Báo cáo khoa học: Lipid-induced conformational transition of the amyloid core fragment Ab(28–35) and its A30G and A30I mutants docx

Lipid-induced conformational transition of the amyloid
core fragment Ab(28–35) and its A30G and A30I mutants
Sureshbabu Nagarajan
1
, Kirubagaran Ramalingam
2
, P. Neelakanta Reddy
1
, Damiano M. Cereghetti
3
,
E. J. Padma Malar
4
and Jayakumar Rajadas
1
1 Bio-Organic and Neurochemistry Laboratory, Central Leather Research Institute, Adyar, Chennai, India
2 National Institute of Ocean Technology, Pallikaranai, Chennai, India
3 Department of Medicine, Stanford University, CA, USA
4 National Centre for Ultrafast Processes, University of Madras, Chennai, India
Keywords
amyloid core fragment Ab(28–35);
hydrophobicity and sheet propensity;
membrane disruption and neurotoxicity;
mutation; negatively charged lipids
Correspondence
J. Rajadas, Bio-Organic and Neurochemistry
Laboratory, Central Leather Research
Institute, Adyar, Chennai 600 020
Fax: +91 44 24911589
Tel: +91 44 24911386 extn 324
E-mail:

(Received 9 November 2007, revised
8 February 2008, accepted 5 March 2008)
doi:10.1111/j.1742-4658.2008.06378.x
The interaction of the b-amyloid peptide (Ab) with neuronal membranes
could play a key role in the pathogenesis of Alzheimer’s disease. Recent
studies have focused on the interactions of Ab oligomers to explain the
neuronal toxicity accompanying Alzheimer’s disease. In our study, we
have investigated the role of lipid interactions with soluble Ab(28–35)
(wild-type) and its mutants A30G and A30I in their aggregation and con-
formational preferences. CD and Trp fluorescence spectroscopic studies
indicated that, immediately on dissolution, these peptides adopted a ran-
dom coil structure. Upon addition of negatively charged 1,2-dipalmitoyl-
syn-glycero-3-phospho-rac-(glycerol) sodium salt (PG) lipid, the wild-type
and A30I mutant underwent reorganization into a predominant b-sheet
structure. However, no conformational changes were observed in the
A30G mutant on interaction with PG. In contrast, the presence of zwit-
terionic 1,2-dipalmitoyl-syn-glycero-3-phosphatidylcholine (PC) lipid had
no effect on the conformation of these three peptides. These observations
were also confirmed with atomic force microscopy and the thioflavin-T
assay. In the presence of PG vesicles, both the wild-type and A30I mutant
formed fibrillar structures within 2 days of incubation in NaCl ⁄ P
i
, but not
in their absence. Again, no oligomerization was observed with PC vesi-
cles. The Trp studies also revealed that both ends of the three peptides
are not buried deep in the vesicle membrane. Furthermore, fluorescence
spectroscopy using the environment-sensitive probe 1,6-diphenyl-1,3,5-hex-
atriene showed an increase in the membrane fluidity upon exposure of the
vesicles to the peptides. The latter effect may result from the lipid head
group interactions with the peptides. Fluorescence resonance energy trans-

fer experiments revealed that these peptides undergo a random coil-to-
sheet conversion in solution on aging and that this process is accelerated
by negatively charged lipid vesicles. These results indicate that aggregation
depends on hydrophobicity and propensity to form b-sheets of the amy-
loid peptide, and thus offer new insights into the mechanism of amyloid
neurodegenerative disease.
Abbreviations
AFM, atomic force microscopy; Ab, b-amyloid peptide; DPH, 1,6-diphenyl-1,3,5-hexatriene; FRET, fluorescence resonance energy transfer;
PC, 1,2-dipalmitoyl-syn-glycero-3-phosphatidylcholine; PG, 1,2-dipalmitoyl-syn-glycero-3-phospho-rac-(glycerol) sodium salt; PrP, prion protein;
Tht, thioflavin-T.
FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS 2415
One of the neuropathological features in the brain of
patients with Alzheimer’s disease is the presence of
extracellular amyloid plaques that are primarily com-
posed of a 39–43 residue peptide known as the b-amy-
loid peptide (Ab) [1–3]. Intermixed with this are also
shorter fragments of peptides [4]. Administration of
Ab and its fragments to cultured cells and living tis-
sues damages their functionality and compromises
their viability [5–7]. The b-amyloid peptides are known
to interact strongly with the lipid bilayer [8] as well as
metal cations [9,10], thereby possibly initiating cyto-
toxic events. The conversion of soluble b-amyloid pep-
tides into amyloid fibrils in vitro has been shown to
occur via a nucleation-dependent mechanism [11,12].
However, another pathway is likely to be followed in
the presence of lipids [13,14].
Many misfolded proteins that are produced during
normal protein processing become capable of inducing
cytotoxic effects via interaction with cytosolic mem-

branes [15–18]. Such cytotoxic proteins tend to contain
a significant number of exposed hydrophobic residues,
and are often classed as hydrophobic or amphipathic
proteins [19]. In addition to their hydrophobic nature,
these peptides are often positively charged, and this
enables them to interact with negatively charged lipid
membranes [20]. We have chosen a key hydrophobic
region of Ab that extends over residues 28–35
(KGAIIGLM) [Ab(28–35)] and displays amphipathic
properties. This region is thought to play an important
role in determining the secondary structure and the
neurotoxicity of the protein [21]. Using H ⁄ D exchange
NMR spectroscopy, Ippel et al. have shown that this
fragment forms a rigid amyloid core [22].
New insights into the components that mediate the
self-assembly of various polypeptides into amyloid
fibrils will help to answer questions of medical as well
as technological interest. Thus, much attention has
been devoted to the study of minimal amyloid-forming
fragments [23]. As short peptides are easy to design
and synthesize, they serve as an excellent model system
for studying amyloid fibril formation in particular and
biological self-assembly processes in general. It has
been shown that mutations within Ab(25–35) reduce
the b-sheet content and fibrillogenic properties of the
peptide [24,25]. Pike et al. showed that Ab(28–35)
modulated both secondary structure and neurotoxicity.
Hence, we decided to investigate the effects of substi-
tuting Gly and Ile for Ala on the aggregation behavior
of the peptide, both in the presence and in the absence

of charged and zwitterionic lipids.
Substantial evidence has been provided suggesting
that electrostatic interactions between the positively
charged residues of Ab and the negatively charged
membranes might be responsible for the toxic effect of
the former on neuronal cells [26,27]. However, there is
also provisional evidence that, due to their hydropho-
bicity, the amino acids are most likely embedded in
the membrane, thereby causing membrane destabiliza-
tion and leakage [28–30]. In order to address the rela-
tionship between hydrophobicity and propensity to
form b-sheets in the presence of biological membranes,
we investigated the structure of Ab(28–35) and its
mutants A30G and A30I, using various biophysical
techniques. Hence, our study was focused on the con-
formational transitions occurring in A b(28–35) as a
function of both the lipid nature (either negatively
charged or zwitterionic) and the degree of hydropho-
bicity of the peptide. We employed CD, Trp fluores-
cence and acryl amide quenching to evaluate the
peptide interaction with the lipids. These results illus-
trate that peptide sequence and membrane composition
dramatically influence protein assembly (or misassem-
bly) at membrane interfaces.
Results
Induction of b-sheet conformation by acidic
phospholipids and the effect on b-sheet
formation of substituting Ile and Gly for Ala
The far-UV CD spectra of freshly prepared, soluble
wild-type (WT) Ab(28–35) and its mutants A30G and

A30I (Table 1) in NaCl ⁄ P
i
support a random coil con-
formation with negative minima around 197 nm and a
shoulder peak at 225 nm (Fig. 1A). This shoulder peak
results from the minor contributions of b-sheet and
b-turn structures. Addition of negatively charged 1,2-
dipalmitoyl-syn-glycero-3-phospho-rac-(glycerol) sodium
salt (PG) vesicles induced conformational changes in
the WT peptide and its mutants. The CD spectra of
the WT and A30I peptides showed a b-sheet structure
in the presence of PG vesicles. For the A30G mutant,
a reduction of the coil peak at 197 nm and a concomi-
tant increase of the helix and sheet peak at 222 nm
were observed (Fig. 1B). Interestingly, the increase in
b-sheet percentage was linearly proportional to the
increase in hydrophobicity (Gly < Ala <Ile)
(Fig. 1B). It should be noted that the CD spectra of
the three peptides are not identical in the aggregated
state, suggesting that the three peptides form different
types of b-structures. The crossover points from ran-
dom coil to b-sheet are also different, with
k = 195 nm and h
217
= )9443 for the WT peptide,
and k = 208 nm and h
217
= )14 109 for the A30I
mutant. The facts that all of these peptides form ran-
dom coils in solution and are positively charged, and

Interactions of Ab(28–35) and its mutants with lipids S. Nagarajan et al.
2416 FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS
that they show b-sheet structure in the presence of neg-
atively charged lipids, suggest that these peptides are
membrane-bound. That this association is not transient
is hinted at by the observed changes in the membrane
fluidity upon exposure of the vesicles to the peptides
[see the 1,6-diphenyl-1,3,5-hexatriene (DPH) studies
below].
Confirming the involvement of electrostatic charges
in the conformational transitions of the peptides are
the observations made with zwitterionic phospholipid
vesicles. In this case, no effect on structure was
observed, and the peptides adopted a predominantly
random coil conformation with negative minima at
197 nm (Fig. 1C). We must note, however, that the
possibility exists that the corresponding CD spectra
result from a polyproline type II b-turn.
Similar experiments were performed with both Trp
derivatives of the WT peptide and its two mutants (see
Table 1 for the sequences). The resulting CD spectra
showed that an additional Trp residue at either termi-
nus did not have any significant effect on the physico-
chemical properties exhibited by the parent molecules
(data not shown). These results therefore excluded the
possibility that the Trp-modified peptides used in the
subsequent experiments (see below) behaved differently
from the WT peptide and its two mutants.
Thioflavin-T (ThT) assay
The presence of large aggregates can be detected by

monitoring the binding of dyes such as Congo red or
ThT [31]. These dyes are known to bind specifically to
the cross-b-structure in a variety of amyloids, yet they
do not bind to monomers. Quantification of the aggre-
gation extent was done here by the ThT assay. In
agreement with the CD results, the ThT fluorescence
intensity increased drastically when the WT peptide
was dissolved in the presence of PG vesicles (Fig. 2A).
This effect was more pronounced for the A30I mutant
(Fig. 2C). In contrast, the A30G ThT peak did not
show any significant increase when compared to the
signal generated by PG vesicles alone (Fig. 2B).
Freshly prepared, soluble WT, A30G and A30I pep-
tides, either alone or in the presence of 1,2-dipalmi-
toyl-syn-glycero-3-phosphatidylcholine (PC) vesicles,
did not have any significant effect on the ThT fluores-
cence (Fig. 2D–F).
Atomic force microscopy (AFM) studies
To determine whether PG vesicles promote assembly
of the WT, A30G and A30I peptides into amyloid
fibers, we employed AFM to examine the structural
patterns obtained upon exposure of these three pep-
tides to lipids (Fig. 3). In the presence of PC vesicles,
the WT, A30G and A30I peptides did not form any
detectable amyloid fibers within 2 days of incubation.
10 000
A
B
C
190 210 230

Wavelength (nm)
Peptides alone
Peptides + PG
Peptides + PC
Wavelength (nm)
Wavelength (nm)
250
190 210 230 250
190 210 230 250
40
000
30 000
20
000
10 000
0
20
000
10 000
0
–10
000
[θ] deg cm
2
dmol
–1
[θ] deg cm
2
dmol
–1

[θ] deg cm
2
dmol
–1
–20 000
–10 000
–20 000
–30
000
–10
000
–20 000
–30
000
–40
000
–50
000
0
Fig. 1. CD spectra were acquired for 50 l M WT ( ), A30G ( ) and
A30I (
) peptides in NaCl ⁄ P
i
(pH 7.4) alone (A) and in the presence
of PG (B) or PC (C) at a 1 : 30 peptide ⁄ lipid ratio. Differences in the
CD spectra demonstrate that WT, A30G and A30I peptides adopt a
random structure in NaCl ⁄ P
i
alone and in the presence of PC lipid.
However, a conformational transition is observable in the presence

of the PG lipid.
Table 1. Peptides used in this study.
No. Peptide
Primary
sequence
Relative molecular
mass
Theoretical Found
1Ab(28–35) KGAIIGLM 802.0 803.9
1a Ab(28–35) ⁄ W27 WKGAIIGLM 988.3 988.6
1b Ab(28–35) ⁄ W36 KGAIIGLMW 988.3 988.7
1c Dansyl-Ab(28–35) ⁄
W36
Dansyl–
KGAIIGLMW
1035.7 1036.9
2Ab(28–35) A30G KGGIIGLM 788.0 789.6
2a Ab(28–35) A30G ⁄
W27
WKGGIIGLM 974.2 974.6
2b Ab(28–35) A30G ⁄
W36
KGGIIGLMW 974.2 974.7
2c Dansyl-Ab(28–35)
A30G ⁄ W36
Dansyl–
KGGIIGLMW
1022.9 1023.4
3Ab(28–35) A30I KGIIIGLM 844.1 845.5
3a Ab(28–35) A30I ⁄

W27
WKGIIIGLM 1030.3 1031.1
3b Ab(28–35) A30I ⁄
W36
KGIIIGLMW 1030.8 1031.5
3c Dansyl-Ab(28–35)
A30I ⁄ W36
Dansyl-
KGIIIGLMW
1078.8 1079.6
S. Nagarajan et al. Interactions of Ab(28–35) and its mutants with lipids
FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS 2417
Instead, globular structures with an average radius of
10–50 nm were observed (Fig. 3A–C,G–I). On the
other hand, abundant fibers were detected after 2 days
for the WT and A30I peptides in the presence of PG
vesicles, with an average extension of 200–500 nm
(Fig. 3D,F). As expected from the experiments
described above, the AFM images of the A30G mutant
did not reveal any fibrils but only amorphous
aggregates with an average radius of 150–200 nm
(Fig. 3E).
Effect of site mutation on Trp fluorescence
The characteristic fluorescence emission of Trp is
highly sensitive to changes in the environment, and it
has therefore been widely used to monitor the interac-
tion of proteins with lipid membranes [32]. We studied
changes of the intrinsic Trp fluorescence with six pep-
tides derived from N-labeling and C-labeling of the
original peptides (WT, A30G and A30I peptides; see

Table 1). When no lipid vesicles were used and the
peptides were mainly in a monomeric state, the Trp
emission spectra of both peptide derivatives showed
maxima at 364, 362 and 365 nm for the WT ⁄ W27,
A30G ⁄ W27 and A30I ⁄ W27 peptides, respectively
(Fig. 4A–C), indicating that the Trp was highly
solvent-exposed. On the contrary, important spectral
changes were observed in the presence of the nega-
tively charged lipid vesicles (Fig. 4A–C). These changes
were characterized by a decreased fluorescence inten-
A
WT
No
lipid
500 nm
500 nm
500 nm
500 nm
500 nm
500 nm
500 nm
500 nm
500 nm
PG
PC
A30G A30l
BC
DEF
GHI
Fig. 3. AFM images showing the formation of fibrils and aggre-

gates of WT, A30G and A30I peptides in NaCl ⁄ P
i
alone (A–C) and
in the the presence of PG (D–F) and PC (G–I). After 2 days of incu-
bation in the absence of lipid vesicles, oligomeric species were visi-
ble for all peptides (A–C). Peptide fibrils formed in the presence of
PG vesicles (D–F), whereas globular aggregates were visible in the
presence of PC vesicles (G–I).
200
180
60
50
40
30
20
10
0
450 500 550
160
140
120
100
80
60
40
20
450
450
20
AB

C
18
Tht
A30G
A30l
WT
16
14
12
10
8
6
4
2
0
500 550
Wavelength (nm)
Wavelength (nm)
Peptides + PC
Peptides + PGPeptides in solution
Tht fluorescence (a.u.)
Tht fluorescence (a.u.)
Tht fluorescence (a.u.)
Wavelength (nm)
500
PG
WT+PG
WT+PC
A30G+PC
A30l+PC

PC
A30G+PG
A30l+PG
550
0
Fig. 2. ThT fluorescence spectra. WT, A30G and A30I peptides
were incubated at a concentration of 10 l
M with 5 lM ThT
for 30 min in NaCl ⁄ P
i
alone or in the presence of PG (A–C) or PC
(D–F). Formation of amyloid fibrils, as shown by increased ThT
absorption, is particularly evident in the presence of the PG lipids.
150
150
100
50
0
125
100
75
50
25
Tryptophan fluorescence
intensity (a.u.)
Wavelength (nm)
A B
D
C
WT/W@27

A30l/W@27
W@36
A30G/W@27
Wavelength (nm)
Wavelength (nm) Wavelength (nm)
Tryptophan fluorescence
intensity (a.u.)
Tryptophan fluorescence
intensity (a.u.)
Tryptophan fluorescence
intensity (a.u.)
0
300 350 400 450
400
300
140
120
100
80
60
40
20
0
200
100
0
300 350 400 450 300 350 400 450
300 350 400 450
Fig. 4. Trp fluorescence spectra of the N-modified WT ⁄ W27 (A),
A30G ⁄ W27 (B) and A30I ⁄ W27 (C) peptides in NaCl ⁄ P

i
alone ( )
and in the presence of PG vesicles (
). Data for the C-modified
peptides are summarized in (D). The excitation wavelength was set
to 280 nm, and the fluorescence emission was monitored between
300 and 500 nm. The PG vesicles cause a blue shift of the fluores-
cence emission maximum and either a decrease (A–C) or increase
(D) in the fluorescence intensity.
Interactions of Ab(28–35) and its mutants with lipids S. Nagarajan et al.
2418 FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS
sity and a spectral blue shift of the peak maxima of
14, 8 and 19 nm for the WT ⁄ W27, A30G ⁄ W27 and
A30I ⁄ W27 peptides, respectively (Fig. 4A–C). On the
other hand, the spectra of the C-labeled peptides
showed an increase in the Trp fluorescence and a less
marked blue shift of about 8 nm (Fig. 4D).
The more pronounced blue shift for the N-modified
peptides suggests that Trp experiences a less polar
environment at the N-terminus than at the C-terminus,
and that it is located in an environment with increased
microviscosity. The increased fluorescence intensity
observed with the Trp36 peptides is probably due to
the reduced degrees of freedom that the peptides
experience in association with the membrane, thereby
leading to increased quantum yields. The fact that we
see a diminished signal in the N-labeled series may
be due to internal quenching by the Lys next to the
Trp [33].
Acrylamide quenching studies

A prerequisite for any understanding of the interaction
of the peptide with the membrane is the knowledge of
its location on the membrane. Hence, the location of
Trp in negatively charged lipid vesicles was studied by
adding increasing amounts of an acrylamide solution
and monitoring the resulting quenching of fluorescence
in the absence and presence of lipid vesicles.
High Stern–Volmer constant (K
sv
) values of 5.71,
5.13 and 5.16 (WT ⁄ W27–WT ⁄ W36), A30G ⁄ W27–
A30G ⁄ W36 and A30I ⁄ W27–A30I ⁄ W36, respectively)
were obtained for the six N-labeled and C-labeled pep-
tides in aqueous solution (Fig. 5). When the N-labeled
peptides were incubated with PG lipids, the K
sv
values
decreased to 2.81, 2.74 and 2.63 (WT ⁄ W27,
A30G ⁄ W27 and A30I ⁄ W27 peptides, respectively). In
the case of the C-labeled probes, the K
sv
values (3.89,
4.14 and 3.16 for the WT ⁄ W36, A30G ⁄ W36 and
A30G ⁄ W36 peptides, respectively) were between those
of the former two experiments. These data show that
upon interaction with the vesicles, the Trp is shielded
from the surrounding aqueous environment and it is
not easily reached by the quencher. The differences
observed between N-labeled and C-labeled peptides
may be ascribed to different factors. For instance, it is

possible that the two amino groups in the WT ⁄ W27,
A30G ⁄ W27 and A30I ⁄ W27 peptides bring the Trp
moiety closer to the solution–lipid interface, where it is
less accessible to acrylamide. An effect due to internal
quenching in the WT ⁄ W27, A30G ⁄ W27 and
A30I ⁄ W27 series, due to the closer proximity of the e-
amino and a-amino groups, can be ruled out on the
basis of the results obtained in the absence of vesicles.
Effect of Ab(28–35) on DPH anisotropy in acidic
phospholipids
It has been reported that the binding of the amyloid
protein of AD to lipid membranes can change their
fluidity [34]. Therefore, we examined the effect of bind-
ing of the WT, A30G and A30I peptides on the fluid-
ity of PG lipid vesicles. The relative fluidity of PG
vesicles was considered to be gel-like, as indicated by
an r-value close to 0.21. However, the DPH anisotropy
constant measured after 1 h of incubation with the
WT, A30G and A30I peptides significantly decreased,
hence pointing to an enhanced internal fluidity of the
bilayer (Fig. 6).
Fluorescence resonance energy transfer (FRET)
assays
FRET has been used as a so-called spectroscopic ruler
to monitor self-association and to measure distances
within proteins and other macromolecules [35]. In
order to understand the folding and unfolding of
peptides, both in the presence and the absence of PG
lipids, FRET measurements were carried out. As there
is no intrinsic fluorophore in Ab(28–35), we chose Trp

2
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
1.8
AB
C
y = 5.7143x + 1.0114
y = 5.1286x + 1.0211
y = 4.1429x + 1.0064
y = 5.1571x + 1.0232
y = 3.1686x + 0.9911
y = 2.63x + 0.9762
y = 2.7429x + 09786
y = 3.8857x + 1.0057
y = 2.8143x + 0.9989
WT A30G
A30l
1.6
1.4
2
1.8
1.6

1.4
1.2
1
0 0.05
[Acrylamide] (m
M) [Acrylamide] (mM)
[Acrylamide] (m
M)
0.1 0.15
0 0.05 0.1 0.15
0 0.05 0.1 0.15
1.2
1
l
0
/l
l
0
/l
l
0
/l
Fig. 5. Stern–Volmer plots for the acrylamide-mediated quenching
of the fluorescence signal in the Trp derivatives of WT (A), A30G
(B) and A30I (C) peptides. The fluorescence emission was mea-
sured at either about 360 nm (in the absence of PG) or at 348 nm
(in the presence of PG). In the absence of PG, both N-modified and
C-modified peptides gave similar curves, and only that of the Trp27
peptide (r) is shown. Major differences were observed when the
peptides were incubated with PG, as shown by the curves

obtained for theTrp36 peptide (
) and the Trp27 peptide ( ).
S. Nagarajan et al. Interactions of Ab(28–35) and its mutants with lipids
FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS 2419
and dansyl as the donor–acceptor pair. Samples were
prepared such that labeled and unlabeled peptides were
present in a 1 : 8 ratio to ensure that the FRET was
predominantly intramolecular. For peptides in
solution, the Trp fluorescence of the dansyl-Ab
(28–35) ⁄ W36 derivatives dansyl-WT ⁄ W36, dansyl-
A30G ⁄ W36 and dansyl-A30I ⁄ W36 was less than that
of the corresponding peptides not conjugated to the
dansyl group, WT ⁄ W36, A30G ⁄ W36, and A30G ⁄ W36
(Fig. 7D–F and A–C, respectively). Upon aging, and
with no vesicles present, the Trp fluorescence intensity
increased, with a concomitant decrease in dansyl fluo-
rescence, indicating an increased end-to-end distance.
Generally, formation of b-sheet is accompanied by an
increase in the intramolecular fluorophore distance.
Surprisingly, upon binding to PG vesicles, an increase
in energy transfer was also noted, as indicated by a
decrease in Trp intensity and an increase in dansyl flu-
orescence as compared to peptides in solution (Fig. 8).
Only a marginal change in the energy transfer was
observed upon prolonged incubation. The very high
dansyl ⁄ Trp fluorescence intensity ratio obtained in the
latter case questions the formation of a b-sheet. How-
ever, the data can be reconciled by considering anti-
parallel b-sheet aggregates. In this event, peptides
would self-associate along the surface in an alternate

way, whereby a peptide bound to the membrane via its
two amino groups is flanked by a peptide in the oppo-
site direction, and so on. This organization would
bring donor and acceptor in close proximity, thus
explaining the increased dansyl ⁄ Trp fluorescence inten-
sity ratio. This mechanism would be particularly plau-
sible with the dansyl derivatives, as the presence of the
aromatic fluorophore at the N-terminus removes
a basic group and introduces a sulfamoyl group. The
latter may lead to a destabilized interaction with the
phosphate heads in the bilayer. The tertiary amine
present in the fluorophore is probably not ideally posi-
tioned and ⁄ or strong enough to efficiently bind to the
membrane and therefore counterbalance the desta-
bilizing effect.
Discussion
A prerequisite for a peptide to interact with lipids is
the presence of an exposed hydrophobic region that
can be stabilized by an amphipathic environment such
as a lipid membrane. In this study, we designed and
tested the properties of the amyloid core fragment
Ab(28–35) and two of its mutants, A30G and A30I, to
understand the peptide–membrane interactions, and
especially the contributions of the hydrophobic resi-
dues Ala, Gly and Ile to this process. Previous studies
have demonstrated that acidic phospholipid and phos-
phoinositides promote a conformational transition in
Ab from a random to a b-structure [36–41]. Figures 1
and 2 show that mutants with a more hydrophobic
character have higher propensity to aggregate than the

WT sequence in the presence of negatively charged PG
vesicles. The greater hydrophobicity of Ile relative to
Ala at position 30 presumably accounts for this
enhanced aggregation. Likewise, for the A30G mutant,
the lower hydrophobicity of Gly may account for the
decreased aggregation of this mutant relative to the
wild-type. These results indicate that the hydrophobic-
ity of the amino acid at position 30 is a major contrib-
utor to the enhanced amyloidogenicity. These
observations strongly argue that hydrogen bonding
within b-structures and hydrophobic interactions
between side-chains are likely to be major stabilizing
interactions within aggregates. Therefore, increases in
the propensities for such interactions are likely to
enhance the rate at which aggregation occurs. Hence,
these interactions would presumably be very sensitive
to the size and character of the hydrophobic residue.
The link between structural alteration and membrane
destabilization is confirmed by point mutations in the
prion protein (PrP) hydrophobic region. The decrease
in b-sheet content closely corresponds to decreased
cytoxicity. Jobling et al. [42] substituted the hydropho-
bic residues Ala and Val with the hydrophilic residue
Ser in the PrP(106–126) hydrophobic core (resi-
dues 113–122). This substitution induced a reduction
in the hydrophobicity of region 113–122 and dramati-
cally reduced the neurotoxicity of PrP(106–126).
Our investigation provides insights into the role of
Trp in peptide aggregation and interactions with lipids.
According to Fig. 9, the kinetic changes in Trp

0.17
0.18
0.19
0.2
0.21
0.22
0.23
Steady state anisotropy
Control WT
A30G A30I
Fig. 6. Effect of WT, A30G and A30I peptides on membrane fluid-
ity of PG vesicles as determined by DPH anisotropy. The addition
of 10 l
M peptides to lipid vesicles increases the membrane fluidity.
Interactions of Ab(28–35) and its mutants with lipids S. Nagarajan et al.
2420 FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS
emission for the WT ⁄ W36, A30G ⁄ W36 and
A30G ⁄ W36 peptides follow a sigmoidal shape, indicat-
ing a cooperative process in solution. However, inter-
action with PG lipids decreases the activation barrier
via favorable electrostatic and hydrophobic forces.
This is clearly seen for the WT and A30I peptides,
where the fluorescence intensity increases during the
first 24 and 48 h, and then gradually decreases. The
less hydrophobic A30G mutant reached a maximum
only after 5 days (Fig. 9). Furthermore, the recruit-
ment of the peptide to the surface of the PG lipid is
rapid, as evidenced by an immediate increase in ThT
fluorescence. Terzi et al. have reported on the impor-
tance of electrostatic interactions for Ab(25–35) bind-

ing to negatively charged liposomes [27]. By forming a
b-sheet scaffold structure, Ab can reside on the surface
of the lipid head group and self-associate to form the
critical fibril nucleus. After nucleation, the fibril grows
through the lipid bilayer, ultimately destabilizing the
membrane, as indicated by the increased membrane
fluidity (Fig. 6). The information on the orientation of
the peptide relative to the membrane was acquired by
using two probes labeled with Trp at two different
positions: the N-terminus and the C-terminus, respec-
tively. The differences noticed between the Trp fluores-
cence emission blue shifts and the K
sv
values of the
A B
C D
E F
150
0 h
WT/W@36
A30G/W@36
A30l/W@36
Dan– WT/W@36
Dan–A30G/W@36
Dan–A30l/W@36
24 h
48
h
72
h

96
h
120
h
0
h
24
h
48
h
72
h
96
h
120
h
0
h
24
h
48
h
72
h
96
h
120
h
0
h

24
h
48
h
72
h
96
h
120
h
0
h
24
h
48
h
72
h
96
h
120
h
0
h
24
h
48
h
72
h

96
h
120
h
160
140
120
100
80
60
40
20
0
100
100
140
120
200
180
160
140
120
80
70
60
50
40
30
20
10

0
100
80
60
40
20
0
100
80
60
40
20
0
50
0
300 350 400 450 300 400 500
300 350 400 450 300 400 500
50
300 350
Wavelength (nm)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Wavelength (nm)
Wavelength (nm) Wavelength (nm)
Wavelen
g

th (nm) Wavelen
g
th (nm)
400 450 300 400 500
0
Fig. 7. Time dependence of FRET for the
dansyl-Trp peptide derivatives dansyl-
WT ⁄ W36, dansyl-A30G ⁄ W36, and dansyl-
A30I ⁄ W36 (D–F), and for their negative
controls WT ⁄ W36, A30G ⁄ W36, and
A30I ⁄ W36 (A–C). Spectra were acquired at
different time intervals (0, 24, 48, 72, 96
and 120 h) by exciting Trp at 280 nm and
recording the emission between 300 and
535 nm at 298 K.
S. Nagarajan et al. Interactions of Ab(28–35) and its mutants with lipids
FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS 2421
WT ⁄ W27–WT ⁄ W36, A30G ⁄ W27–A30G ⁄ W36 and
A30I ⁄ W27–A30I ⁄ W36 peptides are reported in Figs 4
and 5. Trp at the C-terminus is more solvent accessible
and exists in a relatively less apolar environment as
compared to the N-terminus homolog. According to
these results, the Trp at the N-terminus is likely to
bind to the polar–apolar interface via electrostatic
binding between the positively charged Lys and the
negatively charged lipids. These measurements hint
that the C-terminus may reside in the aqueous phase.
Insertion of hydrophobic residues into the lipid
bilayer is generally accompanied by a decrease in mem-
brane fluidity and a corresponding increase in the

anisotropy constant. Such changes are typically
observed after insertion of hydrophobic peptides into
the membrane [43]. For example, functional mutant
OmpA signal peptides that possess high hydrophobic
contents insert into membranes and increase DPH
anisotropy [44]. Similarly, a peptide fragment from the
cytotoxic protein a-sarcin penetrates into the hydro-
phobic core of the bilayer and substantially increases
DPH anisotropy at a temperature above the phase
transition [45].
In contrast, the opposite was observed here, i.e.
decreased anisotropy constant and increased mem-
brane fluidity, possibly because of membrane destabili-
zation by the formation of b-aggregates. This
observation is consistent with a recent report that the
A B
C D
E F
300
300
250
200
150
100
250
600
500
400
300
200

100
0
200
150
180
350
300
250
200
150
100
50
0
160
140
120
100
80
60
40
20
0
100
50
0
50
0
350 400 450 300
600
500

400
300
200
100
0
400 500
300 350 400 450 300 400 500
300 350 400 450 300 400 500
Wavelength (nm)
WT/W@36 + PG Dan–WT/W@36 + PG
Dan–A30G/W@36 + PG
Dan–A30l/W@36 + PG
A30G/W@36 + PG
A30l/W@36 + PG
Wavelength (nm)
Wavelength (nm) Wavelength (nm)
Wavelen
g
th (nm) Wavelen
g
th (nm)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
0 h
24 h
48 h

72 h
96 h
120 h
0 h
24 h
48 h
72 h
96 h
120 h
0 h
24 h
48 h
72 h
96 h
120 h
0 h
24 h
48 h
72 h
96 h
120 h
0 h
24 h
48 h
72 h
96 h
120 h
0 h
24 h
48 h

72 h
96 h
120 h
Fig. 8. Effect of PG lipids on the time
dependence of FRET for the dansyl-Trp
peptide derivatives dansyl-WT ⁄ W36, dansyl-
A30G ⁄ W36, and dansyl-A30I ⁄ W36 (D–F),
and for their negative controls WT ⁄ W36,
A30G ⁄ W36, and A30I ⁄ W36 (A–C). Spectra
were acquired at different time intervals (0,
24, 48, 72, 96 and 120 h) by exciting Trp at
280 nm and recording the emission
between 300 and 535 nm at 298 K.
Interactions of Ab(28–35) and its mutants with lipids S. Nagarajan et al.
2422 FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS
membrane ultrastructure is similarly disrupted by islet
amyloid polypeptide and polymyxin B [46], Ab in the
presence of Golgi bilayers [47], transthyretin amyloid
binding to the plasma membrane, and aggregated Ab
on the synaptic plasma membrane [48]. Our results are
in agreement with the study of Terzi et al. (1997) and
are in partial agreement with Dante et al. [49]. In that
case, Ab(28–35) bound electrostatically to the nega-
tively charged membrane only under physiological con-
ditions.
From the above results, we propose that the peptide
N-terminus interacts with the negatively charged lipids,
whereas the C-terminal portion is oriented away from
the membrane surface. This association must result in
close intermolecular contact between the hydrophobic

residues of the peptide. Hence, the kinetic barriers for
the association of peptides into aggregates are greatly
reduced by the binding of the peptides to the mem-
brane surface. This study is supported by previous
studies showing an immediate increase in membrane
disruption when soluble Ab(25–35) was added to nega-
tively charged membranes [50]. As membrane fluidity
is known to be important for normal cell function and
viability [51], this phenomenon of membrane disrup-
tion ⁄ destabilization may be a crucial mechanism of
amyloid neurotoxicity. Some studies suggest that the
b-amyloid peptides bind electrostatically only to the
polar head groups, i.e. do not become embedded
within the hydrophobic interior [52]. Our results pro-
vide firm evidence that these smaller peptides bind to
negatively charged lipids through electrostatic interac-
tions and disturb the membrane by forming a b-sheet
scaffold at the membrane–water interface.
In summary, our results indicate that electrostatic
interactions are responsible for the initial binding of
negatively charged lipids and positively charged pep-
tides. We conclude from this comparison that the
b-sheet preferences that were observed for the peptides
in negatively charged lipid depends on the intrinsic
b-sheet propensities, and side-chain–side-chain and
side-chain–backbone interactions. This study also gives
an understanding of the specific role played by hydro-
phobic residues in membrane lipid binding and can be
exploited for the development of specific therapeutic
drugs to prevent amyloid peptide neuronal membrane

toxicity.
Experimental procedures
Peptide synthesis and characterization
All Fmoc amino acids were purchased from Nova Biochem
(San Diego, CA, USA). Pentafluorophenol was obtained
from Spectrochem Ltd (Bombay, India). Wang resin was
purchased from SRL Ltd (Bombay, India). All analytical
grade organic solvents used in the present study were pro-
cured from Merck Ltd (Bombay, India) and S.D. Fine
chemicals (Bombay, India). Ab(28–35) and the A30G and
A30I peptides (Table 1) were synthesized manually by stan-
dard solid-phase synthesis using Wang resin and amino
acids protected by the pentafluorophenyl ester of Fmoc, as
previously reported [53]. For the peptides labeled with Trp,
Trp was added at positions 27 and 36, respectively, using
the same procedure. To synthesize compounds, addition of
a dansyl group to the N-terminus of the WT ⁄ W36,
A30G ⁄ W36 and A30I ⁄ W36 peptides was effected by incu-
bating the protected, resin-bound peptide with 1.5 equiva-
lents of dansyl chloride and three equivalents of
triethylamine for 45 min at room temperature. The resin
was washed several times with dichloromethane and dried
under vacuum. A Kaiser test was performed to check the
completion of the reaction. The peptides were purified
by HPLC and characterized by 500 MHz proton NMR
A B C
600
500
400
Tryptophan maximum fluorescence

Intensity (a.u.)
Tryptophan maximum fluorescence
Intensity (a.u.)
Tryptophan maximum fluorescence
Intensity (a.u.)
300
200
0 50
Time (h) Time (h) Time (h)
100
WT/W@36
WT+PG/W@36
A30G/W@36
A30l+PG/W@36
A30l/W@36
A30G+PG/W@36
150 0 50 100 150 0 50 100 150
100
0
600
450
400
350
300
250
200
150
100
50
0

500
400
300
200
100
0
Fig. 9. Time dependence of the Trp maximum emission fluorescence for the WT ⁄ W36 (A), A30G ⁄ W36 (B) and A30I ⁄ W36 (C) peptides in
the absence (r) and presence (
) of PG vesicles. Spectra were acquired at different time intervals (0, 24, 48, 72, 96 and 120 h). Trp-labeled
peptides were excited at 280 nm, and the emission was measured from 300 to 500 nm.
S. Nagarajan et al. Interactions of Ab(28–35) and its mutants with lipids
FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS 2423
spectroscopy. The purity of the peptides obtained was 88%.
Furthermore, the molecular masses of the peptides were
confirmed by MALDI-TOF MS and compared with theo-
retical molecular masses (Table 1).
Liposome preparation
Lyophilized PG and PC lipids (Sigma-Aldrich) were dis-
solved in 3 : 1 chloroform ⁄ methanol, and then left to dry
in air and then in vacuum for 6 h to remove any traces of
solvent. Millipore water was added to the lipid film and
sonicated using an ultrasonicator bath, until an optically
clear solution was obtained. The phosphate concentration
was determined by the method of Ames [54].
Peptide dissolution
WT, A30G and A30I peptides were pretreated with trifluo-
roacetic acid ⁄ trifluoroethanol as previously described [55].
Briefly, peptides were incubated with trifluoroacetic
acid ⁄ trifluoroethanol (1 : 5) for 2 h. Then, trifluoroacetic
acid ⁄ trifluoroethanol was removed under a stream of nitro-

gen gas. This was followed by the immediate addition of
10% acetic acid in water, sonication, and lyophilization.
The lyophilized peptides were dissolved in NaCl ⁄ P
i
and
used immediately for the studies carried out in this work.
CD
Lipid ⁄ peptide ratios were maintained at 30 : 1, with final
peptide concentrations of 50 lm. The effect of various lip-
ids on peptide conformation was determined by adding an
aliquot of freshly prepared peptide stock solution to pre-
formed lipid vesicles under continual stirring. The contribu-
tion of lipid vesicles to the CD signal was removed by
subtracting the CD spectra of pure lipid vesicles from the
CD spectra of peptide ⁄ lipid suspensions. Spectra were
acquired by means of a JASCO J-715 spectropolarimeter
(Jasco, Tokyo, Japan) equipped with a thermostated cell
holder, using a quartz cell of 1 mm path length at 25 °C.
This equipment was calibrated using ammonium-d
10
-cam-
phor sulfonic acid as recommended by the instrument man-
ufacturer. WT, A30G and A30I peptides were dissolved in
10 mm NaCl ⁄ P
i
(pH 7.4), at a concentration of 50 lm. The
CD quartz cell was placed near the photomultiplier tube to
reduce the scattering from the lipid vesicles. Spectra were
collected over the wavelength range 260–190 nm and
smoothed from the buffer spectra. The CD value was

expressed as molar ellipticity.
AFM
The samples were imaged with a Shimadzu-5500 atomic
force microscope (Shimadzu, Kyoto, Japan), using tapping
mode scanning and an Si
3
N
4
tip. The tube scanner was a
30 lm scan master. The images shown were taken in the
noncontact AFM imaging mode. Samples were prepared
for AFM imaging by drying a 10 lL sample from the reac-
tion mixture on freshly cleaved mica with nitrogen gas. The
buffer was washed from the surface of the mica with dou-
ble-distilled water, and the mica was dried again.
Steady-state fluorescence anisotropy
Anisotropy experiments were performed on a Perkin Elmer
fluorimeter equipped with manual polarizers. Excitation
and emission wavelengths were set at 360 and 425 nm, with
slit widths of 1 and 4 nm, respectively. Our system was ini-
tially calibrated using DPH in mineral oil, which should
give an anisotropy equal to 1. The g-factor was calibrated
using horizontally polarized excitation and subsequent com-
parison of the horizontal and vertical emissions, which for
our machine is 0.88. Lipid vesicles were diluted to 500 lm
with NaCl ⁄ P
i
, incubated for 20–30 min in the presence and
absence of Ab, and then incubated for a further 30 min
with DPH at a 1 : 500 probe ⁄ lipid ratio. Fluorescence

intensity was measured with the excitation polarizer in (I
vv
)
and horizontal (I
vh
) positions, and anisotropy, r, was calcu-
lated using Eqn (1) [56]:
r ¼ I
vv
À gI
vh
=I
vv
þ 2gI
vh
ð1Þ
Lipid vesicles in the absence of DPH were measured in
order to evaluate the effect of light scattering on our mea-
surements.
Fluorescence measurements
Intrinsic fluorescence
The kinetics of aggregation were monitored for peptides
N-labeled and C-labeled with Trp by exciting at 280 nm
and detecting between 300 and 540 nm both in the absence
and in the presence of PG and PC vesicles. Peptides were
used at 10 lm in 10 mm NaCl ⁄ P
i
(pH 7.4). Measurements
were performed on a Perkin Elmer LS 45 fluorimeter
equipped with a xenon lamp and a thermostatically con-

trolled cuvette holder using a semi-microquartz cuvette
(1 cm path length; excitation and emission bandpass of
2 nm). Spectra were plotted, and the wavelength and inten-
sity at the maximum emission were recorded. All the fluo-
rescence studies were carried out at 25 °C.
Acrylamide quenching
Acrylamide was added to the Trp-labeled peptide solutions,
both in the absence and in the presence of PG and PC vesi-
cles. Fluorescence intensities were corrected for dilution
effects. Fluorescence quenching data were analyzed using
the general form of the Stern–Volmer equation (Eqn 2)
Interactions of Ab(28–35) and its mutants with lipids S. Nagarajan et al.
2424 FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS
I
0
=I ¼ð1 þ K
sv
½QÞ ð2Þ
where I
o
and I are the fluorescence intensities in the absence
and presence of the quencher, respectively, K
sv
is the
dynamic quenching constant, and [Q] is the quencher con-
centration. The K
sv
value was determined by titrating acryl-
amide from a 5 mm stock solution to 10 lm peptide in
either the presence or the absence of lipids.

FRET
Dansyl-WT ⁄ W36 and WT ⁄ W36, dansyl-A30G ⁄ W36 and
A30G ⁄ W@36, and dansyl-A30I ⁄ W36 and A30I ⁄ W36 pep-
tide pairs were used to study folding and unfolding of lipid-
free and lipid-bound peptides. Intramolecular FRET was
minimized by mixing the labeled peptides with nonlabeled
peptides at a molar ratio of 1 : 8 (total 9 lm)in10mm
NaCl ⁄ P
i
. Emission spectra were recorded between 300 and
550 nm, using an excitation wavelength of 280 nm.
Acknowledgements
We thank Dr A. B. Mandal, Director in Charge,
CLRI, for his kind support in this work. One of the
authors, N. Sureshbabu, would like to acknowledge
CSIR for supporting funds in the form of SRF. We
thank Dr S. Kathiroli, Director, National Institute of
Ocean Technology, for providing the AFM facility.
We also thank Mr Kumaran and Dr V. Ramoorthi
(National Centre for Ultra Fast Processes, Chennai,
India) for helping with the DPH anisotropy study.
References
1 Selkoe DJ (2006) Amyloid beta-peptide is produced by
cultured cells during normal metabolism: a reprise.
J Alzheimers Dis 9, 163–168.
2 Hardy J & Selkoe DJ (2002) The amyloid hypothesis of
Alzheimer’s disease: progress and problems on the road
to therapeutics. Science 297, 353–356.
3 Selkoe DJ (1999) Translating cell biology into therapeu-
tic advances in Alzheimer’s disease. Nature 399, A23–

A31.
4 Tan SY & Pepys MB (1994) Amyloidosis. Histopathol-
ogy 25, 403–414.
5 Yankner BA, Dawes LR, Fisher S, Villa-Komaroff L,
Oster-Granite ML & Neve RL (1989) Neurotoxicity
of a fragment of the amyloid precursor
associated with Alzheimer’s disease. Science 245, 417–
420.
6 Pike CJ, Burdick D, Walencewicz AJ, Glabe CG &
Cotman CW (1993) Neurodegeneration induced by
beta-amyloid peptides in vitro: the role of peptide
assembly state. J Neurosci 13, 1676–1687.
7 Shearman MS, Ragan CI & Iversen LL (1994) Inhibi-
tion of PC12 cell redox activity is a specific, early indi-
cator of the mechanism of beta-amyloid-mediated cell
death. Proc Natl Acad Sci USA 91, 1470–1474.
8 McLaurin J & Chakrabartty A (1996) Membrane dis-
ruption by Alzheimer b-amyloid peptides mediated
through specific binding to either phospholipids or
gangliosides. J Biol Chem 271, 26482–26489.
9 Lau TL, Ambroggio EE, Tew DJ, Cappai R, Masters
CL, Fidelio GD, Barnham KJ & Separovic F (2006)
Amyloid-beta peptide disruption of lipid membranes
and the effect of metal ions. J Mol Biol 356, 759–770.
10 Miura T, Suzuki K, Kohata N & Takeuchi H (2000)
Metal binding modes of Alzheimer’s amyloid beta-
peptide in insoluble aggregates and soluble complexes.
Biochemistry 39, 7024–7031.
11 Wogulis M, Wright S, Cunningham D, Chilcote T,
Powell K & Rydel RE (2005) Nucleation-dependent

polymerization is an essential component of amyloid-
mediated neuronal cell death. J Neurosci 25, 1071–1080.
12 Fezoui Y & Teplow DB (2002) Kinetic studies of amy-
loid beta-protein fibril assembly. Differential effects of
alpha-helix stabilization. J Biol Chem 277, 36948–36954.
13 Zhu M, Souillac PO, Ionescu-Zanetti C, Carter SA &
Fink AL (2002) Surface-catalyzed amyloid fibril forma-
tion. J Biol Chem 277, 50914–50922.
14 Yamamoto N, Matsuzaki K & Yanagisawa K (2005)
Cross-seeding of wild-type and hereditary variant-type
amyloid beta-proteins in the presence of gangliosides.
J Neurochem 95, 1167–1176.
15 Arispe N, Pollard HB & Rojas E (1993) Giant multilev-
el cation channels formed by Alzheimer disease amyloid
beta-protein [A beta P-(1–40)] in bilayer membranes.
Proc Natl Acad Sci USA 90, 10573–10577.
16 Arispe N, Rojas E & Pollard HB (1993) Alzheimer dis-
ease amyloid beta protein forms calcium channels in
bilayer membranes: blockade by tromethamine and alu-
minum. Proc Natl Acad Sci USA 90, 567–571.
17 Hirakura Y, Lin MC & Kagan BL (1999) Alzheimer
amyloid Ab1–42 channels: effects of solvent, pH, and
Congo red. J Neurosci Res 57, 458–466.
18 Lin H, Bhatia R & Lal R (2001) Amyloid b-protein
forms ion channels: implications for Alzheimer’s disease
pathophysiology. FASEB J 15, 2433–2444.
19 Kourie JI & Shorthouse AA (2000) Properties of cyto-
toxic peptide-formed ion channels. Am J Physiol 278,
1063–1087.
20 Bateman A, MacLeod RJ, Lembessis P, Hu J, Esch F

& Soloman S (1996) The isolation and characterization
of a novel corticostatin ⁄ defensin-like peptide from the
kidney. J Biol Chem 271, 654–659.
21 Pike CJ, Walencewicz-Wasserman AJ, Kosmoski J,
Cribbs DH, Glabe CG & Cotman CW (1995) Struc-
ture–activity analyses of beta-amyloid peptides: contri-
S. Nagarajan et al. Interactions of Ab(28–35) and its mutants with lipids
FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS 2425
butions of the beta 25–35 region to aggregation and
neurotoxicity. J Neurochem 64, 253–265.
22 Ippel JH, Olofsson A, Schleucher J, Lundgren E &
Wijmenga SS (2002) Probing solvent accessibility of
amyloid fibrils by solution NMR spectroscopy. Proc
Natl Acad Sci USA 99, 8648–8653.
23 Tenidis K, Waldner M, Bernhagen J, Fischle W, Berg-
mann M, Weber M, Merkle ML, Voelter W, Brunner
H & Kapurniotu A (2000) Identification of a penta-
and hexapeptide of islet amyloid polypeptide (IAPP)
with amyloidogenic and cytotoxic properties. J Mol Biol
295, 1055–1071.
24 Sato K, Wakamiya A, Maeda T, Noguchi K, Takashi-
ma A & Imahori K (1995) Correlation among second-
ary structure, amyloid precursor protein accumulation,
and neurotoxicity of amyloid beta (25–35) peptide as
analyzed by single alanine substitution. J Biochem 118 ,
1108–1111.
25 Ma B & Nussinov R (2006) The stability of monomeric
intermediates controls amyloid formation: Abeta25–35
and its N27Q mutant. Biophys J 90, 3365–3374.
26 Del Mar Martinez-Senac M, Villalain J & Gomez-

Fernandez JC (1999) Structure of the Alzheimer
beta-amyloid peptide (25–35) and its interaction with
negatively charged phospholipid vesicles. Eur J Biochem
265, 744–753.
27 Terzi E, Holzemann G & Seelig J (1995) Self-associa-
tion of b-amyloid peptide (1–40) in solution and bind-
ing to lipid membranes. J Mol Biol 252, 633–642.
28 Jarrett JT, Berger EP & Lansbury PT Jr (1993) The car-
boxy terminus of the beta amyloid protein is critical for
the seeding of amyloid formation: implications for the
pathogenesis of Alzheimer’s disease. Biochemistry 32 ,
4693–4697.
29 Pillot T, Drouet B, Queille S, Labeur C, Vandekerchkh-
ove J, Rosseneu M, Pincon-Raymond M & Chambaz J
(1999) The nonfibrillar amyloid beta-peptide induces
apoptotic neuronal cell death: involvement of its C-ter-
minal fusogenic domain. J Neurochem 73, 1626–1634.
30 Brasseur R (2000) Tilted peptides: a motif for mem-
brane destabilization (hypothesis). Mol Memb Biol 17,
31–40.
31 Cellmer T, Douma R, Huebner A, Prausnitz J &
Blanch H (2007) Kinetic studies of protein L aggrega-
tion and disaggregation. Biophys Chem 125, 350–359.
32 Raghuraman H, Ganguly S & Chattopadhyay A (2006)
Effect of ionic strength on the organization and dynam-
ics of membrane-bound melittin. Biophys Chem 124,
115–124.
33 Hasselbacher CA, Rusinova E, Waxman E, Rusinova
R, Kohanski RA, Lam W, Du Guha AJ, Lin TC &
Polikarpov I (1995) Environments of the four trypto-

phans in the extracellular domain of human tissue fac-
tor: comparison of results from absorption and
fluorescence difference spectra of tryptophan replace-
ment mutants with the crystal structure of the wild-type
protein. Biophys J 69, 20–29.
34 Muller E, Kirsch C & Eckert GP (2001) Membrane-dis-
ordering effects of b-amyloid peptides. Biochem Soc
Trans 29, 617–623.
35 Phu MJ, Hawbecker SK & Narayanaswami V (2005)
Fluorescence resonance energy transfer analysis of apo-
lipoprotein E C-terminal domain and amyloid beta pep-
tide (1–42) interaction. J Neurosci Res 80, 877–886.
36 Choo-Smith LP & Surewicz WK (1997) Acceleration of
amyloid fibril formation by specific binding of Abeta-
(1–40) peptide to ganglioside-containing membrane vesi-
cles. FEBS Lett 40, 295–298.
37 Mason RP, Jacob RF, Walter MF, Mason PE, Avdulov
NA, Chochina SV, Igbavboa U & Wood WG (1999)
Distribution and fluidizing action of soluble and aggre-
gated amyloid beta-peptide in rat synaptic plasma mem-
branes. J Biol Chem 274
, 18801–18807.
38 McLaurin J & Chakrabartty A (1997) Characterization
of the interactions of Alzheimer b-amyloid peptides with
phospholipid membranes. Eur J Biochem 245, 355–363.
39 McLaurin J, Franklin T, Fraser PE & Chakrabartty A
(1997) Structural transitions associated with the interac-
tion of Alzheimer b-amyloid peptides with gangliosides.
J Biol Chem 273, 4506–4515.
40 Mirzabekov T, Lin MC, Yuan WL, Marshall DJ, Car-

man M, Tomaselli K, Leiberburg I & Kagan BL (1994)
Channel formation in planar lipid bilayers by a neuro-
toxic fragment of the b-amyloid peptide. Biochem
Biophys Res Commun 202, 1142–1148.
41 Pillot T, Goethals M, Vanloo B, Talussot C, Brasseur
R, Vandekerckhove J, Rosseneu M & Lin L (1996)
Fusogenic properties of the C-terminal domain of the
Alzheimer b-amyloid peptide. J Biol Chem 271, 28757–
28765.
42 Jobling MF, Stewart LR & White AR (1999) The
hydrophobic core sequence modulates the neurotoxic
and secondary structure properties of the prion peptide.
J Neurochem 73, 106–126.
43 Jo E, Darabie AA, Han K, Tandon A, Fraser PE &
McLaurin J (2004) alpha-Synuclein–synaptosomal mem-
brane interactions: implications for fibrillogenesis. Eur J
Biochem 271, 3180–3189.
44 Hoyt DW, Cyr DM, Gierasch LM & Douglas MG
(1991) Interaction of peptides corresponding to mito-
chondrial presequences with membranes. J Biol Chem
266, 21693–21699.
45 Mancheno JM, Gasset M, Albar JP, Lacadena J, Marti-
nez del Pozo A, Onaderra M & Gavilanes JG (1995)
Membrane interaction of a beta-structure-forming syn-
thetic peptide comprising the 116–139th sequence region
of the cytotoxic protein alpha-sarcin. Biophys J 68,
2387–2395.
46 Porat Y, Kolusheva S, Jelinek R & Gazit E (2003)
The human islet amyloid polypeptide forms transient
Interactions of Ab(28–35) and its mutants with lipids S. Nagarajan et al.

2426 FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS
membrane-active prefibrillar assemblies. Biochemistry
42, 10971–10977.
47 Chochina SV, Avdulov NA, Igbavboa U, Cleary JP,
O’Hare EO & Wood WG (2001) Amyloid b-peptide
1–40
increases neuronal membrane fluidity: role of choles-
terol and brain region. J Lipid Res 42, 1292–1297.
48 Hou X, Richardson SJ, Aguilar M-I & Small DH
(2005) Binding of amyloidogenic transthyretin to the
plasma membrane alters membrane fluidity and induces
neurotoxicity. Biochemistry 44, 11618–11627.
49 Dante S, Hauss T & Dencher NA (2002) Beta-amy-
loid 25 to 35 is intercalated in anionic and zwitterionic
lipid membranes to different extents. Biophys J 83,
2610–2616.
50 Mattson MP, Begley JG, Mark RJ & Furukawa K
(1997) Abeta25–35 induces rapid lysis of red blood cells:
contrast with Abeta1–42 and examination of underlying
mechanisms. Brain Res 771, 147–153.
51 Simons K & Toomre D (2000) Lipid rafts and signal
transduction. Nat Rev Mol Cell Biol 1, 31–39.
52 Jefferson D, Knight JD & Miranker AD (2004) Phos-
pholipid catalysis of diabetic amyloid assembly. J Mol
Biol 341, 1175–1187.
53 Shanmugam G & Jayakumar R (2004) Structural analy-
sis of amyloid b-peptide fragment (25–35) in different
microenvironments. Biopolymers 76, 421–434.
54 Ames BC (1966) Assay of inorganic phosphate, total
phosphate. Methods Enzymol 8, 115–118.

55 Ma K, Talafous J, Orlando R & Zagorski MG (1997)
Trifluoroacetic acid pretreatment reproducibly disaggre-
gates the amyloid b-peptide. Int J Exp Clin Invest 4,
240–252.
56 Jo E, Darabie AA, Han K, Tandon A, Fraser PE &
McLaurin J (2004) alpha-Synuclein–synaptosomal mem-
brane interactions: implications for fibrillogenesis. Eur J
Biochem 15, 3180–3189.
S. Nagarajan et al. Interactions of Ab(28–35) and its mutants with lipids
FEBS Journal 275 (2008) 2415–2427 ª 2008 The Authors Journal compilation ª 2008 FEBS 2427