
have assumed a working model that allows the peptide
to either partially penetrate the lipid membrane with
its hydrophobic tail (i.e. residues 29–40), or to stay on
the surface of the bilayer membrane without significant
penetration. This generates two separate layers, com-
posed of peptide and of the lipid bilayer. Using this
two-layer model we have been able to quantify how
much of the peptide molecule protrudes beyond the
bilayer–water interface, and how much the lipid
Fig. 5. Binding and aggregation of Ab in a DOPC ⁄ SM ⁄ cholesterol (1 : 1 : 1) bilayer in the presence of 1 mM zinc ions. Spectra were obtained
using a high-resolution sensor. Other experimental conditions were as in Fig. 1. (A) and (B) p-Polarized PWR spectra obtained for the bilayer
in the presence of 1 m
M Zn ions, as a function of time, after the addition of A b (5 lM bulk concentration in the sample cell). Initial binding

occurred mainly in the SM-rich microdomain (data not shown). With time, additional resonance shifts occurred, mainly in the SM-rich micro-
domain, along with large changes in amplitude. The resonance for the DOPC-rich domain diminished in intensity, whereas that for the SM-
rich domain increased in intensity and large shifts to longer angles occurred. Note that the t ¼ 208 min resonance is repeated in both panels
for reference purposes. (C,D) Plots of initial peptide binding, resulting in shifts to larger angles (inset to C) and binding at higher concentra-
tions resulting in shifts to smaller angles. Binding to the SM-rich domain is shown in (C) and to the DOPC-rich domain in (D). Data were fit
with three hyperbolic curves (solid lines) to yield affinity constants and spectral shifts as given in Table 1. Initial insertion into the SM-rich
domain was followed by incorporation into both microdomains.
S. Devanathan et al. Factors affecting Ab binding in lipid bilayers
FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1395
membrane properties have been changed by the pep-
tide–bilayer interaction. This is accomplished by evalu-
ation of the optical parameters for the lipid layer
before and after binding of peptide and subsequent
aggregation, and of the layer composed of peptide (or
a portion of the peptide) that extends beyond the lipid
surface. Both p- and s-polarized data were used in such
simulations. Figure 7 shows an example of the simula-
ted spectra superimposed on the experimental spectra,
obtained with s-polarized red light (k ¼ 632.8 nm).
The optical parameters resulting from such simulations
for bilayers composed of DOPC, of SM and of
SM ⁄ cholesterol are summarized in Tables 3–5 for the
binding and aggregation processes. The error limits
shown in these tables correspond to the standard
errors obtained from the fitting procedure. It must be
noted that we have not been able to satisfactorily
simulate the spectra obtained with the DOPC ⁄
SM ⁄ cholesterol bilayers, with and without zinc, using
the high-resolution resonator. This requires additional
information about the effects of varying amounts of

cholesterol on DOPC and SM bilayers, which we do
not have at present. Thus, further experiments are nee-
ded in order to accomplish this. Therefore, for the time
being we have limited our discussion to qualitative
aspects of Ab binding and aggregation in this system,
as presented above.
There are several important conclusions that can be
obtained from the values in Tables 3–5. First, the
bilayer membranes consisting of DOPC, SM, or
SM ⁄ cholesterol have quite different optical parameters
before peptide has been bound (Table 3). This has
been observed previously for DOPC and SM bilayers
[35] and discussed in the context of the membrane
structure and properties. The present work has now
extended this to an SM bilayer containing 0.35 mole
percent cholesterol. Thus, the refractive indices clearly
indicate that the bilayer consisting of DOPC is much
less densely packed with lipid molecules than that of
either SM or SM ⁄ cholesterol (note that refractive
index is proportional to mass density, i.e. molecules
per unit surface area). The lipid-packing density influ-
ences the ordering of hydrocarbon tails, resulting in a
higher degree of order in the latter two membranes,
and this difference is reflected in an increased thickness
of both membranes. In addition, it is clear that the
presence of cholesterol significantly increases both
the packing density of lipid molecules (as shown by
the increase in refractive index) as well as the thickness
Fig. 6. Plot of the time course of spectral changes occurring in the
SM-rich domain. The solid line is a fit to the data with three expon-

entials; half-times and spectral shifts are given in Table 2.
61.54 61.64 61.74
0.75
0.85
0.95
Incident an
g
le, de
g
Reflectance
1
2
3
Fig. 7. Examples of simulated spectra (solid lines) superimposed
on experimental spectra (symbols), obtained with a SM ⁄ cholesterol
membrane using s-polarized red light. Spectra are shown for the
membrane before (curve 1) and after (curve 2) addition of peptide.
Curve 3 was obtained after aggregation.
Table 3. Optical parameters of lipid bilayers prior to Ab binding.
DOPC, dioleoylphosphatidylcholine; n
p
, p-polarized refractive index;
n
s
, s-polarized refractive index; SM, sphingomyelin; t
av
, average
thickness.
Bilayer n
p

(± 0.003) n
s
(± 0.002) t
av
(nm) (± 0.1)
DOPC 1.460 1.445 5.0
SM 1.520 1.450 5.8
SM ⁄ cholesterol 1.555 1.522 6.2
Factors affecting Ab binding in lipid bilayers S. Devanathan et al.
1396 FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS
of the SM bilayer membrane. Therefore, peptide bind-
ing is occurring to three different membrane structures
in these studies.
The data presented in Table 4 show that the binding
interaction of the peptide with the three membranes
creates different effects within the lipid layer and in
the peptide layer. Although the peptide layers in both
the DOPC and SM membranes are similar in thick-
ness, indicating that the peptide protrusions from the
bilayer surface are similar, they differ in the values of
the refractive indices, indicating a higher peptide sur-
face mass density in the case of the SM membrane as
compared with the DOPC membrane. It is also
important to note that, in both of these cases, the val-
ues of the refractive indices are the same for both
polarizations. This strongly supports the idea that the
internal structures of the peptide layers are quite iso-
tropic, without any significant degree of long-range
molecular order. Based on this, one can conclude that
these layers are formed from anisotropic molecular

structures which are arranged in the peptide layer in a
random way, thereby creating an average isotropic dis-
tribution of conformations. It is worth noting that
such anisotropies can result from either molecular con-
formation or molecular orientation, or both; these can-
not be distinguished by the present methods.
In none of the three membrane systems did the ini-
tial binding of peptide cause any significant changes in
the lipid bilayer parameters (Table 4). This suggests
that the peptide was not anchored deeply within these
membranes, indicating that the bulk of the peptide
mass remained largely on the surface of the membrane.
However, the peptide layer was appreciably different
for the SM ⁄ cholesterol membrane, having a higher
mass density, a significant anisotropy (n
p
> n
s
), and a
larger thickness. Thus, more peptide was bound when
cholesterol was included in the bilayer, and the struc-
ture of the peptide layer was different. In order to
explain the anisotropic nature of the peptide layer, it
seems reasonable to assume that some of bound pep-
tides had an extended conformation (b-sheet like) and
bound with their long axis perpendicular to the plane
of the lipid membrane. Furthermore, the 4.7 nm thick-
ness of the peptide layer indicates that the hydropho-
bic tail of the peptide must be buried within the lipid
bilayer. This type of arrangement of the bound peptide

is in agreement with previous studies showing that the
addition of cholesterol to a DOPC ⁄ SM bilayer conver-
ted a b-sheet Ab form into a-helix structures, a change
that was necessary to allow incorporation of the C-ter-
minal tail into the membrane [40]. Such incorporation
is probably driven by hydrophobic interactions caused
by the high nonpolar amino acid content in the C ter-
minus of Ab. A hydrophobic peptide segment would
be expected to have an orientation and anisotropic
refractive index values similar to both the fatty acyl
tails of the lipid and the cholesterol molecule, and thus
would not produce much change in the lipid portion
of the bilayer. However, the peptide layer would be
appreciably altered by such a conformational change
of the peptide.
It is generally accepted that carefully prepared fibril-
free Ab in an aqueous environment exists mainly as
monomers, dimers, trimers, and tetramers, in a rapid
equilibrium, within which the predominant secondary
structural element is random coil with smaller amounts
Table 4. Optical parameters of lipid and peptide layers after amyloid b
1)40
peptide (Ab) binding. chol, cholesterol; DOPC, dioleoylphosphat-
idylcholine; n
p
, p-polarized refractive index; n
s
, s-polarized refractive index; SM, sphingomyelin; t
av
, average thickness.

Bilayer
Lipid layer (after binding) Peptide layer (after binding)
n
p
(± 0.003) n
s
(± 0.002) t
av
(nm) (± 0.2) n
p
(± 0.005) n
s
(± 0.005) t
av
(nm) (± 0.2)
DOPC 1.460 1.445 5.0 1.340 1.340 3.0
SM 1.520 1.450 5.8 1.365 1.365 3.0
SM ⁄ chol 1.555 1.522 6.2 1.420 1.400 4.7
Table 5. Optical parameters of lipid and peptide layers after amyloid b
1)40
peptide (Ab) aggregation. chol, cholesterol; DOPC, dioleoylphos-
phatidylcholine; n
p
, p-polarized refractive index; n
s
, s-polarized refractive index; SM, sphingomyelin; t
av
, average thickness.
Bilayer
Lipid layer (after aggregation) Peptide layer (after aggregation)

n
p
(± 0.003) n
s
(± 0.002) t
av
(nm) (± 0.2) n
p
(± 0.005) n
s
(± 0.005) t
av
(nm) (± 0.2)
DOPC No aggregation No aggregation No aggregation No aggregation No aggregation No aggregation
SM 1.535 1.465 5.8 1.365 1.350 3.0
SM ⁄ chol 1.470 1.460 6.2 1.420 1.400 4.7
S. Devanathan et al. Factors affecting Ab binding in lipid bilayers
FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1397
of b-sheet, and even smaller amounts of a-helix [41].
We therefore assume that the peptide layer on the
DOPC and SM membrane surface consists of such a
mixture of oligomers and secondary conformations.
This accounts for the isotropic nature of this layer in
these two systems. It is interesting, however, to note
that both the binding affinity and the amount of accu-
mulated peptide (i.e. mass density) were significantly
higher with the SM bilayer than with the DOPC
bilayer. This is presumably a result of differences in
both the type of lipid molecule (i.e. sphingolipid versus
glycerolipid), as well as the topology and morphology

of the surfaces of each of the bilayers. This requires
further study.
The differences between the membranes increased
further with time after the initial binding process. As
noted above, and as shown in Table 5, after 15 h there
were no significant changes in either the bilayer or the
peptide layer with the DOPC membrane (i.e. no pep-
tide aggregation occurred). In contrast, changes
occurred in both of these layers with the SM mem-
brane. Thus, the surface mass density of the peptide
layer decreased, as reflected by the smaller value of n
s
,
and a significant refractive index anisotropy was
induced (n
p
> n
s
). A corresponding increase in the
mass density in the lipid bilayer occurred, without a
significant change in anisotropy. These results clearly
indicate that some peptide mass was inserted into the
lipid membrane, and that the mass distribution within
the peptide layer was altered. In order for the latter to
occur, a significant conformational rearrangement
must take place, involving either creating more aniso-
tropic conformations or reorganizing the existing
anisotropic conformations so as to create long-range
molecular order, or both. A plausible interpretation of
these effects is that some of the surface-bound peptide

(possibly monomeric forms with a b-sheet structure)
created higher oligomeric structures that inserted their
hydrophobic tails into the interior of the membrane.
We suggest that this process was favored, in the case
of the SM membrane, because the surface mass density
(i.e. concentration) of peptide was much higher than
with the DOPC membrane.
As can be seen from the data presented in Table 5,
the aggregation of the peptide in the lipid membrane
consisting of SM ⁄ cholesterol resulted in large decreases
in the refractive index parameters for the lipid layer.
This indicates a significant expulsion of lipid mass
(presumably SM molecules) from the bilayer. Thus,
the aggregates formed in the presence of cholesterol
must have a structure that occupies a larger fraction of
the bilayer volume than the unaggregated peptide, in
contrast to those formed in the absence of cholesterol.
This suggests that cholesterol may have been incorpor-
ated into these aggregated peptide structures, which
requires further study. It is also worth noting that the
insertion and aggregation of Ab with membranes con-
taining at least 30% cholesterol has been shown to
form channel-like structures [42]. This is consistent
with our observation of lipid removal from the bilayer
upon peptide insertion and aggregation in the
SM ⁄ cholesterol system.
In summary, the data presented here provide strong
support for a mechanism in which peptide aggregation
requires an SM-rich environment and occurs largely
on the bilayer surface in the absence of cholesterol.

The presence of cholesterol facilitates peptide insertion
into the bilayer and promotes the aggregation process.
This leads to the formation of a less densely packed
bilayer. The presence of Zn
2+
also enhances insertion
and aggregation, and promotes large bilayer structural
changes by peptide aggregates resulting in a more por-
ous membrane. The large effects of SM and cholesterol
on peptide insertion and aggregation are consistent
with the reported occurrence of the amyloid precursor
protein and Ab in neuronal membrane rafts [20–
23,29,30].
Experimental procedures
Materials
Solid-supported lipid bilayers (DOPC, SM, cholesterol and
their mixtures; Avanti Polar Lipids, Birmingham, AL,
USA) were made using solutions of either the single lipid
components or mixtures containing various molar ratios
of lipids (10 mgÆmL
)1
total lipid concentration) in buta-
nol ⁄ squalene (10 : 0.1, v ⁄ v). The buffer solution in contact
with the bilayer in the sample cell for all the experiments
was 10 mm Tris (pH 7.4) at 25 °C, either in the absence of
additional salt ions or in the presence of 1 mm Zn
2+
ions.
The 40-residue amyloid peptide (Ab
1)40

) was synthesized by
standard solid-phase Fmoc chemistry, subsequently purified
by HPLC and found to be over 90% pure, and character-
ized by MALDI-TOF mass spectroscopy. It was stored in
a lyophilized form by a combined trifluoroacetic acid
(TFA) ⁄ trifluoroethanol (TFE) protocol to keep it in a
monomeric form prior to further use [19,28].
PWR spectroscopy
The principles of PWR spectroscopy have been thoroughly
described in previous publications [34–36,43,44]. Here, we
will briefly review those aspects that are especially relevant
to the present work. Figure 8 shows a schematic diagram
of a PWR spectrometer. Resonances can be excited with
Factors affecting Ab binding in lipid bilayers S. Devanathan et al.
1398 FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS
light whose electric vector is polarized either perpendicular
(p) or parallel (s) to the resonator surface, and can be used
to probe the properties (refractive index, n, extinction coef-
ficient at the excitation wavelength, k, and thickness, t)of
molecules immobilized on the silica layer that coats a thin
silver film deposited on the surface of a glass prism [33].
This allows the characterization of the optical properties of
uniaxially ordered anisotropic systems, such as proteolipid
membranes, that are deposited on this surface, involving
changes in surface mass density (i.e. the amount of mass
per unit surface area), in the spatial mass distribution (i.e.
the internal organization of the membrane, including
molecular anisotropy and the long range molecular order
of the bilayer), and in protein ⁄ lipid conformation occurring
as a consequence of molecular interactions.

The PWR spectrum can be described by the depth, the
half-width and the angular position of the resonances,
which are determined by the optical characteristics of the
sensor and the immobilized molecules at the plasmon
excitation wavelength. Molecular interactions or composi-
tion changes occurring at the surface are detected as
changes in these spectral characteristics in real time.
Thus, PWR provides a means to directly measure the
binding, insertion and aggregation of molecules, either at
the lipid bilayer surface or upon incorporation into the
bilayer.
Formation of lipid membranes and peptide
incorporation
The principles of creating a self-assembled single solid-sup-
ported planar lipid bilayer membrane on a PWR resonator
surface have been described in previous publications
[45,46]. Here, we will briefly review those principles. Bilay-
ers were prepared by spreading a small amount of the
appropriate lipid solution in the butanol ⁄ squalene solvent
onto a 2 mm orifice in a Teflon block, separating the silica
surface of the PWR resonator from the aqueous compart-
ment. The hydrated silica surface attracts the polar head
groups of the lipid to form a monolayer with the hydrocar-
bon tails oriented towards the excess lipid solution. Bilayer
formation is spontaneously initiated when the sample com-
partment is filled with an aqueous buffer solution, resulting
in a thinning process to form the second monolayer. A
plateau-Gibbs border, consisting of an annulus of excess
lipid solution, anchors the bilayer to the Teflon block. This
border allows lipid molecules to be transferred into or out

of the orifice to accommodate structure ⁄ conformation
changes that occur upon peptide insertion into the bilayer
and subsequent aggregation.
The resonator surfaces used in these studies were of two
designs – low resolution and high resolution – which varied
in the intrinsic linewidth of the PWR resonances. In order
to observe membrane microdomains with appropriate lipid
mixture compositions (containing equimolar amounts of
DOPC, SM and cholesterol), a resonator that was specific-
ally designed to obtain higher-resolution spectra was used
[35]. With this PWR system, the three-component bilayer
spectra displayed two overlapping resonances. These were
shown previously, by spectral simulation [35], to be the
result of lateral segregation of lipid molecules that sponta-
neously form membrane microdomains, with the more
ordered thicker domains (SM-rich) producing resonances at
higher-incident angles compared with the more disordered
thinner domains (DOPC-rich), which produced resonances
at lower-incident angles [35]. The microdomains were esti-
mated to be larger than 100 nm in diameter (note that the
laser beam is  0.1 mm in diameter and the bilayer is
 2 mm in diameter). It is also important to note that these
microdomains consisted of lipid mixtures, with the majority
component as indicated. Bilayers composed of a single lipid
generated a single resonance that could be observed using
the standard low-resolution PWR resonator system. When
the standard resonator was used with the mixed bilayers,
again a single resonance was observed, as a consequence of
the lower spectral resolution of the system.
Some variability is expected in the self-assembling process

and in the bilayer distribution of such microdomains from
one experiment to another (i.e. in the ratio between the
lipid ordered and disordered phases), as well as in the
sampling of such microdomains by the laser beam [35]. This
can result in variability in the spectral line shape from
Fig. 8. Schematic diagram of a PWR apparatus. A lipid bilayer with
inserted peptides is shown immobilized on the resonator surface.
The presence of a silica layer allows excitation by both p-polarized
and s-polarized light. Excitation produces an evanescent electro-
magnetic field localized on the outer silica surface; molecules
immobilized on this surface interact with the field causing spectral
changes. Adapted from a previous publication [44].
S. Devanathan et al. Factors affecting Ab binding in lipid bilayers
FEBS Journal 273 (2006) 1389–1402 ª 2006 The Authors Journal compilation ª 2006 FEBS 1399
experiment to experiment (compare, for example, Fig. 4A
with Fig. 5A). The diffusion of the microdomains within
the bilayer is relatively slow and occurs in the order of min-
utes [47,48]. PWR spectra were taken at regular intervals to
follow the equilibration processes involved in bilayer forma-
tion occurring on the resonator surface.
After bilayer membrane equilibration, lyophilized TFA ⁄
TFE-pretreated Ab, freshly dissolved in aqueous buffer,
was added in small (microliter) aliquots to the aqueous
compartment of the PWR cell (total volume  1 mL), and
binding of peptide to the bilayer was observed as a change
in the resonance position minimum, with time, for both
p- and s-polarization. In all experiments, the total Ab con-
centration in the sample cell after these additions was 5 lm.
The binding process reached equilibrium  5 min after pep-
tide addition. Inasmuch as time-dependent aggregation of

Ab is known to occur in aqueous media [41], it is not clear
what the oligomeric state of the peptide was prior to being
bound to the bilayer. However, in a control experiment, the
peptide was initially dissolved in dimethylsulfoxide and
freshly prepared aliquots of this solution were added to the
PWR cell, without producing much change in the subse-
quent behavior from that observed with the aqueous pep-
tide solutions. As previous work has shown [49],
TFA ⁄ TFE-pretreated Ab is monomeric in dimethylsulfox-
ide. Apparent peptide K
D
values were obtained from hyper-
bolic fits to plots of the resonance shift versus added
peptide concentration; values reported in Table 1 corres-
pond to averages of the data for p- and s-polarization,
which are expected to give the same values. For membranes
obtained with lipid mixtures containing DOPC ⁄ SM ⁄ choles-
terol, Ab at lower concentrations preferentially inserted into
the SM-rich microdomains, although at higher concentra-
tions the peptide incorporated into both microdomains.
After the peptide concentration in the sample compartment
reached 5 l m, the experiment was allowed to proceed over-
night (‡ 15 h) and monitored to follow aggregation of the
peptide; PWR spectra were taken at regular intervals. Note
that the aggregation process occurred much more slowly
(hours) than the initial peptide binding (minutes). The
observed time course of aggregation was consistent with
previous observations using other technologies [17,20,29].
Note that PWR spectral changes do not directly monitor
peptide aggregate formation; rather, aggregation is detected

by changes in the anisotropic ordering of peptide molecules
and influences on the lipid bilayer structure. This has the
advantage of allowing information to be obtained concern-
ing such structural modifications. In order to determine
whether aggregation required additional peptide to be
recruited from the aqueous medium, some experiments were
carried out in which the excess peptide in solution was
removed by washing with fresh buffer subsequent to pep-
tide binding to the bilayer. These experiments resulted in
spectral shifts (owing to aggregation) with time that were
approximately the same magnitude as those obtained with-
out washing. Thus, we conclude that aggregation proceeded
using peptide that was already bound to the bilayer. All
PWR experiments reported here were carried out using a
Beta-PWR instrument (Proterion Corp., Piscataway, NJ,
USA) using either 543.5 nm or 632.8 nm wavelength excita-
tion. Both wavelengths yielded similar results. As performed
in previous studies [35,36], spectral simulation was used to
analyze some of the PWR data to provide insights into the
structural consequences of peptide binding and aggregation.
Acknowledgements
This work was supported, in part, by grants from the
US National Institutes of Health (GM 59630 to G.T.
and Z.S.), from the Amgen Corporation (to G.T., Z.S.
and S.D.), from the Swedish Research Council (to
G.L. and G.G.), from the Umea
˚
University Biotechno-
logy Fund (to G.G.) and from the Alzheimer Founda-
tion (to G.G.).

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