(Received for publication, July 29, 1994; and in revised form, October 11, 1994)
From the
The effect that altering amphipathicity has on the folding
process and self association of melittin, a small model protein, has
been investigated using single amino acid substitutions of lysine 7, a
residue distant from the contact residues involved in the hydrophobic
core of tetrameric melittin. While substitutions of such a residue were
not expected to interfere with the packing process, the largest
alterations in the potential overall amphipathicity of melittin were
found to prevent the folding into an -helical conformation to
occur and, in turn, to prevent the self association. Amphipathic
-helices were found to be a key determining feature in the early
folding process of the self association of peptides and protein
segments. Those substitutions, which prevented the inducible
amphipathic folding ability, were also found to result in a loss in
hemolytic and antimicrobial activity. These results, combined with
studies of the binding to artificial liposomes and to polysialic acids,
indicate that the losses in activity were due to an initial inability
to be induced into an amphipathic
-helix and to self associate.
Ultimately, melittin's self association is proposed to be
required to penetrate the carbohydrate barrier present in biological
membranes.
Amphipathic -helices are a ubiquitous structural feature
found in many proteins and biologically active peptides. This
structural motif has been found to play multiple roles in protein
folding, protein-protein recognition, protein-membrane interactions,
and protein and peptide biological activity. Amphipathic
-helices,
in turn, have a pronounced tendency to self associate in defined
structural arrangements (for review, see (1) ). However, due to
the complexity and compound variables involved in such interactions,
the identities of the amino acid residues and/or sequences that
contribute to the folding, self association, stability, and activity of
the native proteins are not well understood. In an effort to study this
problem, a number of groups using protein mutants (2) and guest
peptides(3, 4, 5) are investigating the
propensity of individual amino acid residues to induce specific
secondary structures, regardless of the resulting alteration of the
amphipathicity of peptide and protein domains.
The role and
importance that altering the amphipathicity plays in relation to the
folding process was studied in the present work using melittin, a
model, small tetrameric self assembling protein. Melittin is a
26-residue peptide (GIGAVLKVLTTGLPALISWIKRKRQQ-NH), which
is known to adopt an amphipathic
-helical conformation in the
presence of biological membranes, micelles, and
surfactants(6) . Furthermore, at high peptide concentration or
at high ionic strength, melittin is known to self associate into a
tetrameric structure driven by the formation of a hydrophobic
core(7) . Reduction of electrostatic repulsion between the
positive charges occurring at high pH and/or high salt concentration
was also found to favor the self association of
melittin(7, 8) . Variations of residues distant from
the hydrophobic core and clearly solvent-exposed are not expected to
interfere with the protein packing process and, in turn, should result
in the formation of a similar self aggregational state. Thus, the lack
of self association following substitution or omission of specific
residues in a peptide sequence would tend to imply that the alteration
of the amphipathicity of the modified region and/or the nature of the
mutation are important elements in the early stages of the folding
process. Using x-ray crystallography, the lysine at position 7 in
melittin's sequence has been shown to be fully solvent exposed
following tetrameric self association(9, 10) , and its
microenvironment has been shown to be essentially unaltered upon
tetramerization(11) . Single substitution analogues at position
7 with amino acids having varying chemical properties were synthesized
in the present study to determine the effect of such alterations on
melittin's folding and self association capability, using
circular dichroism (CD) spectroscopy.
In addition to its importance
in the folding process, the role of amphipathic -helices self
association on melittin's and other peptides' biological
activity remains unclear. Thus, the relationship between the tetrameric
self assembly of melittin or mastoparan in solution and their lytic
activities has not been defined, nor has the role of the trimerization
of glucagon. The results obtained in the current structural studies
using melittin's substitution analogues were therefore related to
the differences in the biological activities (hemolytic and
antimicrobial activity) of these substitution analogues in order to
elucidate the effect amphipathic
-helices have on the self
association in relation to the biological activity of peptides.
The concentration dependence of the CD spectra of the peptides in 5
mM MOPS-NaOH buffer at pH 7.0 was analyzed for size-defined
oligomer formation, nM &cjs0635; L, K =
[M]
/[L], where
[M] and [L] are the monomeric and
self-assembled concentrations of the peptides, respectively, n is the degree of association, and K
is a
dissociation constant. The experimentally determined mean molar
ellipticity/residue at 222 nm for each peptide at varying
concentrations was analyzed using the formalism proposed by Taylor and
Kaiser (16) with the software Graphpad (ISI, San Diego, CA).
The guanidinium hydrochloride denaturation studies were carried out by preparing mixtures of a stock solution of peptide in buffer (5 mM MOPS-NaOH, pH 7.0), buffer alone, plus a solution of 7.2 M guanidine HCl in buffer. The ratios of buffer and 7.2 M guanidine HCl solutions were varied to give the appropriate final guanidine HCl concentrations. The samples were allowed to equilibrate for 30 min at room temperature prior to CD measurement. The thermal dependence of ellipticity at 222 nm was analyzed using the program TTSCAN (Jasco) with a step resolution of 0.1 °C and a temperature slope of 20 °C/h. The ellipticity values obtained from both, guanidine HCl and thermal denaturation studies were analyzed using the software Graphpad.
Microdilution assays were
carried out against Escherichia coli ATCC 25922 and E.
coli BAS 849 in 96-well tissue culture plates as described
elsewhere(19) . Following overnight incubation at 37 °C,
the optical density at 620 nm of the bacterial suspension in
Muller-Hinton broth was measured in the presence of serial 2-fold
dilution of peptides. The concentration of peptide necessary to inhibit
50% of the bacterial growth (IC) was then determined for
each peptide using a sigmoidal curve fitting method (Graphpad).
Figure 1:
Concentration-dependent ellipticities
at 222 nm. The CD spectra were recorded in MOPS buffer at varying
peptide concentrations (from 4 to 500 µM).
[]
is plotted for A,
,
melittin;
, subK7D; and &cjs2132;, subK7E; B, +,
subK7A;
, subK7G,
, subK7I;
, subK7L;
,
subK7V; and
, subK7W. Computer generated monomer-tetramer curves
are shown superposed on the data.
It has been shown that melittin is in an unfolded
state at micromolar concentrations under conditions of low ionic
strength, but adopts a tetrameric helical structure under conditions of
high ionic strength (7, 23) . Folding of the eight
melittin substitution analogues as a function of ionic strength was
therefore investigated further (Fig. 2). In agreement with the
concentration-dependent results described above (Fig. 1A), melittin, subK7D, and subK7E were found to
fold into an -helical conformation with increasing NaCl
concentration (Fig. 2A). The NaCl concentration
required to induce 50% folding ([NaCl]
) was
0.73, 0.47, and 0.15 M for melittin, subK7D, and subK7E,
respectively. The CD spectra of each peptide in the presence of varying
NaCl concentration exhibited an isodichroic point at 203 nm, indicating
a two-state equilibrium between a random coil and an
-helix.
Furthermore, the shape of each analogues' CD spectra in the
presence of a high salt concentration indicates the presence of
-helical content.
Figure 2:
Ionic strength-dependent ellipticities.
The CD spectra were recorded in MOPS buffer for a peptide concentration
of 20 µM in the presence of NaCl.
[]
is plotted for A,
,
melittin;
, subK7D; and
, subK7E; B, +,
subK7A;
, subK7G;
, subK7I;
, subK7L;
,
subK7V; and
, subK7W.
In contrast to the concentration dependent
behavior of the three analogues described above (Fig. 1B), two distinctly different behaviors were
observed for the remaining six analogues (Fig. 2B). It
should be noted that these six peptides differ from the preceding ones
by their lack of a charged amino acid at position 7. The -helical
content of subK7A and subK7G increased with increasing ionic strength (Fig. 2B), but not with increasing peptide
concentration ([NaCl]
= 0.5 and 1.0 M, respectively). On the other hand, in the presence of 1 M NaCl, the CD spectra of subK7I, subK7L, subK7V, and subK7W
indicated a predominantly
-sheet structure (minimum around 217 nm;
the shape of the CD spectra is similar to the recently reported spectra
for a predominantly
-sheet protein(24) ). Minimal or a
lack of
-helicity was indicated by the absence of a clearly
defined minimum at 208 nm.
The degree of self assembly of the peptides was also investigated using size exclusion chromatography. Two separate solvent systems were used to mimic the environment used in our CD studies: 5 mM MOPS containing 0.05% trifluoroacetic acid in order to minimize the nonspecific interactions between the peptides and the chromatographic support (no change in conformation for melittin appeared in CD upon decreasing the pH); and 5 mM MOPS-NaOH buffer, pH 7.0, containing 1.5 M NaCl. Both conditions are expected to favor self association. The elution times of the nine peptides, as well as a range of molecular weight standards, are shown in Table 2. In 5 mM MOPS, 0.05% trifluoroacetic acid, all of the peptides had elution times similar to insulin chain B, indicating that they were monomeric. The presence of a minor peak (for subK7I, subK7L, and subK7V) was found at an elution time close to the void volume of the column and is believed to indicate the presence of large nonspecific aggregated structures. In the presence of 1.5 M NaCl, however, melittin, subK7A, subK7D, subK7E, and subK7G appear to be tetramers, as described for melittin(9, 10) . Only monomeric forms were indicated in both chromatographic systems for the other four peptides (subK7I, subK7L, subK7V, and subK7W).
Figure 3:
Guanidine HCl denaturation curves. The CD
spectra were recorded for a peptide concentration of 20 µM in 5 mM MOPS buffer. A, containing 1.2 M NaCl for +, subK7A; , subK7D; and
, subK7E; and
2 M NaCl for
, melittin; and
, subK7G; B, containing 1.2 M NaCl (without NaCl in the inset for subK7I (
), subK7L (
), subK7V (
),
and subK7W (
). Computer generated sigmoidal curves are shown
superimposed on the data.
In a monomer-to-tetramer transition,
the unfolding process should follow a two-state equilibrium model
between folded tetramers (F) and unfolded monomers (U): F
&cjs0635; 4U, with K
=
[U]
/[F
]
= 4Pt3(fu4/(1 - f
)(3, 7) . K
represents the tetramer dissociation constant; P
represents the total peptide concentration; and f
represents the molar fraction of unfolded peptide as determined
from the ellipticity at 222 nm. The formula f
= (
-
)/(
-
) is used to calculate f
, where
represents the observed
ellipticity at a given guanidine HCl concentration, and
and
are the ellipticities of the tetrameric
folded and monomeric unfolded states, respectively. The value K
was calculated in the transition zone for
each guanidine HCl concentration tested. The K
were found to increase logarithmically with guanidine HCl
concentration for the five peptides (mel, subK7A, subK7D, subK7E, and
subK7G). These results suggest the presence of an invariant tetramer
structure, which, upon increasing guanidine HCl concentration, vary in
stability.
The free energy variation in the unfolding process
(G
) was determined at each guanidine HCl
concentration using the formula
G
=
-RT lnK
. The free energy of the unfolding
process in the absence of denaturant (
GuH
O)
was estimated by linear extrapolation of the
G
values to a guanidine HCl concentration equal to zero using the
equation
G
=
GuH
O
- m [guanidine HCl](25) . The
GuH
O and m values for all the peptides are
listed in Table 3. Based on the crystal structure of tetrameric
melittin(9, 10) , position 7 is fully exposed to the
solvent. If one envisions that the stability of a four-bundle or
tetrameric melittin array results solely from the hydrophobic
interactions occurring in the hydrophobic core, one would expect to
find the same stability for all the peptides studied. However, at 25
°C in the absence of denaturant, tetrameric self assembly of subK7E
was found to be approximately 3 kcal/mol more favorable than the
tetrameric self assembly of melittin. SubK7E also had the highest
transition midpoint, indicating greater stability. In contrast, subK7G
had the lowest transition midpoint ([guanidine HCl]
= 0.54 M), which can be related to its elevated m
value. As proposed by Matouschek et al.(25) , an m value of this magnitude suggests a significant exposure to the
solvent of subK7G on denaturation.
The denaturation curves for
subK7I, subK7L, subK7V, and subK7W were determined in 5 mM MOPS-NaOH buffer, pH 7, in the presence or absence of 1.0 M NaCl. As shown in Fig. 3B, the denaturation
process was virtually independent of the initial buffer conditions. In
all cases, the [guanidine HCl] values were found
to be approximately 3 M (Table 3). These results,
combined with those derived from the CD aggregation studies and size
exclusion experiments described above, require a different analysis of
the guanidine HCl denaturation curves for these peptides. If one
assumes that the intrachain interactions or the orientation of given
amino acid residues are determinant factors in the folding of these
analogues, then the unfolding reaction should follow a two-state
equilibrium model between folded and unfolded monomers: F &cjs0635; U, with K
=
[U]/[F] = f
/(1 - f
)(26) . K
represents the equilibrium constant for the
unfolding process, and f
represents the molar
fraction of unfolded peptide as determined from the ellipticity at 222
nm described above (similar K
values were obtained
using f
values generated from ellipticities at 217
nm). The free energy of unfolding in the absence of denaturant
(
GuH
O) was estimated in a similar manner by linear
extrapolation of
G
at each guanidine HCl
concentration to a final guanidine HCl concentration equal to zero (Table 3). The four analogues were found to have virtually
identical stability.
Figure 4: Thermal stability. The ellipticity at 222 nm of each analogue was recorded at a peptide concentration of 20 µM in 5 mM MOPS buffer containing 1.2 M or 2 M NaCl as described in Fig. 3, upon first increasing the temperature from 4 to 90 °C (solid line) and then, using the same sample, decreasing the temperature from 90 to 4 °C (dashed line) for A, subK7D; B, subK7G; and C, subK7W.
For melittin, subK7D, and subK7E (first group of
peptides), the CD spectral changes seen upon thermal denaturation in
the presence of NaCl reflect the occurrence of a transition from a
salt-induced folded state to an unfolded state. This denaturation
process appears to be almost completely reversible upon lowering the
temperature back to its initial value (Fig. 4A). No
significant cold denaturation was observed upon cooling from 25 to 4
°C for melittin and its analogues under our experimental conditions
(2 M NaCl for melittin and subK7G, and 1.2 M NaCl for
the other analogues). The smooth cooperative transition obtained for
these three peptides under the conditions studied suggests the
occurrence of a thermodynamic equilibrium(27, 28) .
The calculated T values for helix-coil transition (i.e. the temperature at which 50% of the peptide is in its
unfolded form) was approximately 60 °C, indicating a stable
-helical array for these peptides (Table 3).
Cooperative
denaturation profiles were also observed for subK7A and subK7G (Fig. 4B) when these peptides were heated from 4 to 90
°C. The lower T values found for subK7A and
subK7G relative to the first group of peptides (41 and 43 °C
respectively) reflect a pronounced loss of thermal stability.
Furthermore, the initial folded state was not recovered upon lowering
the temperature from 90 to 4 °C; rather a nonspecific aggregation
process was observed.
Finally, for those peptides that have a bulky
hydrophobic amino acid at position 7, the change in ellipticity as a
function of temperature was almost linear, indicating a noncooperative
denaturation process (Fig. 4C). These results can be
understood by the peptides' lack of folding into -helices in
aqueous solution(29) .
Figure 5:
CD spectra in the presence of vesicles
for (A) subK7E, (B) subK7G, and (C) subK7I.
The CD spectra were recorded at a peptide concentration of 20
µM in 5 mM MOPS buffer in the presence of egg
PC/PS with R equal to 0 (solidline), 4 (dottedline), 20 (dashedline),
and 50 (dot-dashline).
The binding of each
analog to SUVs was quantitatively analyzed using spectral
enhancement(30) . The ellipticity enhancement is defined as
( -
)/
, where
is
the ellipticity at 222 nm for the peptide in the presence of SUVs and
is the ellipticity for R
= 0. The ellipticity of the fully bound peptide
(
) and the lipid concentration at which half of the
peptide is bound to the vesicles (L
) were
determined using the equation: (
-
)/
= [(
-
)/
]
[L]/([L] + L
), where [L] is the
concentration of lipid (Table 4). Thus, subK7E was found to have
a high binding affinity to SUVs, which resulted in 76%
-helical
content at R
= 16 (value corresponding to
50% melittin bound to the SUV) as estimated using the curve-fitting
procedure describe by Yang et al. ((14) ; Table 4). At this R
value, 45 and 37%
-helical content was estimated for melittin and subK7D,
respectively. All of the other analogues showed approximately 20%
-structure under these conditions. At R
= 50, none of the peptides showed significant
-structure content (Table 4).
The initial driving force
for melittin's interaction with phospholipid membranes is
generally thought to be an electrostatic interaction between the highly
positively charged C-terminal of melittin and the negatively charged
phospholipid head groups(31) . Since each of the peptides
studied here have the same unmodified positively charged C-terminal,
they should all show similar affinity to phospholipid membranes if the
primary binding force is due to electrostatic interactions. However,
each of the peptides has different L values, as
well as different
-helical content upon binding to SUVs (Table 4). Thus, it is unclear if these differences are due to
the method used to determine the binding affinity, since it is assumed
for the calculation of the L
that the peptides
fold into an
-helical conformation upon binding to SUVs. As an
alternative approach, multilamellar vesicles (not sonicated) were used
to determine the binding affinity of melittin and three analogues
(subK7D, subK7G, and subK7L). Although this method is not as accurate
as for the determination of affinity through the use of SUVs, and even
though multilamellar vesicles have slightly different physical
properties than SUVs, this method allows for the evaluation of the
percent of each peptide that did not bind to the vesicles regardless of
the peptide folding propensity. Thus, at an R
of
16, the percentage of nonbound peptide ranged from 9 to 30% (Table 4), which suggests that the amount of peptide bound to the
vesicles is virtually the same for melittin as for the three analogues.
These results can be interpreted if one assumes that, for a determined
peptide density on the bilayer surface, a complex equilibrium between
peptide-lipid, peptide-peptide, and peptide-aqueous solvent exists, and
furthermore that the peptides' ability to disrupt the
phospholipid bilayer depends on which of these interactions is the
predominant one. The finding of a high
-helical content at R
= 50, i.e. when the peptide
surface density was lowered, which, in turn, means that peptide-lipid
interactions were dominant, supports these premises.
Finally, the
ability of melittin and its analogues to bind to cholesterol present in
biological membranes was investigated using egg PC/PS/cholesterol
(62:5:33, mol/mol/mol) liposomes. Overall, lowered affinity parameters (i.e. higher L values; Table 4)
were found in the presence of cholesterol relative to the L
values obtained using SUVs that did not contain
cholesterol. Only subK7W had a similar L
in the
presence of SUVs with and without cholesterol, which may be due to the
known ability of tryptophan residues to assume a defined orientation
when binding to cholesterol (32) .
The CD spectra
of melittin and its analogues were measured in the presence of
colominic acid (poly-2,8-N-acetylneuraminic acid) at different
concentration ratios (R = C/P; where C represents the colominic acid
concentration, and P represents the peptide concentration in
mg/ml). Melittin was induced into an
-helical conformation upon
increasing R
to 1, with no further changes in
ellipticity found upon increasing R
beyond 1 (Fig. 6A). A small amount of turbidity appeared at
ratios lower than 1. In contrast, no helix formation was observed for
melittin in the presence of the monomeric sialic acid (data not shown).
Interestingly, the presence of a negative charge at position 7 in
subK7E or subK7D did not affect the induced conformation in the
presence of negatively charged colominic acid relative to melittin (i.e. increases in helicity upon increasing R
; Fig. 6B). SubK7E showed higher
-helical content than melittin and subK7D under these conditions
in a manner similar to that found in the presence of salt or SUVs. An
-helical conformation was also observed for subK7G and subk7W in
the presence of colominic acid. In contrast, the shape of the CD
spectra of subK7A, subK7I, subK7L, and subK7V revealed a significant
-structure content, with, however, a much wider band at the
-structure minima (Fig. 6C), which suggests that
other structures such as a distorted
-helix were also present.
Figure 6:
CD spectra in the presence of colominic
acid for (A) melittin, (B) subK7E, and (C)
subK7I. The CD spectra were recorded at a peptide concentration of 20
µM, in 5 mM MOPS buffer, in the presence of
colominic acid at ratio R = 0 (solidline), 1 (dottedline), 2 (dashedline), and 6 (dot-dashline).
The antimicrobial activity against E. coli was also determined for melittin and its analogues using two different strains, a strain used in standard assays (ATCC 25922) and a drug more permeable mutant strain (BAS 849). In a manner similar to the activity against treated RBCs, the three peptides (subK7I, subK7L, and subK7V) exhibited no activity against the standard strain of E. coli, while they became highly active against the mutant strain (Table 5). It is also noteworthy that, while subK7W was as active as melittin against the untreated RBCs, it had lower antimicrobial activity against the standard strain of E. coli. This may be due to the occurrence of tryptophan/cholesterol interactions with RBCs, as proposed above in the study of the binding affinity of this analog to SUVs containing cholesterol. The absence of cholesterol in bacterial membranes may account for these differences in hemolytic and antimicrobial activities.
The effects of altering the degree of amphipathicity on the
folding process, as well as the membrane binding ability of peptides,
was examined in the present work using closely related model peptides.
Since position 7 of melittin's sequence is fully exposed to the
solvent in melittin tetramer and, in turn, is distant from the
hydrophobic core(9, 10) , the use of single
substitution analogues at position 7 was expected to provide insight
into the early steps of the folding and self association processes.
Thus, differences in the overall stability of the self-assembled
tetramers resulting from such substitutions should be attributable to
changes in the initial coil to helix transition and not in the
interhelical packing interactions. Furthermore, this alteration affects
only the first -helix in folded melittin, while the hinge, second
-helix, and the basic C-terminal remain unchanged for all the
analogues. This was confirmed by the adoption of a fully
-helical
conformation by all of the analogues in the presence of
trifluoroethanol.
Two approaches are commonly used to study folding
in amphipathic peptides: variation in peptide concentration and
induction by increasing salt concentration. An increase in peptide
concentration results in the folding of amphipathic peptides, driven by
hydrophobic interactions, which in turn are favored by the closer
proximity of peptide molecules. On the other hand, folding in the
presence of increased salt concentration is predominantly driven by the
weakening of charge repulsions. In agreement with the hydrophobic
moment values (<µ>), only those analogues that
remained amphipathic when induced into an
-helix were found to
fold when their concentration was increased (i.e. subK7E and
subK7D). The free energies of tetramerization in salt-free buffer for
subK7D and subK7E (-17.9 and -18.3 Kcal/mol, respectively;
-17.0 kcal/mol for melittin) appear to be consistent with an
electrostatic stabilization in an anti-parallel arrangement between the
negative charge at position 7 and the basic C-terminal region.
Preliminary molecular modeling studies, based on the crystal structure
of tetrameric melittin (9) and generated from the
crystallographic dimer (Protein Data Bank entry 2MLT) using symmetry
and residue replacement operations, suggested that electrostatic
interactions may occur between glutamic acid-7 and the positively
charged residues of the C terminus of the anti-parallel chain of the
same dimer. In contrast, in the case of subK7D, such interactions
appeared to be more favorable between aspartic acid-7 and the basic
C-terminal residues of the equivalent chain in the second dimer. These
potential differences in the amino acids involved in such interactions
may be responsible for the higher stability observed for the tetrameric
form of subK7E. As determined by the guanidine HCl denaturation
studies, the presence of NaCl was found to stabilize the tetrameric
structure of these two analogues, as well as that of melittin, by more
than 7 kcal/mol. These results are in agreement with the earlier
reported stabilization of tetrameric melittin by 5-6 kcal/mol in
the presence of 0.5 M NaCl(8) . These results suggest
that besides the hydrophobic interhelix stabilizing interactions, the
amino acids located in the N- and C-terminal regions in anti-parallel
peptide bundles are involved in overall protein stability.
While the
two analogues subK7A and subK7G remained random at high peptide
concentrations, they folded upon increasing the NaCl concentration.
Both analogues were also found to have lower hydrophobic moments than
melittin, subK7E, or subK7D, and higher changes in entropy than
melittin as determined by the van't Hoff method (S = 62 cal/K
mol and 152 cal/K
mol for subK7A and
subK7G relative to melittin, respectively). It appears that these
losses in amphipathicity are compensated by the decrease in charge
repulsion that occurred at high ionic strength. This results in an
increased propensity for folding induction and is supported by the
occurrence of an additional folding energy in the presence of salt
similar to the one calculated for subK7E and subK7D. The poor folding
capability of subK7A and subK7G was confirmed by their nonreversible
thermal denaturation. Upon lowering the temperature, these peptides
aggregated in a disordered manner at temperatures higher than the T
values, as evidenced by precipitation. However,
the relationship between their inability to refold upon decreasing the
temperature and their inability to fold upon increasing peptide
concentration is uncertain.
In contrast to the above peptides, no
tetrameric folding was observed for subK7I, subK7L, and subK7V at
either high peptide concentration or high ionic strength. These results
indicate that the intermolecular hydrophobic interactions are not
strong enough to compensate for the energetically unfavorable
positioning of these hydrophobic amino acids in the hydrophilic face of
the -helix. A small amount of distorted
-structure was
instead observed in the presence of salt. We believe this results
mainly from the occurrence of intramolecular hydrogen bonding, as
determined by size exclusion chromatography. These results show that
amphipathicity is a determining feature in the early folding process, i.e. in the induction of the appropriate molten globule or
intermediate structures, and, in turn, in the self association ability
of peptides or protein segments.
Artificial liposomes are commonly
used as a model system to study the binding affinity of peptides or
proteins to cell membranes(44, 45) . However, the
binding affinity to SUVs found for melittin's substitution
analogues could not be directly related to the variation in their
hemolytic or antimicrobial activities. The peptide density on the
aqueous/lipid interface also appears to be involved in the adoption of
the active structure, as indicated by the increase in folding
capability observed upon increasing the SUV/peptide ratio (R). The differences observed in folding ability
in the presence of salt and in the presence of SUVs for those analogues
having hydrophobic residues at position 7 lead to the conclusion that
an ionic strength-dependent feature was responsible for the lack of
activity found for these peptides. The increases in activity observed
for these peptides upon removing the charged sugars from the RBCs,
combined with their lack of folding in the presence of colominic acid,
support this hypothesis. The sugar present in biological membranes can
thus be seen as assisting in the induction of self association
(tetramerization in the present case), which would prevent hydrophobic
residues from being exposed to the sugar charges. Such structures can
be expected to be stabilized by the surrounding high ionic strength.
The aggregate would then pass through this first biological barrier to
reach the phospholipid bilayers, and, finally, may act on the membrane
following a still controverted mechanism (either in an aggregate form
or in a folded monomeric form(6, 46, 47) ).