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INTRODUCTION |
Simian immunodeficiency virus
(SIV)1 entry in target cells
is mediated by the viral envelope glycoproteins, designated gp120 and
gp32, which are derived by proteolytic cleavage of the gp160 precursor.
SIV gp120 and gp32 play an equivalent role to that of gp120 and gp41 in
the human immunodeficiency virus, type 1 (HIV-1), which has a structure
and biological properties very similar to SIV (1-3). gp32 and gp41
appear to possess one transmembrane domain and are thought to exhibit a
multi-role function that involves anchoring the envelope glycoprotein
complex to the viral membrane, oligomerization of the envelope
glycoprotein, and for the putative membrane fusion between viral and
cell membranes.
During the entry of the virus into the target cell, gp120 is known to
bind to CD4 that serves as a primary receptor for the virus on a target
membrane surface (4, 5). Members of the chemokine receptor family are
also known to be necessary to facilitate the entry of the virus (6, 7).
Thus consistent with this model, direct interactions have been
demonstrated between gp120-CD4 complexes and specific chemokine
receptors (8). The binding of gp120 to CD4 appears to induce major
conformational changes of the gp120 complex that leads to the exposure
of the gp32 N-terminal fusogenic domain in the case of SIV or gp41 in
the case of HIV (2, 9). Exposure of this structure therefore, is
thought to facilitate the fusion of the juxtaposed viral and plasma
membranes and leads to intracellular infection.
Membrane fusion, therefore, is one of the key events during viral
infection and is thought to facilitate the incorporation of the viral
capsid into the cytoplasm of host cell. Many studies using model
membranes, such as liposomes and synthetic peptides corresponding to
the fusion regions of enveloped virus proteins, have revealed the
essential role of the secondary structure and the orientation of the
peptides when inserted in a lipid bilayer (for a review see Ref. 10).
The data presently available suggest that the HIV/SIV fusion peptide
assumes extended and disordered forms in the non-fusogenic state and
transforms into an 
-helix as it penetrates into the target cell
membrane as a prelude to or actually during the fusion process. There
are strong indications that unique oblique orientations of the viral
fusion peptides modify the average orientation of phospholipid acyl
chains, giving rise either to inverted lipid phases or to intermediates
in membrane destabilization and lipid mixing (11-14). Although a lot
of information on the correlation of the viral synthetic fusion
peptides lytic and fusogenic activity with the peptide secondary
structure is available (see e.g. Ref. 10), detailed studies
on the nature of the sequence of specific interactions of fusion
peptides with simple phospholipid membranes are still lacking. Thus
information relating to the initial interactions of fusion peptides
with membranes may shed more light on the fusion process itself. Many
membrane binding assays of viral peptides involve radio derivatives or their conjugation to a chromophoric indicator (15-17). In the former case no kinetic data are accessible, and inevitably, elements of doubt
exist in the latter case that such chemical modification may interfere
with the interaction of the peptide with the membrane surface. It has
been established, however, that localization of fluorescent probes such
as fluorescein phosphatidylethanolamine (FPE) at the membrane surface
offers the possibility of measuring, in real time, the interactions of
peptides or proteins with membranes in a virtually non-invasive manner
(18, 19). Thus, we report studies of fusion peptide-membrane
interactions approached by a utilization of this novel fluorescence
technique. The technique involves labeling membranes with very small
amounts (<1 mol%) of FPE which is sensitive to the membrane surface
electrostatic potential (
s). Changes in s caused
by the net addition or removal of charged species induce a
corresponding increase or decrease in the fluorescence intensity of the
probe. This simple but highly sensitive technique allows determination of the time evolution of the binding of such peptides to membranes, but
additional interactions such as conformational changes of the peptides
may also be identified (18, 19). Whereas the present study is directed
toward simple membrane systems, the additional virtue of the FPE-based
technique is that it facilitates almost identical experiments with
lymphocytes (20).
On the other hand, we have shown recently (21) that peptide-membrane
interactions can be affected by variations of the membrane dipole
potential. This is a relatively recently understood membrane property
that is generated by the presence of electrical dipoles on the
phospholipid molecules and the presence of orientated water molecules
at the membrane-water interface (22). Following a dual-wavelength
fluorescence method, it has been shown that the potential sensitive dye
di-8-ANEPPS can be used to measure changes in the dipole potential
produced by dipolar compounds, such as phloretin or ketocholestanol,
interacting with the membrane (23-25) and to monitor the peptide
membrane interaction and its effect and dependence on the magnitude of
the dipole potential (21).
A number of complications are evident with studies of the free fusion
peptides as opposed to whole viral particles. It is known that the SIV
fusion peptides, which sequence is highly hydrophobic, are very
insoluble in water and not even totally soluble in Me2SO, according the spectroscopic data reported by Martin et al.
(26). This is a situation very different from that of the fusion
peptides on the virus, where for example in the case of HIV the fusion peptide constitutes just the N terminus of the gp41 protein, which is
attached to the rest of the virus and seems to form trimeric complexes
(27). Thus, it is important to try to assess the influence of the
aggregation state of the fusion peptide in the Me2SO
suspension on the interaction of the peptide with the lipidic membranes
and take into account the existence of peptide-peptide interactions in
addition to the lipid-peptide interactions. In recent years, the use of
the dye thioflavin T (ThT) for the study of the fibrillogenesis process
triggered by the 
-amyloid peptide of Alzheimer's disease (28) has
opened the possibility of using such dyes to study other
aggregation-disaggregation processes.
In the present study, membrane systems with well defined lipid
compositions were used in an attempt to characterize the sequence of
interactions of the membrane binding and insertion of the simian viral
fusion peptide. The synthetic peptide corresponding to the 12-residue
N-terminal region of SIV- gp32
(NH2-Gly-Val-Phe-Val-Leu-Gly-Phe-Leu-Gly-Phe-Leu-Ala) was
added to the lipid membrane of specified phospholipid compositions (50 mol % PC and 50 mol % PE), and their interactions were followed using
the FPE-, ThT-, and di-8-ANEPPS-based techniques (18). A number of
factors such as peptide concentration and lipid composition, variation
of the dipole potential, and peptide aggregation were investigated. The
implications of these studies for the biological activity of the
immunodeficiency virus are discussed.
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EXPERIMENTAL PROCEDURES |
Egg phosphatidylethanolamine (PE) and thioflavin T (ThT) were
purchased from Sigma. FPE was synthesized as described previously according to Wall et al. (18).
1-(3-Sulfonatopropyl)-4-[[2-(di-n-octylamino)-6-naphthyl]vinyl] pyridinium betaine (di-8-ANEPPS) was purchased from Molecular Probes Inc.
High pressure liquid chromatography-purified synthetic peptides
prepared with the C terminus in amide form were purchased from Quality
Controlled Biochemical, Inc. (Hopkinton, MA). Stock solutions of these
peptides were made up in Me2SO, typically at a
concentration of 4 mg ml
1.
Membrane Preparations and Labeling with FPE and
Di-8-ANEPPS--
Phospholipids dissolved in chloroform, di-8-ANEPPS
(when required), and the appropriate additive (6-ketocholestanol or
phloretin in methanol) were mixed in a round bottom flask, and the
solution was dried under a stream of nitrogen to deposit a thin lipid
film on the inside of a glass tube. Large unilamellar vesicles (LUV) were prepared by hydrating the dried lipid film with the sucrose buffer
(280 mM sucrose, 10 mM Tris, pH 7.5), then
repeatedly freezing and thawing the suspension 5 times, and finally
extruding it 10 times through two polycarbonate filters of pore size
0.1 µm (Nucleopore Corp., Pleasanton, CA) using an extruder (Lipex
Biomembranes Inc., Vancouver, Canada) according to the extrusion
procedure of Mayer et al. (29). LUVs were labeled
exclusively in the outer bilayer leaflet with FPE as described by Wall
et al. (18). Briefly, LUVs were incubated with FPE dissolved
in ethanol (never more than 0.1% of the total aqueous volume) at
37 °C for 1 h in the dark. Any remaining unincorporated FPE was
removed by gel filtration on a PD10 Sephadex column equilibrated with
the appropriate buffer. Such a procedure leads to the incorporation of
30-50% of the externally added FPE to the preformed LUV. Furthermore,
there was no observed transmembrane "flipping" of the FPE, at least
over time scales of 1 week, FPE-liposomes were stored at 4 °C until use.
Fluorescence Measurements and Analysis--
Fluorescence time
courses were obtained by adding the desired amount of peptide to 2-ml
lipid suspensions (200 µM lipid) on an SLM-Aminco model
spectrofluorimeter. For FPE experiments excitation and emission
wavelengths were set at 490 and 518 nm, respectively.
Dual wavelength recordings with the dye di-8-ANEPPS were obtained by
exciting the samples at two different wavelength (460 and 520 nm) and
measuring their emission intensity ratio, R(460/520), at 580 nm (21, 23).
Assessment of peptide aggregation was determined using thioflavin T at
a dye concentration of 35 µM in the fluorescence cuvette containing buffer or a membrane suspension (200 µM lipid).
Typically, spectroscopic data were downloaded in ASCII file format and
analyzed with the aid of commercial data analysis packages such as
EasyplotTM by Stuart Karon copyright by Spiral Software & MIT (for Windows NT 32-bit (version 4) published by Cherwell
Scientific), e.g. both the FPE and ThT fluorescence time
courses were found to be best described by double exponential processes
according to Equation 1.
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(Eq. 1)
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where A1 and A2
are the amplitudes, and k1 and
k2 are the rate constants of the biexponential process.
The contribution of light scattering to the fluorescence signals was
corrected by recordings made with vesicles without the respective
fluorescence dyes at the same vesicle concentration and subtracting
from the traces obtained with the dye present.
Lipid Mixing-Fusion Assay--
Lipid mixing was determined by
measuring the fluorescence intensity change resulting from the
fluorescence resonance energy transfer (FRET) between two probes,
NBD-PE and rhodamine-PE, inserted into the lipid bilayer as described
by Struck et al. (30). Fluorescence was monitored by using
an SLM 8000 spectrofluorimeter with excitation and emission slits at 4 nm. Probes were added to the lipid film, and membrane vesicles were
prepared as described above.
Liposomes containing both probes at 0.6% (molar ratio) each were mixed
with probe-free liposomes at 1/9 molar ratio at a final lipid
concentration of 300 µM. The initial fluorescence at the 1/9 (labeled/unlabeled) suspension was taken as 0% fluorescence, and
the 100% fluorescence was determined by using an equivalent concentration of vesicles prepared with 0.06% fluorescent phospholipid each. The suspensions were excited at 470 nm, and any NBD fluorescence resulting from FRET was recorded at 530 nm.
Infrared Spectroscopy--
Phospholipid vesicles (with 15 mol % phloretin or 6-ketocholestanol when required) were prepared as
described previously (21), using D2O-based media containing
280 mM sucrose, 10 mM Tris, pD 7.5. 300 µl of
liquid suspension containing SIVwt 100 µM (9 µl SIVwt
3.3 mM in Me2SO added to 291 µl of PC/PE
vesicles 2 mM) were placed in a SeZn plate (SpectraTech
contact sampler, HATR) for attenuated total reflectance (ATR) data acquisition.
ATR infrared spectra were acquired on a Nicolet 410 or 710 spectrometer
equipped with an MTC detector, working at an instrumental resolution of
2 cm1. Typically, a total of 1000 scans were averaged at
room temperature, apodized with a triangle function, and
Fourier-transformed. To obtain the pure spectra of the protein, spectra
of the solvent were collected under identical conditions, and
subtractions were done with the computer. Residual water vapor bands
were also subtracted using a water vapor spectrum.
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RESULTS |
Interaction of SIVwt with FPE-labeled Membranes--
Following the
addition of SIVwt to FPE-labeled PC/PE vesicles, a significant increase
of the fluorescence occurred, indicating the interaction of the fusion
peptide with the membrane surface, as shown in Fig.
1. SIVwt was manufactured in its amide
form and is composed of uncharged amino acids; the only charge existing at pH 7.5 on the peptide is that arising from the positive N terminus. An increase of the fluorescence of the FPE-labeled vesicles is consistent with the increased electropositive surface potential caused
by the binding of the positively charged peptide to the membrane
surface. The incremental phase of the trace shown in Fig. 1 was found
to fit a double exponential process (Equation 1), and the calculated
rate constants are reported in Table
I.
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Fig. 1.
Time course of the interaction of SIVwt with
100-nm diameter phospholipid vesicles as revealed by fluorescence of
FPE-labeled membranes. Inset, effect of peptide
concentration on the fluorescence signal amplitude following
normalization by background subtraction. Lipid concentration was 200 µM. Vesicles composition was 50 mol % PC, 50 mol % PE.
Temperature 37 °C. SIVwt was kept in a Me2SO solution (4 mg/ml), and small volumes were added to the suspensions containing the
vesicles to achieve the desired final concentration as indicated in the
main figure.
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Table I
Summary of the rates of membrane interaction of the SIV peptide
Rate constants are derived from fitting of data in Figs. 1 (FPE
measurements), 3 (ThT measurements), and 5A (di-8-ANEPPS
measurements) to Equation 1 (see "Experimental Procedures"
section).
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In contrast with several other peptides we have studied
(e.g. Refs. 19, 21, 31), no slow fluorescence decrease
(following the initial increase) indicating insertion of the positive
charge into the membrane was observed for SIVwt. The positively charged N terminus must remain at the membrane surface but does not preclude the possibility of a more remote segment (i.e. with no net
charge) of the peptide penetrating the lipidic bilayer as reported for other such amphipathic peptides.
The inset in Fig. 1 shows the titration of PC/PE vesicles
with the SIVwt peptide. An interesting observation from attempts to
determine the dose-response characteristics was the fact that above 30 µM for PC/PE membranes, it was not possible to acquire more experimental points as above such concentrations significant peptide aggregation takes place.
The results shown in Fig. 1 clearly indicate that SIVwt interacts with
the membrane surface of PC/PE vesicles. The aggregation observed above
the critical concentration described directed our attention to the
aggregation state of the peptide. SIVwt is highly hydrophobic,
relatively insoluble in water, and even forms soluble aggregates in
Me2SO (the infrared spectrum indicates -sheet structure typical of protein aggregates), according to the structural data reported in Martin et al. (26). When added to the vesicle
suspensions, these peptide aggregates present in the Me2SO
solution have to partition between the aqueous medium and the
membranes. To try to obtain some information about the evolution of the
peptide aggregates when they interact with the membranes we used the
fluorescent dye thioflavin T (ThT) as an indicator of soluble and
colloidal peptide aggregates.
It is worth emphasizing that the addition of Me2SO had
virtually no effect on the fluorescence originating from FPE. This indicates that given the nature of Me2SO, there appears to
be no significant interaction between the solvent and the various membrane moieties.
Interaction of ThT with Peptide Aggregates--
Thioflavin T has
been shown to indicate the presence of -amyloid peptide aggregates
(i.e. so-called fibril formation) (28). The dye appears to
intercalate within the -sheet type secondary structure of the
aggregates, and this interaction causes an increase of its
fluorescence. Fig. 2 shows how the
fluorescence of ThT dissolved in aqueous buffer changed when different
amounts of SIVwt were added into the buffer from a Me2SO
solution. The addition of the peptide was always followed by a very
fast fluorescence increase, the magnitude of which increased with the
peptide concentration. This fluorescence increase results from the very
fast interaction of ThT with the peptide aggregates. Up to SIVwt 5 µM after the initial increase the fluorescence remains
stable. At peptide concentration of 10 µM or higher,
however, a fluorescence decrease follows the initial increase. The
higher the peptide concentration, the faster the decrease and the
noisier became the signal. The noisiest part of the traces corresponds
to the formation of huge aggregates which, as was the case for Fig. 1
(inset), are clearly visible by the naked eye and are
buoyant.
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Fig. 2.
Variation of ThT fluorescence at a
concentration of 35 µM in aqueous
medium following addition of SIVwt at 5, 10, and 30 µM.
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In Fig. 3, the ThT fluorescence time
evolution when 10 µM SIV was added to a PC/PE vesicle
suspension (200 µM lipid) is shown. The most striking
feature is that in the presence of vesicles, after the initial
fluorescence increase, the fluorescence decays in an exponential
fashion, reaches a stable final level without the formation of large
aggregates. Some care must be employed, however, for as shown in Fig. 4
if the concentration of peptide is increased beyond 30 µM
(at a constant lipid concentration) then aggregation dominates the
behavior of the peptide.
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Fig. 3.
ThT fluorescence variation after addition of
10 µM peptide to a suspension
containing 50 mol % PC, 50 mol % PE vesicles (200 µM lipid). ThT was 35 µM. Other conditions as in Fig. 1.
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The ThT fluorescence decay in the presence of membranes was found to
fit a double exponential rate equation (Equation 1), as reported in
Table I. The two rate constants are found to be very similar to those
calculated for the binding of the peptide to the membrane surface (Fig.
1 and Table I). It is interesting to note the fact that after the
initial fast fluorescence increase the fluorescence decreases to the
same level of fluorescence prior to the peptide addition (Fig. 3).
Since the ThT fluorescence reflects the degree of aggregation of the
peptide, it can be deduced from Fig. 3 that in the presence of PC/PE
membranes the peptide dis-aggregates completely. Previous structural
studies with SIVwt or similar peptides (HIV fusion peptides) have
shown, however, that a significant amount of aggregated -structure
is detected when the peptides are mixed with vesicles (11, 26, 32, 33).
To clarify this further and in order to complement the ThT measurements
with appropriate structural information, SIVwt in the absence and
presence of membranes was studied with ATR-FTIR spectroscopy.
In order to keep the conditions for ATR-FTIR measurements as similar as
possible to those of the FPE and ThT experiments, the lipid/peptide
ratio was maintained within the same ranges. Fig.
4A shows the infrared spectrum
of the SIV peptide in the presence of PC/PE membranes. The spectrum in
the region of the amide I (34, 35) shows a band centered at 1627 cm1 that is characteristic of -sheet aggregated
structures.
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Fig. 4.
A, ATR-FTIR spectrum of SIVwt peptide
(100 µM) mixed 50 mol % PC, 50 mol % PE membranes (2 mM lipid). B, ThT fluorescence variation after
adding 100 µM SIV wt to a 2 mM lipid, PC/PE
vesicle suspension (trace 1), and 10 µM SIVwt
to a 200 µM lipid, PC/PE suspension (trace 2).
ThT was 35 µM. Temperature was 25 °C.
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The ThT fluorescence time course when identical conditions to those of
the infrared measurements apply (100 µM peptide, 2 mM lipid) is shown in Fig. 4B. After the initial
fast increase, the fluorescence decreases, but the excursion does not
return to the initial fluorescence level (before peptide addition).
This indicates that peptide aggregates are still present in the
suspension and is confirmed by the infrared spectrum. These results
support the fact that the ThT dye is effectively detecting the presence of aggregates and allows us to be confident that no aggregates are
present after 10 µM peptide has been added to 200 µM lipid vesicles and the ThT fluorescence has recovered
the initial level (Fig. 4B). This implies that, despite the
equivalent lipid/peptide ratio, the two experimental circumstances in
Fig. 4B reflect two different structural states of the peptide.
The ThT and IR results indicate that when the SIVwt (in
Me2SO) is added and therefore diluted into the vesicle
suspension, the aggregates bind to the membranes and disaggregation
takes place presumably on the membrane surface. This occurs typically until a critical peptide concentration is reached (30 µM
peptide at 200 µM lipid; i.e. at a
lipid/peptide ratio around 6), at which the membranes appear to
saturate and the peptide remains in the buffer, existing as large
essentially colloidal aggregates.
Interaction of SIVwt with Membranes Labeled with Di-8-ANEPPS,
Effect of the Membrane Dipole Potential--
The use of di-8-ANEPPS
permits the study of the same peptide-membrane interaction process as
the FPE-based technique following the variation of a different physical
property of the membrane called the dipole potential (21). Fig.
5 illustrates the dual wavelength
ratiometric method used to detect the variations of the dipole
potential. Upon SIVwt addition, the parameter R(460/520), which is sensitive solely to variations of the local electrical field
due to dipolar molecular properties, exhibited a decrease (Fig.
5A). This means that the interaction of the SIVwt peptide with the membrane promotes a decrease in the dipole potential. In Fig.
5B an excitation difference spectra is shown, exhibiting a
minimum around 450 nm and a maximum around 520 nm. The shape of the
difference spectrum is equivalent to that obtained using compounds such
as phloretin, known to decrease the membrane dipole potential (21, 23).
The fluorescence time-dependent decays shown in Fig.
5A were found to be most closely fitted to a double exponential rate process. The corresponding rate constants are reported
in Table I and may be compared with those calculated from respective
studies with FPE (Fig. 1) and ThT (Fig. 3).
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Fig. 5.
A, dual-wavelength ratiometric
measurement of the dipole potential variation in di-8-ANEPPS-labeled
vesicles, after addition of SIVwt peptide to PC/PE vesicles. The
sample, containing 200 µM lipid, was excited at 460 and
520 nm. The fluorescence was read at 580 nm, and the ratio
R(460/520) was calculated. B, fluorescence
difference spectra obtained by subtracting the excitation spectrum
( em = 580 nm) of di-8-ANEPPS-labeled PC/PE vesicles from
the spectrum of PC/PE vesicles plus SIVwt. Before subtraction the
spectra were normalized to the integrated areas so that the difference
spectra would reflect only spectral shifts. The dye concentration was
10 µM. Temperature was 37 °C.
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To analyze further the reciprocal influence between the dipole
potential and the extent of the peptide interaction, use was made of
the possibility of preparing membranes with compounds such as phloretin
and KC, known to decrease and increase, respectively, the dipole
potential (21, 23, 24 36). The effect of phloretin and KC on the
membrane dipole potential is shown in Fig.
6A, where it can be observed
that the membranes containing phloretin or KC exhibit, respectively, a
lower and a higher R(460/520) value. The initial magnitude
of the membrane dipole potential affects the extent of the dipole
potential variation caused by the peptide. As illustrated in Fig.
6B, the observed variation of R(460/520) is
presented as a function of peptide concentration for PC/PE membranes
containing different amounts of phloretin and KC.
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Fig. 6.
A, time course variation of the ratio
R(460/520) after mixing SIVwt 10 µM with PC/PE
vesicles (trace 1) and PC/PE vesicles containing 15 mol % KC (trace 2) and 15 mol % phloretin (trace 3).
B, variation of the ratio R(460/520) as a
function of SIVwt concentration:  , PC/PE vesicles containing 30 mol
% KC;  , PC/PE vesicles containing 15 mol % KC;  , PC/PE
vesicles;  , PC/PE vesicles containing 15 mol % phloretin.
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Influence of the Magnitude of the Dipole Potential on the Extent of
gp32-dependent Membrane Fusion--
It has been reported
previously that the presence of PE is a prerequisite for SIVwt to
trigger membrane fusion (11, 26, 37) in the manner illustrated in Fig.
7A. In Fig. 7B, the
percentage of fusion is shown to increase as a function of the peptide
concentration in the same range (0-30 µM) in which the
peptide has been shown to interact with the membranes (Fig. 1,
inset, and Fig. 6B). To study the effect of
variations of the dipole potential on fusion, membranes were
supplemented with phloretin or KC (Fig. 7A). The presence of
15 mol % phloretin in the membrane bilayer clearly decreased (by 50%)
the amplitude of the fluorescence signal, indicating that the reduction
of the dipole potential decreases the extent of the fusion process.
Further increasing the phloretin molar fraction did not further
decrease the fluorescence. This is in agreement with the fact that an
increasing concentration of phloretin in the bilayer causes a variation
of the parameter R(460/520) which saturates at about 15 (phloretin/phospholipid) mol % (23, 24). On the other hand, the
presence of KC in the membrane evidently enhanced the percentage of
measured membrane fusion. The results show a clear effect of the
magnitude of the dipole potential on the extent of the membrane fusion
process and perhaps point to a mechanistic relationship.
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Fig. 7.
A, fusion of vesicles induced by SIVwt.
At time 0 the peptide dissolved in Me2SO was added, and the
decrease in fluorescence energy transfer following liposome-liposome
fusion was monitored at 530 nm. Trace 1, PC/PE membranes;
trace 2, PC/PE membranes conatining 30 mol % cholesterol;
trace 3, PC/PE membranes containing 15 mol % phloretin.
Peptide and lipid concentration was 10 and 200 µM,
respectively. B, extent of fusion as a function of peptide
concentration. Lipid concentration was 200 µM.
Temperature was 37 °C.
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DISCUSSION |
Considerable progress has been made over the last few years in
understanding the events that lead to fusion of viruses, such as those
involved in immunodeficiency syndromes, with target membranes, a step
that allows the penetration of the virus in the host cell (52). Much of
this work has focused on defining the role played by the so-called
membrane-fusion peptides and their interactions with the membrane
lipids in this process. SIVwt, the N-terminal fusion peptide of the
gp32 protein of the simian immunodeficiency virus, is a highly
hydrophobic peptide that when dissolved in Me2SO yields an
FTIR spectrum very similar to those of other proteinaceous aggregates,
known to form -pleated sheet structures (26). The existence of such
aggregates in Me2SO is further corroborated in the present
study by the rapid increase in the fluorescence of ThT, a dye known to
intercalate within the -structures of the aggregates (51), observed
when the peptide is added into an aqueous solution. If dissolved in
aqueous buffer, the high insolubility of the peptide triggers the
formation of colloidal aggregates.
The presence of membranes in the solution prior to the addition of
peptide causes disaggregation as the aggregates interact with the
membranes, binding to the surface (FPE measurements) and causing a
variation in the magnitude of the surface and dipole potentials,
according to the results outlined in Figs. 1, 3, and 5.
These results are best understood with the aid of the mechanistic
scheme shown in Fig. 8 which helps
explain the differences observed in the peptide structure when
increasing its concentration from 10 to 100 µM despite
keeping a constant lipid/peptide ratio (i.e. infrared and
ThT measurements shown in Fig. 4). In fact, keeping the lipid/peptide
ratio constant favors the formation of aqueous aggregates; at a peptide
concentration of 10 µM (lipid 200 µM), the
binding of the peptide aggregates (from the added Me2SO
solution) to the membrane and the subsequent disaggregation seems to be
more rapid than the dissociation of the aggregates in water and
subsequent formation of large colloidal aggregates. This seems the most
likely explanation of the complete disaggregation of the peptide
measured with ThT under these conditions. Once the peptide
concentration is increased to 100 µM, the equilibrium corresponding to the dissociation of the peptide in water (leading to
the formation of colloidal aggregates) appears to be shifted to the
right in Fig. 8, whereas the equilibrium with the membranes does not
appear to be affected due to the fact that the experimental lipid
concentration has also been increased to 2 mM,
i.e. keeping the lipid/peptide ratio constant. This appears
to explain the fact that under these experimental conditions the
peptide does not completely disaggregate when added to the vesicle
suspension, as observations with ThT and infrared spectroscopy have
indicated (Fig. 4). These results underline the necessity of paying
particular attention when structural and functional data, derived from
quite different experimental conditions in which the peptide
concentrations are largely different, are compared and may in part
explain the different views taken by different laboratories on the
peptide structures involved in membrane fusion (26, 33).
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Fig. 8.
Schematic mechanism for the interaction of
SIVwt with PC/PE membranes in an aqueous solution.
Pag, peptide aggregates from the Me2SO
stock solution; P(H2O),
peptide diluted in the buffer from the Me2SO concentration
solution; and Pag(H2O),
colloidal aggregates that form in water; M, membranes;
PagM, peptide aggregates interacting with
the membranes; PM, peptide monomers (forming) on the
membranes.
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TOP
ABSTRACT
INTRODUCTION
