The Alzheimer b-peptide shows temperature-dependent
transitions between left-handed 3
1
-helix, b-strand and
random coil secondary structures
Jens Danielsson, Ju
¨
ri Jarvet, Peter Damberg and Astrid Gra
¨
slund
Department of Biochemistry and Biophysics, Stockholm University, Sweden
The amyloid b-peptide (Ab) is the major component of
the amyloid plaques found in the extracellular com-
partment in the brains of patients suffering from
Alzheimer’s disease. The Ab-peptide is a 39–42-residue
peptide with the sequence: DAEFRHDSGYEVHHQ
KLVFFAEDVGSNKGAIIGLMVGGVVIA(1–42). It
is cleaved from the Alzheimer’s precursor protein by
the proteases b- and c-secretase [1,2]. The Ab(1–40)
peptide has a hydrophilic N-terminal region and a
more hydrophobic C-terminal region. The peptide con-
tains a central hydrophobic cluster, residues 17–21,
which is suggested to play an important role in peptide
aggregation [3]. There is experimental evidence that
soluble oligomeric aggregates have toxic effects on
neurons and synapses [1,4]. The aggregation involves a
conformational change of the peptide structure to
b-sheet. Solid state NMR spectroscopy has shown that
fibrils of Ab contain parallel b-sheet structure, whereas
shorter fragment fibrils consist of antiparallel b-sheet
structure [5,6]. In vitro, the Ab monomer is in a domi-

tive for the shortest N-terminal fragment Ab(1–9) and weakly cooperative
for Ab(1–40) and the longer fragments. By analysing the temperature-
dependent
3
J
HNHa
couplings and hydrodynamic radii obtained by NMR
for Ab(1–9) and Ab(12–28), we found that the structure transition includes
more than two states. The N-terminal hydrophilic Ab(1–9) populates PII-
like conformations at 0 °C, then when the temperature increases, confor-
mations with dihedral angles moving towards b-strand at 20 °C, and
approaches random coil at 60 °C. The residues in the central hydrophobic
(18–28) segment show varying behaviour, but there is a significant contri-
bution of b-strand-like conformations at all temperatures below 20 °C. The
C-terminal (29–40) segment was not studied by NMR, but from CD differ-
ence spectra we concluded that it is mainly in a random coil conformation
at all studied temperatures. These results on structural preferences and
transitions of the segments in the monomeric form of Ab may be related
to the processes leading to the aggregation and formation of fibrils in the
Alzheimer plaques.
Abbreviations
Ab-peptide, amyloid b-peptide; PII, polyproline II.
3938 FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS
An NMR study at 8 °C of the Ab(1–40) and Ab(1–
42) peptide with oxidized Met35 showed deviations
from random coil behaviour, but only limited informa-
tion about the solution structure could be derived [10].
The details of the high resolution structure or structure
propensity of Ab are still not well known. In order to
understand the early aggregation process and oligo-

CD experiments have led to estimations of the propen-
sity of the amino acids for the PII-helix [14]. Using
these results to predict the secondary structure of the
Ab-peptide shows that the PII content of the full
length peptide should be % 40% at low temperature
and predominantly in the N-terminal half of the pep-
tide [14]. The PII-helix is generally more stable at low
temperatures. The fraction of PII-helix increases as the
temperature decreases, an observation valid both for
true polyproline helices and other left-handed 3
1
-heli-
ces [11,13]. Raising the temperature induces a struc-
tural transition. This transition has been suggested to
be noncooperative for short peptides [19]. For longer
peptides molecular dynamics simulations of polyala-
nine suggest a cooperative transition [18]; however, the
theoretical results are dependent of the force field used
[20]. We have earlier observed that the Ab-peptide is
more soluble at low temperatures and is stable when
kept at low temperature [11,21] suggesting that PII-
helix prevents aggregation of the Ab-peptide.
The general properties of a PII-helix have been
determined by various spectroscopic methods such as
CD, NMR, FTIR and Raman optical activity [13]. In
CD spectroscopy a characteristic positive band appears
in the 210–230 nm region [13]. This positive band cor-
responds to an n–p* transition and is at 229 nm in
pure polyproline. It is shifted towards shorter wave-
lengths when other residues are involved [22]. The CD

ted to the persistence length, which depends on the
structural state of the peptide [23,24].
In the present investigation, we have explored the
structure propensities of Ab(1–40) and selected frag-
ment peptides. Using varying temperatures, the energy
landscape close to the solution structure is explored
and information on the structural transitions of the
peptide is obtained. The temperature-induced struc-
tural transitions also yield information on the back-
ground for a potential mechanism for the transition
from soluble monomer to aggregated multimer. We
have used CD as well as NMR at physiological pH, in
a temperature range from 0 °Cto60°C, to study the
solution structure of the peptide as well as the PII to
coil structural transition. The results show that the full
length peptide monomer partially adopts a PII-helix
structure at low temperatures, particularly in an N-ter-
minal region. However, the central hydrophobic clus-
ter, residues 17–21 and particularly the phenylalanine
residues 19 and 20, have a tendency towards b-strand
formation also at low temperature.
Results
Circular dichroism (CD) spectroscopy
Temperature dependence
We recorded CD spectra at varying temperatures for
the full length Ab(1–40)-peptide, as well as the frag-
ments Ab(1–9), Ab(1–16), Ab (1–28), Ab(12–28),
Ab(16–21), Ab(25–35) and the variant fragment
J. Danielsson et al. Structural transitions of Alzheimer b-peptide
FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS 3939

The shape of the spectrum recorded at 0 °C in Fig. 1H
suggests antiparallel b-sheet secondary structure. Mole-
cular mass studies confirmed the aggregated state of
the peptide as described below. This peptide was not
studied further.
PII content estimation
Quantification of the PII content of the CD spectra can
be carried out in several ways [26,27]. Here, the popula-
tion of PII-helix was calculated from the CD amplitude
of the local maximum around 220 nm, [h]
max
. Because
the wavelength of this maximum is dependent on the
sequence [13] we determined an individual [h]
max
for
each series of recorded spectra. From the spectral
intensities at [h]
max
the PII population, x
PII
was esti-
mated using the relation published by Kelly et al. [27]:
x
PII
¼
h½
max
þ 6100
13700

dmol
-1
240220200
0
-10
-20
A
β
(1-40)
A
β
(1-28)
A
β
(1-16)
A
β
(12-28)
AB
CD
240220200
0
-10
[θ]/10
-3
deg cm
2
dmol
-1
240220200

19
G
19
A
β
(25-35)
Fig. 1. Far UV CD spectra of the fragments in 10 mM sodium phos-
phate buffer at pH 7.4. The concentration was 10 l
M for all frag-
ments. Spectra were recorded at 0, 10, 20, 30, 40, 50 and 60 °C.
Generally the population of PII-helix decreased as the temperature
increased. (A) The full length peptide Ab(1–40); (B) Ab(1–28); (C)
Ab(1–16); (D) Ab(12–28); (E) Ab(1–9); (F) the central hydrophobic
cluster KLVFFA, Ab(16–21); (G) the variant fragment Ab(12–
28)G19G20. In (H) the C-terminal fragment Ab(25–35) fragment at
0 °C is shown, indicating antiparallel b-sheet secondary structure.
Structural transitions of Alzheimer b-peptide J. Danielsson et al.
3940 FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS
46% for the Ab(12–28) to almost 60% for the Ab(1–9)
fragment. The calculated populations are in good
agreement with the predicted populations based on
structural propensities of different residues [14]. The
structure transition is fully reversible for all peptides
(Fig. 1A–G). This was shown by lowering the tempera-
ture to 0 °C after the temperature increase and then
recording a new spectrum at 0 °C. This new spectrum
was identical to the original low temperature spectrum
(data not shown), indicating that no irreversible pro-
cesses are present.
We could estimate the location of the PII-helix on

Eqns (5) and (6) to the data of Fig. 2, a transition
temperature T
m
, an enthaply change DH and a cooper-
ativity r was obtained for each peptide, Table 1. The
parameter T
m
is the temperature when 50% of the
secondary structure of the peptide is populated as
PII-helix.
The full length peptide and the longer fragments
exhibit a certain degree of cooperativity in the trans-
ition. The shorter peptides show a lower degree or no
cooperativity. The shortest fragment Ab(1–9) shows
no cooperativity at all (r ¼ 1.00) in agreement with
earlier observations that short segments do not exhibit
cooperativity in the transition from PII to random coil
[19].
The shorter N-terminal fragments have a higher T
m
,
i.e. are in a more stable PII conformation than the
full length peptide. The central hydrophobic stretch
Ab(16–21) shows a somewhat different pattern and the
temperature dependent population curve cannot be fit-
ted to Eqn (5). These results suggest that this peptide
deviates in its behaviour from the others. It should be
pointed out that these parameters were obtained using
a full Zimm–Bragg model. The results are not very
sensitive to absolute concentrations of the peptides. On

behaviour of Ab(16–21) suggested that more structural
information might be obtained from NMR studies of
the peptides.
1
H NMR
J couplings and hydrodynamic radii
1
H NMR spectroscopy was used to study two shorter
fragments, Ab(1–9) and Ab(12–28) at varying tempera-
tures at 500 lm concentration and pH 7. Assignment
was based on standard procedures. The aim was to
obtain information on the temperature dependence of
the / angles along the peptide chain via
3
J
HNHa
cou-
plings. We also determined the temperature depend-
ence of the overall hydrodynamic radii for the
peptides. These experiments were carried out at higher
concentrations than those used for CD. However,
these shorter fragments are monomeric under the pre-
sent conditions, as verified by the diffusion experi-
ments. The diffusion coefficients were in good
agreement with what was expected from the molecular
masses of the monomers [8].
3
J
HNHa
couplings

HNHa
couplings using
Bax’s parameters (A ¼ 1.60 Hz, B ¼ )1.76 Hz and
C ¼ 6.51 Hz) [29]. The shorter N-terminal fragment
Ab(1–9) shows a homogenous behaviour (Fig. 3A).
At low temperature all residues have angles corres-
ponding to a high population of PII-helix. When
raising the temperature the / angle moves towards
b-strand conformation (more negative values of /),
until a minimum of / is reached, after which the
values start to increase again. A random coil secon-
dary structure represents a weighted average of all
allowed dihedral angles and gives rise to
3
J
HNHa
couplings around 7 Hz. Formally this corresponds to
a single / angle of )80°. However, there are resi-
due-specific variations in the nature of the random
coil state [30]. These results suggest that NMR is
able to resolve an additional structural state for
certain residues in the Ab(1–9) peptide: besides the
PII-helix dominating at low temperature, there are
significant contributions from b-strand around 20–
30 °C, before random coil takes over around 50 °C.
Ala2 is an outlier, and has a much lower
3
J
HNHa
coupling than the other residues. This is in agree-

cooperativity and r << 1 is high cooperativity. Transition temperature (T
trans
) is in degrees Kelvin. DH is the enthalpy difference between PII-
helix and random coil (kJÆmol
)1
per residue). PII
pred
determined using the method proposed by Eker et al. [13] to predict the structure.
Ab(1–40) Ab(1–28) Ab(12–28) Ab(1–16) Ab(1–9) Ab(12–28)G19G20
r 0.14 0.32 0.26 0.40 1.00 0.21
T
trans
263 285 283 283 296 267
DH )4.0 )5.2 )6.3 )7.3 )8.7 )6.9
PII
max
0.45 0.54 0.46 0.52 0.57 0.44
PII
pred
0.40 0.57 0.47 0.62 0.67 0.52
Structural transitions of Alzheimer b-peptide J. Danielsson et al.
3942 FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS
(data not shown) than the native type peptide. Also
the shift towards random coil appeared at lower tem-
perature. These observations indicate that the trans-
ition towards b-strand occurs at lower temperatures in
the variant peptide than in Ab(12–28). This is also in
agreement with the observed values of T
m
from the

made in the previous section. When raising the tem-
perature, R
H
first increases reflecting the transition
from PII dominated to a b-strand containing state
with a more extended structure. At higher tempera-
tures R
H
decreases again due to higher populations
of random coil.
40200
40
30
20
10
0
N
o
of se
g
ments
R
H
/10
-10
m
Random Coil
β
-strand
Left-handed 3

A
21
L
17
E
22
V
24
F
19
F
20
V
18
T
m
Fig. 3. The temperature dependence of the
/ angle calculated from J
HNHa
couplings.
The transition temperature T
m
calculated
from CD data is indicated by an arrow. (A)
Residues 2–8 of Ab(1–9); (B) residues 17,
18, 19–22, 24 of Ab(12–28).
J. Danielsson et al. Structural transitions of Alzheimer b-peptide
FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS 3943
Discussion
The CD results for Ab(1–40) and the various frag-

rather deep minimum for a region that encompasses
both b-sheet and PII-helix [32–34]. This suggests that
there is a low barrier between a generic b-sheet struc-
ture [35] and PII. Futhermore, the low energy barrier
may provide an explanation for a direct conversion
from one form to the other, concomitant with peptide
aggregation, if the concentration is high enough.
The cooperativity coefficient r of Ab(1–40) was
found to be 0.14, a relatively weak cooperativity com-
pared to, e.g. the a-helix forming 1500 residue
poly(benzyl-l-glutamate), for which a r of 2 · 10
)4
was reported [36]. The origin of the weak cooperativity
in Ab(1–40) may lie in interactions between neighbour-
ing large hydrophobic side chains.
The NMR results refine the segmental model of
Ab(1–40) and suggest three distinguishable segments of
the peptide, and three structural states in the tempera-
ture dependent equilibrium. All structural states have
large contribution of random coil, but some structural
preferences are indicated by the varying / angles for
the residues. A smaller negative / indicates PII-helix
and a larger negative / indicates b-strand conforma-
tion. The studies of the
3
J
HNHa
couplings in the two
fragments Ab(1–9) and Ab(12–28) (Fig. 3) indicate
that residues 2–8 begin in a PII-rich average conforma-

β
(1-9)
A
β
(12-28)
α
-CD
Fig. 5. The hydrodynamic radius as a function of temperature for
the fragment Ab(1–9) (s) and Ab(12–28) (d). The hydrodynamic
radius is calculated from pulsed field gradient NMR diffusion data
obtained at 500 l
M peptide concentration in 10 mM sodium phos-
phate buffer at pH 7. The temperature dependence of the hydrody-
namic radius is similar to that of the J couplings. The hydrodynamic
radius is calculated from the diffusion coefficient via Stoke–
Einstein’s equation. All data is corrected for temperature induced
viscosity changes. The temperature dependence of the hydro-
dynamic radius of a-cyclodextrin is shown as a reference (h).
Structural transitions of Alzheimer b-peptide J. Danielsson et al.
3944 FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS
three structural states of the peptide considered as a
whole.
We have no NMR results directly describing the
structural state of the C-terminal (29–40) segment.
However, the CD difference spectra between Ab(1–40)
and Ab(1–28) at different temperatures suggest that
the (29–40) segment is dominated by random coil
structure at all studied temperatures. There is a small
contribution of PII at 0 °C. Trying to break the struc-
tural states down into three different peptide segments,

scopic observations to give a realistic description of
what may in a very approximate description be called
an unstructured (random coil) peptide. Earlier studies
on the Ab-peptide structure in aqueous solution by
NMR have made use of observations at a single tem-
perature of
1
H
1
H NOEs,
3
J
HNHa
couplings and
15
N
1
H
NOEs. The results were presented as a model described
as a collapsed coil [39] or as a structure deviating from
random coil behaviour by local conformational prefer-
ences of short segments [10]. The present results have
been obtained avoiding NOE observations that may
lead to biased results in a highly flexible system. We
have made combined use of the temperature depend-
ence of the observed CD and NMR parameters for the
full length peptide as well as selected fragments, and
have taken a first step towards characterization with
atomic resolution of the structure transitions that
occur in Ab(1–40). The observations may be helpful

TM
(Karlsruhe, Germany) 400 MHz spectrometer, a Bruker
Avance
TM
500 MHz spectrometer equipped with a cryo-
probe, Varian (Palo Alto, CA, USA) 600 and 800 MHz
DAEFR
5
HDSGY
10
EVHHQ
15
KLVFF
20
AEDVG
25
SNKGA
30
IIGLM
35
VGGVV
40
DAEFR
5
HDSGY
10
EVHHQ
15
KLVFF
20

coil
coil
coil
Fig. 6. A model of three distinct segments of Ab(1–40) as a mono-
mer in aqueous solution, and their dominating structure states at
different temperatures.
J. Danielsson et al. Structural transitions of Alzheimer b-peptide
FEBS Journal 272 (2005) 3938–3949 ª 2005 FEBS 3945
spectrometers. The sample concentrations were 500 l m for
both peptides, Ab(1–9) and Ab(12–28), in 10 mm sodium
phosphate buffer at pH 7.4. The temperature was calibrated
using a standard sample of 100% ethylene glycol. All
experiments were performed with samples in 90% H
2
O and
10% (v ⁄ v) D
2
O or 100% D
2
O (for diffusion experiments).
Diffusion experiments were performed using a pulsed field
gradient sequence with longitudinal storage and eddy cur-
rent delay, PFGLED. Solvent suppression was managed
with presaturation of the water. Diffusion experiments were
performed using a list of 32 gradient strengths, a gradient
pulse-length of 4 ms and a diffusion time of 100 ms. To
account for a nonlinear gradient profile the method des-
cribed by Damberg et al. was used [40].
3
J

the solvent, here water, is highly temperature dependent,
which should be corrected for. To account for the viscosity
effect two different approaches were taken. First, reference
molecules with a known hydrodynamic radius were studied.
Here HDO and a-cyclodextrin were used as references. The
unknown hydrodynamic radius of the peptide was calcula-
ted using:
R
H
(T) ¼
D
ref
(T)
D
ref
ð298KÞ
Á
D
OBS
ð298K)
D
OBS
(T)
R
H;ref
D
ref
(298K) is the diffusion coefficient of the reference mole-
cule at a reference temperature T ¼ 298K, D
OBS

2
O. Different mixtures of D
2
O and H
2
O
were then considered to have the weighted mean of the
viscosities [8].
Peptide aggregation
Aggregation was determined by molecular mass filtering
experiments. Light absorption at 212 nm was measured for
the fragments Ab(1–16) and Ab(25–35). The CD sample
solutions were centrifuged (2500 g,4°C, 20 min) through a
10 kDa cut-off filter and the absorption was measured
again. For the fragment Ab(1–16) no loss of peptide was
observed, but for the Ab(25–35) a loss of > 90%, indica-
ting severe aggregation of the peptide. Repeating this pro-
cedure but at low pH where the peptide is less prone to
aggregate shows significantly less loss of peptide, < 30%.
Structural transition theory
The temperature induced transition between PII-helix and
random coil can be treated as an ordinary helix–coil trans-
ition described by Zimm and Bragg. Here we use a slightly
modified Zimm–Bragg model [36]. Using their matrix
method, the partition function, Q, of a peptide with N resi-
dues is given by:
Q ¼ bM
NÀ1
a
0

0
À sÞþk
N
1
ðs À k
1
Þ
k
0
À k
1
k
0;1
¼
1
2
1 þ s Æ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð1 þ sÞ
2
þ 4rs
q

ð4Þ
Here the eigenvalues k
0,1
of M are introduced. At equili-
brium the fraction of residues in PII conformation is given
by the power of s in the partition function divided by the
total number of residues. The fraction of PII is then x

Nc
1
k
1
þ
c
1
À1
k
1
Às

k
N
1
ðs À k
0
Þ

ðN À1Þðk
N
0
ðk
0
À sÞþk
N
1
ðs À k
1
ÞÞ

>
<
>
:
9
>
=
>
;
ð6Þ
The parameter s is, as mentioned above, related to the equi-
librium constant and thus to the enthalpy change due to
the structural transition.
The temperature dependence of the transition can be
studied, and if the populations of the structural entities can
be determined, the parameters s and r can be determined
from Eqn (5). Close to the transition temperature T
m
the
parameter s may be approximated as a linear function of
T [36].
s ¼ 1 À
DH
RT
2
m
ðT À T
m
Þ
At s ¼ 1 the two states are equally populated.

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