The full length peptide undergoes an overall transition from a state with a prominent population of left-handed 31 polyproline II; PII-helix at 0C to a random coil state at 60C, with an
Trang 1transitions between left-handed 31-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
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-nating random coil secondary structure in solution at room temperature and physiological pH [7–9]
Keywords
amyloid b-peptide; b-strand; left-handed
3 1 -helix; random coil; transition enthalpy
Correspondence
A Gra¨slund, Department of Biochemistry
and Biophysics, Stockholm University,
S-106 91 Stockholm, Sweden
E-mail: astrid@dbb.su.se
(Received 13 April 2005, revised 26 May
2005, accepted 9 June 2005)
doi:10.1111/j.1742-4658.2005.04812.x
The temperature-induced structural transitions of the full length Alzheimer amyloid b-peptide [Ab(1–40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy The full length peptide undergoes
an overall transition from a state with a prominent population of left-handed 31 (polyproline II; PII)-helix at 0C to a random coil state at
60C, with an average DH of 6.8 ± 1.4 kJÆmol)1per residue, obtained by fitting a Zimm–Bragg model to the CD data The transition is noncoopera-tive for the shortest N-terminal fragment Ab(1–9) and weakly cooperanoncoopera-tive for Ab(1–40) and the longer fragments By analysing the temperature-dependent 3JHNHa 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 0C, then when the temperature increases, confor-mations with dihedral angles moving towards b-strand at 20C, and approaches random coil at 60C 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 20C 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.
Trang 2An NMR study at 8C 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 soluinforma-tion 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-merization of the peptide, further knowledge about the
structural energy landscape is needed, and this
motiva-ted the present study
Earlier studies show that for the fragment Ab(12–
28) the secondary structure of the monomer changes
gradually towards a left-handed 31 (polyproline II;
PII)-helix when lowering the temperature [11] Also the
fragment Ab(1–28) has been shown to adopt a
PII-helical structure in acidic solution [12] For many short
peptides, however, not for all, the PII-helix seems to
dominate at low temperatures [13–15] The PII-helix is
an extended structure with a rotational symmetry of
three amino acids per 360-degree turn The torsion
angles are (/,w)¼ ()78, 146) The PII-helix differs
from PI in the x-torsion angle, where PII is all trans
peptide bonds
There are no interresidual hydrogen bonds
stabil-izing the PII structure The stabilstabil-izing factor is
pro-posed to be interactions with the solvent PII can exist
only in water and the structure is more stable in D2O
than in H2O, suggesting that water–peptide hydrogen
bonds are involved in the stabilization [14,16–18]
Dif-ferent residues have difDif-ferent propensities to adopt the
PII-helix conformation FTIR, Raman and different
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 31
-heli-ces [11,13] Raising the temperature indu-heli-ces 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 spectra of PII-helices were earlier often interpreted as random coil spectra However, no positive bands or local maxima should appear in a true random coil CD spectrum [22] The dihedral angles / and w that define the PII structure are also reflected in the J couplings between spins in the residues.3JHNHacouplings can be studied by NMR spectroscopy The 3JHNHa coupling between the amide proton and the a-carbon proton is dependent on the torsion angle /
The hydrodynamic properties of peptides reflect structural properties The hydrodynamic radius is rela-ted to the diffusion coefficient via Stoke–Einstein’s equation, and is dependent on not only the size but also on the structure of the diffusing Ab-peptide and scales with the molecular mass as the power law
RH¼ n1Mn2[8] The hydrodynamic radius is also rela-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 0C to 60 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
Trang 3Ab(12–28)G19G20 For each peptide a CD spectrum
was obtained at a series of temperatures, ranging from
0 to 60C Figure 1 shows the temperature-dependent
CD spectra of all the studied peptides Figure 1H
shows Ab(25–35) at 0C
Among the fragments studied here, all N-terminal
peptides and the full length peptide are in monomeric
form at 10 lm concentrations, as shown earlier with
dif-fusion measurements [8] To ensure that the temperature
dependence of the CD spectra reveals structural transi-tions a control experiment was performed with 10 lm Sml1(50–104) [25], a 54-residue unstructured peptide in
10 mm phosphate buffer at pH 7.2 This peptide shows
no structure transition The CD spectrum shows only small changes, as shown in Fig S1, and these do not follow the same pattern as the Alzheimer fragments From the CD experiments we conclude that the N-terminal Alzheimer peptide fragments, as well as the (1–40) peptide, are in equilibrium between a structural state with a large contribution of PII-helix secondary structure (most prominent at 0C) and random coil structural state (most prominent at 60C) The two state equilibrium is consistent with a relatively well-defined isodichroic region around 208 nm
Ab(25–35) The hydrophobic fragment Ab(25–35) is different from the other peptides in its CD characteristics It is in an aggregated b-sheet form under these conditions at all temperatures between 0C and 60 C, as the CD spec-tra show little temperature variation (data not shown) The shape of the spectrum recorded at 0C 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, xPII was esti-mated using the relation published by Kelly et al [27]:
xPII ¼½ hmaxþ 6100
13700
In the CD spectra of Ab(1–40) in Fig 1A the local maximum at 221 nm, best seen at low temperature, is characteristic of a PII-helix The population is strongly temperature dependent and as the temperature reaches
60C only a small fraction of PII-helix remains The PII content was estimated to 43% at 0C and reduced
to 15% at 60C At 37 C the PII content was 20% The PII population at 0C is higher for the shorter fragments compared to Ab(1–40) and ranges from
240 220 200
0
-10
velength / nm
2 dmol
240 220 200
0
-10
Wavelength / nm
240 220 200
0
-10
2 dmol
240 220 200
0
-10
-20
A β(1-40) A β(1-28)
A β(1-16) Aβ(12-28)
240 220 200
0
-10
2 dmol
240 220 200
40
20
0
-20
240 220 200
0
-10
2 dmol
240 220 200
0
-10
-20
Aβ(1-9) Aβ(16-21)
A β(12-28)G 19 G19 A β(25-35)
Fig 1 Far UV CD spectra of the fragments in 10 m M 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.
Trang 446% 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 0C after the temperature increase and then
recording a new spectrum at 0C 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
the Ab(1–40) peptide by comparing the results on the
different fragments The Ab(1–9) peptide shows high
propensity to form PII-helix, Fig 1E At 0C the
pop-ulation is 58%, and at high temperature it reduces to
42% The central hydrophobic stretch, Ab(16–21)
shows a spectrum that has some distinct differences
compared to the other fragments, Fig 1F The
evalu-ated PII populations of the fragments except Ab(16–
21) are shown in Fig 2
As already mentioned, the Ab(23–35) fragment aggregated under the conditions used here, and could therefore not represent the monomeric form of the C-terminal segment of Ab However, considering the differences observed between Ab(1–40) and Ab(1–28) one can gain some information about the (29–40) seg-ment of Ab Suppleseg-mentary Fig S2 shows the differ-ence spectra at 0C, 20 C and 60 C The difference spectra are dominated by a contribution from random coil structure At 0C there is a small contribution (< 10%) from PII-helix
Cooperativity for longer and noncooperativity for shorter peptides
The amount of PII-helix, hPII, in the peptides is strongly temperature dependent, and the temperature dependence varies within the peptide group From the fitting of hPII according to the linearized model in Eqns (5) and (6) to the data of Fig 2, a transition temperature Tm, an enthaply changeDH and a cooper-ativity r was obtained for each peptide, Table 1 The parameter Tm 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 (r¼ 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 Tm, 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 the other hand, using a simplified Zimm–Bragg model (valid only for very long polymer chains) gives very different values of the cooperativity coefficient r, and
is not applicable here
CD spectroscopy is a low resolution structural method The CD results could be interpreted in terms
of a two state equilibrium between a more structured state with large contributions from a PII-helix and a random coil state However, the small deviations from isodichroic point behaviour, and the anomalous
400 300
200
1
0.8
0.6
0.4
0.2
0
Temperature / K
ω PI
Fig 2 Population of PII-helix as a function of temperature The
fit-ted curves are calculafit-ted assuming the Zimm–Bragg model using a
linearized approximation for s close to the transition temperature.
The fragments measured are Ab(1–40) (h), Ab(1–28 (.), Ab(1–16)
(r), Ab(12–28) (s), Ab(12–28)G19G20 (d) and Ab(1–9) (e) The
shortest fragment shows no cooperativity in the transition, in
con-trast to the full length peptide.
Trang 5behaviour of Ab(16–21) suggested that more structural
information might be obtained from NMR studies of
the peptides
1H NMR
J couplings and hydrodynamic radii
1H 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 3JHNHa
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]
3JHNHacouplings
The / dihedral angles differ for different secondary
structures: typical values are from )100 to )65 for
PII-helix and from )150 to )100 for b-strand The
temperature dependences of the 3JHNHa couplings
(data not shown) were studied for the two peptides
The Karplus’ equation relates the / dihedral angle to
the3JHNHa coupling [28] The / dihedral angle can be
determined from the inverse of Eqn (1) if / is between
)120 and )22, according to
JHNHa¼ A þ B cosð/ 60Þ þ C cos2ð/ 60Þ ð1Þ
Figure 3 shows the results for the / angles for
selec-ted residues obtained from 3JHNHa couplings using
Bax’s parameters (A¼ 1.60 Hz, B ¼)1.76 Hz and
C¼ 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 3JHNHa 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–
30C, before random coil takes over around 50 C Ala2 is an outlier, and has a much lower 3JHNHa coupling than the other residues This is in agree-ment with other studies [19] The small negative val-ues of the evaluated / angles have been interpreted
as an indication of preferred PII conformation for alanine
The longer fragment Ab(12–28) shows a similar pat-tern as Ab(1–9), but with some important differences (Fig 3B) Ala21 mainly follows the same pattern as Ala2 in Ab(1–9) The other residues show a minimum / angle at 10–15C, after which they return towards a random coil average The two phenylalanines in the central hydrophobic cluster, Phe19 and Phe20, have / dihedral angles much shifted towards a b-strand con-formation already at low temperatures Phe19 and Phe20 are believed to play an important role in peptide aggregation Figure 3 also includes the Tm values obtained by the two state interpretation of the CD data These values of Tm appear to fall near the minima of the / angle curves
For comparison we also studied the variant peptide Ab(12–28)G19G20 At low temperature, 277K, the whole Ab(12–28)G19G20 peptide had larger 3JHNHa couplings corresponding to more negative / angles
Table 1 Parameters calculated from the temperature dependence of the PII population Cooperativity factor, given by r, where r ¼ 1 is no cooperativity and r << 1 is high cooperativity Transition temperature (Ttrans) is in degrees Kelvin DH is the enthalpy difference between PII-helix and random coil (kJÆmol)1per residue) PII pred determined using the method proposed by Eker et al [13] to predict the structure.
Trang 6(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 Tm from the
evaluations of the CD results (Table 1)
Hydrodynamic radii from translational diffusion
A structural transition changes the hydrodynamic
properties through the persistence length of the
dif-ferent structural groups A true random coil has a
smaller hydrodynamic radius, RH, than a PII-helix
and PII has a smaller RH than an extended b-strand
Figure 4 shows the calculated hydrodynamic radius
dependence on number of residues for a polypeptide
chain in ideal random coil, b-strand and PII-helix
conformations Measurements of translational
diffu-sion for the Ab(12–28) and Ab(1–9) peptides were
performed at different temperatures The use of
ref-erence molecules and the empirical function of the
viscosity as described below, gave the same results
The hydrodynamic radii, RH, for the fragments
Ab(12–28) and Ab(1–9) as a function of temperature
are shown in Fig 5 These results exhibit a similar
pattern of three structural states as the majority of
the 3JHNHa couplings and support the interpretations
made in the previous section When raising the
tem-perature, RH first increases reflecting the transition from PII dominated to a b-strand containing state with a more extended structure At higher tempera-tures RH decreases again due to higher populations
of random coil
40 20
0
40
30
20
10
0
N o of segments
RH
Random Coil β-strand Left-handed 31-helix
Fig 4 Simulated hydrodynamic radius for short peptide chains with different secondary structures For each chain length and each structure 1500 structures were generated and the hydrodynamic radius was calculated.
60 40
20 0
-60
-80
-100
30 20 10 0
Fig 3 The temperature dependence of the
/ angle calculated from JHNHacouplings.
The transition temperature Tmcalculated
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).
Trang 7The CD results for Ab(1–40) and the various
frag-ments presented in Fig 1 indicate that we may
consi-der the full length peptide as composed of at least two
different segments with at least two different structural
states in a temperature-dependent equilibrium At low
temperature (0C) the N-terminal half displays
domin-ant characteristics of a PII-helix, most prominently
dis-played by, e.g the short N-terminal fragment Ab(1–9),
whereas the C-terminal half is dominated by random
coil already at low temperature seen directly by
com-paring the CD spectra of Ab(1–40) and Ab(1–28),
Fig 1A,B At high temperature (60C) the whole
pep-tide is in a random coil state The CD results presented
here are in agreement with a two-segment model, e.g
with 65% PII propensity in the Ab(1–25) segment and 0% PII propensity in the Ab(26–40) segment at 0 C The anomalous behaviour of Ab(25–35) being in an aggregated b-state already at 0C indicates that the less hydrophobic N-terminus helps to keep this part in
a monomeric state in the full length peptide
The enthalpy change of the transition from PII to random coil is calculated to 6.8 ± 1.4 kJÆmol)1 per residue, an average for all studied peptides This enthalpy change is less than that of a typical hydrogen bond This is in agreement with the lack of hydrogen bonding to stabilize a PII conformation Instead, a PII-helix is considered to be stabilized by solvation in
an aqueous solution [18,31] A conformational energy map calculated for a hydrated alanine residue shows a 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 3JHNHa 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-tion at 0C, move towards a b-strand conformation at 20–30 C, and finally towards random coil at 60 C Residues 17–24 display a more complex temperature behaviour and do not all behave similarly Two of them (Leu17, Glu22) follow a pattern of PIIfi b-strandfi coil similar to residues 2–8, whereas Ala21 goes directly from PII-rich state to random coil Val18, Phe19, Phe20 and Val24 on the other hand seem to start in a b-strand-rich conformation already at 0 C, and go directly from there to random coil This high b-strand propensity is in good agreement with the pre-dicted structures [14] The temperature dependence of the hydrodynamic radius RH supports the existence of
40 30
20 10
0
12
10
8
6
4
Temperature /oC
RH
Aβ(1-9)
Aβ(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 m M sodium
phos-phate buffer at pH 7 The temperature dependence of the
hydrody-namic radius is similar to that of the J couplings The hydrodyhydrody-namic
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).
Trang 8three 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 0C Trying to break the
struc-tural states down into three different peptide segments,
(1–17), (18–24), (25–40), we arrive at a simplified
model with overall characteristics as described in
Fig 6
This structural state model has several interesting
features, in agreement with observed experimental
results The PII contribution in the N-terminus helps
to keep the peptide in solution at low temperatures, in
agreement with our previous observations [21] of the
increased solubility of the peptide at low temperatures
The gradual transition to more b-strand in the whole
N-terminus when the temperature is raised increases
the total content of b-strand in the sequence If the
concentration is high enough this could trigger the
irreversible aggregation concomitant with the
conver-sion into b-structure, the favoured secondary structure
of the aggregates The central segment 18–24, which
has a large contribution of b-strand already at the low
temperatures may be considered as the seed in this
structural transition of the peptide It is already known
that the Ab(1–40)G19G20 variant does not aggregate
like the native sequence [37] From the present results
we may suggest that Val18, Phe19, Phe20 and Val24
contribute considerably to the structural transition
towards b-structure, whereas Ala21 and Glu22
coun-teract this transition by occupying a more
tempera-ture-stable PII conformation Some known mutations,
Flemish (A21G), Dutch (E22Q), Italian (E22K) and
Arctic (E22G) associated with early onset of
Alzhei-mer’s disease are located at these residues [38]
This study is an attempt to combine various
spectro-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 of1H1H NOEs, 3JHNHacouplings and 15N1H 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 for understanding the Alzheimer peptide early aggrega-tion behaviour
Experimental procedures
The peptides, the full length Ab(1–40) and the fragments Ab(1–28), Ab(1–16), Ab(12–28), Ab(1–28)G19G20, Ab(25– 35) and Ab(1–9) were purchased from Neosystem (Stras-bourg, France) The samples were purified by HPLC by the supplier and were used without further purification All pep-tides were nonmodified in the termini The peppep-tides were
experi-ments were performed in 10 mm sodium phosphate buffer
at pH 7.4 at 10 lm peptide concentrations The pH was adjusted using phosphate buffer and was measured using a standard pH-meter The sample preparation was carried out
the experiments were performed The concentrations of the samples were determined by weight
Circular dichroism
CD spectra were obtained with a Jasco (Easton, MO, USA) J-720 spectropolarimeter and the temperature was controlled with a PTC-343 temperature controller A quartz cell with
2 mm optical path was used The spectral range was 190–
250 nm with a resolution of 0.2 nm and a bandwidth of 2 nm
employed The background spectrum was subtracted and the results were expressed as mean residue molar ellipticity [h]
Nuclear magnetic resonance
(Karlsruhe, Germany) 400 MHz spectrometer, a Bruker
cryo-probe, Varian (Palo Alto, CA, USA) 600 and 800 MHz
DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKGA30IIGLM35VGGVV40
DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKGA30IIGLM35VGGVV40
DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKGA30IIGLM35VGGVV40
0 °C
60°C
15-20 °C
β-strand
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.
Trang 9spectrometers The sample concentrations were 500 lm 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
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
cou-plings on the longer fragment Ab(12–28) To obtain the
3
was fitted to the in-phase doublet peaks from the 1D
experiment and the anti-phase doublet peak from the 2D
obtained by using a jump-return sequence to avoid signal
COSY experiments a watergate sequence was used
Viscosity correction in diffusion experiments
The diffusion coefficient is temperature dependent,
primar-ily because the thermal Brownian motion is directly
propor-tional to the temperature but also because the viscosity of
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:
DOBS(T) RH;ref
reference molecule’s hydrodynamic radius at the reference
coeffi-cients at temperature T for the reference molecule and the
studied peptide, respectively In a second approach, empirical
from fitting tabulated values to the empirical function [41]:
g¼ KejðT273Þm
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
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 Repeaindica-ting 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:
assumed to be unstructured, b is a row vector consisting only of ones M is the matrix operator that adds one resi-due to the vector of possible conformations with their
is here, Eqn (2) becomes:
1 0
ð3Þ
conformation r is a cooperativity coefficient The factor
rs is then the statistical weight for a residue PII conforma-tion following after an unstructured residue If r is 1 there
coop-erativity is high Calculating the partition function from Eqn (3) gives:
N
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
q
equili-brium the fraction of residues in PII conformation is given
by the power of s in the partition function divided by the
Performing this calculation we obtain:
Trang 10k 0 þc0 1
k 0 s
k 1 þc1 1
k 1 s
q
8
>
>
9
>
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
parameter s may be approximated as a linear function of
T [36]
m
Hydrodynamic radius from simulations
Simulations were performed to obtain hydrodynamic data As
a model for the peptides a peptide chain with no side chains
was used Three bonds with fixed bond lengths represented
every residue The bond lengths used were 1.33, 1.45 and
1.52 A˚ and the fixed angles between the bonds were: 58, 64
and 75 The rotations around the bonds were controlled: for
a random coil peptide the rotation was free and for defined
structures the rotation was constrained The simulation was
performed using a hard sphere model with a 1.3 A˚ van der
Waals radius of the heavy atoms Fifteen hundred structures
of each length were generated with different distributions of
torsion angles The random coil had free rotation in all three
torsion angles The PII-helix was simulated using equally
with a arbitrary width of 10 for all angles A b-strand was
simulated in the same way with the torsion angles centred at
The hydrodynamic radius was calculated as the radius of
gyration, the mean distance from the centre of mass
Acknowledgements
This study was supported by a grant from the Swedish
Research Council and by the European Commission,
contracts LSHG-CT-2004-51 and
QLK3-CT-2002-01989 We wish to thank Maria Yamout for giving us
the Sml1 peptide sample
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