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-s
Trang 1core fragment Ab(28–35) and its A30G and A30I mutants Sureshbabu Nagarajan1, Kirubagaran Ramalingam2, P Neelakanta Reddy1, Damiano M Cereghetti3,
E J Padma Malar4and Jayakumar Rajadas1
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: karkuvi77@yahoo.co.uk
(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⁄ Pi, 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 amyamy-loid 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.
Trang 2One 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 Ab(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⁄ Pisupport 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 h217=)9443 for the WT peptide, and k = 208 nm and h217=)14 109 for the A30I mutant The facts that all of these peptides form ran-dom coils in solution and are positively charged, and
Trang 3that 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
C
190 210 230
Wavelength (nm)
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
2 dmol
2 dmol
2 dmol
–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 ⁄ Pi (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 ⁄ Pi 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
1c Dansyl-Ab(28–35) ⁄
W36
Dansyl–
KGAIIGLMW
2a Ab(28–35) A30G ⁄
W27
2b Ab(28–35) A30G ⁄
W36
2c Dansyl-Ab(28–35)
A30G ⁄ W36
Dansyl–
KGGIIGLMW
3a Ab(28–35) A30I ⁄
W27
3b Ab(28–35) A30I ⁄
W36
3c Dansyl-Ab(28–35)
A30I ⁄ W36
Dansyl-KGIIIGLMW
Trang 4Instead, 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
Fig 3 AFM images showing the formation of fibrils and
aggre-gates of WT, A30G and A30I peptides in NaCl ⁄ Pi 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
160 120 100 80 60 40 20 450
450
20
C
18
Tht A30G A30l WT
16
12
8
4
2
Wavelength (nm)
Wavelength (nm) Peptides + PC
Peptides + PG Peptides in solution
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 l M ThT
for 30 min in NaCl ⁄ Pi 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
Wavelength (nm)
A B
D
C
WT/W@27
A30G/W@27
Wavelength (nm)
Wavelength (nm) Wavelength (nm)
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 ⁄ Pi 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.
Trang 5sity 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 (Ksv) 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 Ksv 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 Ksv 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-e-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 fluidfluid-ity 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
1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1
1.8
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
A30l
1.6 1.4
2 1.8 1.6 1.4 1.2 1
[Acrylamide] (m M ) [Acrylamide] (m M )
[Acrylamide] (m M )
0.1 0.15
1.2 1
l0
l0
l0
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 ( ).
Trang 6and 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
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.
Trang 7emission 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 formbind-ing 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 Ksv values of the
A B
C D
E F
WT/W@36
A30G/W@36
A30l/W@36
Dan– WT/W@36
Dan–A30G/W@36
Dan–A30l/W@36
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)
Wavelength (nm)
Wavelength (nm) Wavelength (nm)
Wavelength (nm) Wavelength (nm)
400 450 300 400 500
0
Fig 7 Time dependence of FRET for the
Trp peptide derivatives
dansyl-WT ⁄ W36, 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.
Trang 8WT⁄ 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 memmem-brane 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
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
600
500
400
300
200
100
0
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)
Fluorescence intensity (a.u.) Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.) Fluorescence intensity (a.u.)
0 h
24 h
72 h
120 h
0 h
24 h
72 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
72 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.
Trang 9membrane 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 epro-vidence 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
incu-bating the protected, resin-bound peptide with 1.5
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
300
200
0 50
100
WT/W@36 WT+PG/W@36
A30G/W@36
A30l+PG/W@36 A30l/W@36
A30G+PG/W@36
100
0
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.
Trang 10spectroscopy 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
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
nitro-gen gas This was followed by the immediate addition of
10% acetic acid in water, sonication, and lyophilization
used immediately for the studies carried out in this work
CD
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
acquired by means of a JASCO J-715 spectropolarimeter
(Jasco, Tokyo, Japan) equipped with a thermostated cell
-cam-phor sulfonic acid as recommended by the instrument
man-ufacturer WT, A30G and A30I peptides were dissolved in
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
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
absence of Ab, and then incubated for a further 30 min
calcu-lated using Eqn (1) [56]:
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
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
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)