(Received for publication, August 1, 1994; and in revised form, September 26, 1994)
From the
Exchangeable apolipoproteins possess tandem repeating units of
class A amphipathic helical segments and many of them are linked
together by proline residues. To understand the optimal arrangement of
the amphipathic helixes for lipid association, we have studied the
interactions of three model class A amphipathic helical peptides with
lipids. The three peptides are: 37pA, a dimer of 18A
(DWLKAFYDKVAEKLKEAF) linked together by a Pro (18A-Pro-18A); 37aA, a
dimer of 18A linked together by an Ala (18A-Ala-18A); and 36A, a dimer
of 18A without any linker residue (18A-18A). Circular dichroism (CD)
spectra showed that the peptides are predominantly -helical in
aqueous and lipid environments. Temperature dependent CD studies
indicated that in buffer helix stability decreases in the order 36A
> 37aA > 37pA; however, in the presence of dimyristoyl
phosphatidylcholine (DMPC), the above order is reversed. The retention
times of the peptides on a C
reversed-phase high
performance liquid chromatography column decreased in the order 36A
> 37aA > 37pA, consistent with the lengths of the nonpolar faces
of the
-helixes being in the same order; the retention time of the
parent 18A was shorter than 37pA. While 37pA adsorbed to egg
phosphatidylcholine monolayers most strongly, the degree and rate of
association of 36A were significantly lower. Differential scanning
calorimetry indicated that, while 37pA was most effective in reducing
the enthalpy of the gel to liquid-crystalline phase transition of DMPC
multilamellar vesicles, 36A was least effective; 36A was even less
effective than 18A. Fluorescence quenching experiments with iodide and
acrylamide indicated that, in the presence of DMPC, Trp residues in 36A
are most exposed to the quenchers while in 37pA they are least exposed.
In the presence of DMPC, shielding of Trp in 18A from the quenchers was
more than that observed with Trp residues in 36A. The results of this
study suggest that the arrangement of tandem repeating amphipathic
helical units which results in the formation of a class A amphipathic
helix with a nonpolar face longer than five or six turns reduces the
ability of the helix to associate with phospholipid.
Exchangeable apolipoproteins possess the periodic pattern of an
-helix with well demarcated polar and nonpolar faces. This
periodicity of amino acid arrangement to produce multiple amphipathic
-helixes is encoded into the genomic structure of these proteins (1) . All except apolipoprotein A-IV show a remarkable
similarity in having four exons and three introns(1) . The most
striking feature of these exchangeable apolipoproteins is the presence
of internal 11-residue long amino acid repeats. In apolipoproteins A-I,
A-IV and E, the 11-mer repeats have evolved into 22-mer tandem repeats,
with most of these repeats having the periodicity of a class A
amphipathic
-helix(2, 3, 4) . The class
A amphipathic helixes are characterized by the location of positively
charged amino acid residues at the polar-nonpolar interface and
negatively charged amino acid residues at the center of the polar
face(5) . The occurrence of the tandem 22-mer repeats means
that there exists a possibility for amphipathic helixes significantly
longer than 22 residues with possible lack of register of the polar and
nonpolar faces of the two identical tandem amphipathic class A
helixes(4) . Based on the molecular hydrophobicity potential,
Brasseur (6) has shown that the hydrophobic contours are
smaller than the hydrophilic contours in the tandem helixes in apoA-I.
The formation of discoidal particles with phospholipid was explained on
the basis of shielding of the hydrophobic face of the amphipathic helix
at the edge of the lipid bilayer(6) . It follows that the
relative areas of the hydrophilic and hydrophobic faces in the
amphipathic helixes influence the lipid-associating properties of the
exchangeable apolipoproteins. Another factor that might influence the
lipid binding ability of exchangeable apolipoproteins and which has not
been studied in detail so far is the arrangement of tandem repeating
amphipathic helixes with respect to one another.
The amino acid proline is commonly found between tandem repeating amphipathic helixes in plasma exchangeable apolipoproteins; such an arrangement is most noticeable in the exchangeable apolipoproteins A-I and A-IV(3, 4) . Previous studies have shown that if proline is incorporated within a lipid-associating amphipathic helical sequence of 20 residues or less, the lipid-associating ability of the peptide is reduced(7) . It is believed that proline interrupts the helix and thus decreases the lipid-associating ability of a given short amino acid sequence(7) . We and others have shown that proline incorporation between the two amphipathic helixes, which are individually capable of interacting with the lipid, increases the lipid-associating ability, probably due to cooperative effects between the two helixes(8, 9, 10) .
In the present
study, we compare the lipid interactions of three synthetic peptides
containing two tandem repeating units of class A amphipathic helical
segments. The class A amphipathic helical peptide with the sequence
AspTrpLeuLysAlaPheTyrAspLysValAlaGluLysLeuLysGluAlaPhe (referred to as
18A) was designed by us and studied by us and others (11, 12, 13) and has been shown to interact
with phospholipid(8, 11) . The three peptides studied
in the present investigation are 37pA, 37aA, and 36A. The peptide 37pA
is a dimer of 18A punctuated by a Pro (18A-Pro-18A) and was designed to
mimic apo A-I which has tandem amphipathic helical repeats linked
together by Pro residues. It has been shown that 37pA closely mimics
many properties of apo A-I(8, 14, 15) . In
the peptide 37aA, the proline residue of 37pA is replaced by an alanine
residue (18A-Ala-18A); this peptide is less effective than 37pA in
protecting small unilamellar vesicles composed of dioleoyl
phosphatidylethanolamine and oleic acid against bovine serum
albumin-induced lysis(16) . In the present study, we have
further investigated the lipid-associating properties of 37aA and 37pA.
The lipid-associating properties of the above two peptides have been
compared with that of 36A (18A-18A), in which the linker residue (Pro
in 37pA and Ala in 37aA) is deleted. This peptide was designed to form
a longer class A amphipathic -helix in which the polar and
nonpolar faces are aligned along the length of the entire molecule.
Helical wheel representations of the amino acid sequences of the three
peptides in the fully
-helical state indicate that, while in 37pA
and 37aA the faces of the two 18 residue long putative amphipathic
helical segments are not aligned, they are in register in 36A (Fig. 1). The linker residues in 37pA and 37aA cause the faces
of the two 18A
-helical segments to be out of register by 100
° (Fig. 1). The results of this study show that the degree
of alignment of the faces of different tandem repeating amphipathic
helical segments in a peptide can affect its lipid-associating
properties.
Figure 1: Helical wheel diagrams of 37aA (left) and 36A (right) assuming that each molecule is completely helical. The amino acid residues present on the hydrophobic face of the amphipathic helixes are circled heavily. Note that while the hydrophilic and hydrophobic faces in 36A are aligned, they are out of register by 100 ° in the case of 37aA. The arrangement of the amino acids in the case of 37pA is identical with that of 37aA except for the substitution of Ala in 37aA by Pro (indicated by an arrow).
Figure 2: Far-UV CD spectra of the peptides (100 µM) in PBS (pH 7.4) (A), 50% (v/v) TFE/PBS (B), and DMPC (2 mM) (C), at 25 °C. Path length of the optical cell, 0.1 mm; 36A (solid lines), 37aA (dotted lines), 37pA (dashed lines).
To measure the stability of the peptide helixes in aqueous solution and in the presence of DMPC, the CD spectra of the peptides were obtained as a function of temperature(26) . The results are shown in Fig. 3. As estimated from the mean residue ellipticity at 222 nm, the helical contents of the peptides decrease linearly with increasing temperature. The extent of decrease in the helical contents of the peptides was measured by estimating the slopes of the lines obtained by least squares fit of the data points using linear regression analysis (Table 1). A comparison of these slopes indicates that lipid-free 36A forms the most stable helix while 37pA forms the least stable helix. In the presence of DMPC, however, the stability of 37pA is maximum while 36A forms the least stable helix.
Figure 3:
Mean residue ellipticities of the
peptides in PBS (pH 7.4) and in the presence of DMPC (peptide
concentration 10 µM, lipid/peptide molar ratio 20:1) at
222 nm, []
, as a function of temperature.
Path length of the optical cell, 2 mm. 36A in PBS (
),
36A
DMPC complex (
), 37aA in PBS (
), 37aA
DMPC
complex (
), 37pA in PBS (
), 37pA
DMPC complex
(
).
Figure 4: Reversed-phase HPLC chromatograms of the peptides. Details are given under ``Experimental Procedures.'' 36A (dotted line), 37aA (broken line), 37pA (dashed-dotted line), 18A (solid line).
Figure 5:
Interaction of peptides with EYPC
monolayers. A, the increases in surface pressure induced by
penetration of the peptides are plotted as a function of the initial
surface pressure of the EYPC monolayer: 36A (), 18A (
),
37aA (
), 37pA(
). The straight lines are
least-squares fit to the data points. (See ``Experimental
Procedures'' for more detains.) B, the increase in
surface pressure as a function of time are shown for the four peptides:
36A (
), 18A (
), 37aA (
), 37pA(
). The EYPC
monolayer was spread at an initial surface pressure of 10
dynes/cm.
Figure 6: DSC heating endotherms of DMPC multilamellar vesicles (lipid concentration 1.5 mM) and lipid/peptide mixtures (lipid/peptide molar ratio 100:1). DMPC vesicles alone (i), DMPC + 36A (ii), DMPC + 18A (iii), DMPC + 37aA (iv), and DMPC + 37pA (v).
Figure 7:
Fluorescence emission spectra of the
peptides. Peptide concentration = 7 µM,
lipid/peptide molar ratio = 20, slit width = 4 nm,
excitation wavelength = 280 nm. 36A in PBS (),
36A
DMPC complex (
), 18A in PBS (
), 18A
DMPC
complex (
), 37aA in PBS (
), 37aA
DMPC complex
(
), 37pA in PBS (
), 37pa
DMPC complex
(
).
To further probe the location of the tryptophan residues of the peptides in the lipid bilayer, fluorescence quenching experiments were carried out. Two types of aqueous phase quenchers of the tryptophan fluorescence were used, namely, iodide and acrylamide(33) . While iodide is a charged quencher, acrylamide is polar but uncharged. Results of the quenching experiments in the form of Stern-Volmer plots are shown in Fig. 8. In solution as well as in the presence of DMPC, the fluorescence emission of 36A is quenched more by iodide compared to the quenching of 37aA and 37pA peptides (Fig. 8A). In solution, while the fluorescence emission of 37pA is quenched more compared to 37aA by iodide, in the presence of DMPC, this order is reversed (Fig. 8A). Thus, in the presence of DMPC, the accessibility of the tryptophans present in the three peptides decreases in the following order: 36A>37aA>37pA. Acrylamide is a very efficient quencher of tryptophan fluorescence; it has been shown to quench the fluorescence of all but the most deeply buried tryptophan residues(34) . With acrylamide also, in the presence of DMPC, the above mentioned order for the accessibility of the tryptophan residues in the three peptides to the quencher is observed (Fig. 8B). These data suggest that the tryptophan residues in 37pA are most shielded in the lipid bilayer while those in 36A are least shielded. In presence of the lipid, in agreement with the extent of the blue shift, the tryptophan fluorescence of 36A is quenched more than that of 18A (Fig. 8, A and B). The data obtained from the fluorescence studies are summarized in Table 3.
Figure 8:
Stern-Volmer plots of fluorescence
quenching of the peptides by iodide (A) and acrylamide (B). Peptide concentration = 7 µM,
lipid/peptide molar ratio = 20, slit width = 4 nm,
excitation wavelength = 295 nm. F is
fluorescence intensity in the absence of the quencher and F is
fluorescence intensity in the presence of the quencher. N-acetyltryptophanamide in PBS (
), 36A in PBS (
),
36A
DMPC complex (
), 18A in PBS (
), 18A
DMPC
complex, (
) 37aA in PBS (
), 37aA
DMPC complex
(
), 37pA in PBS (
), 37pA
DMPC
complex(
).
In view of the discoidal structure that exchangeable apolipoproteins such as apoA-I forms with DMPC, and given the thickness of the hydrocarbon region of the DMPC bilayer (which varies from 30 Å in the gel phase to about 20 Å in the liquid-crystalline phase), it is believed that amphipathic helixes capable of forming five to six helical turns (18 or 22 residues in length) are of proper length to be able to arrange on the edge of the disc(35, 36) . This being the case, much attention has been paid to the design of 18-22-residue long amphipathic helical peptides. Studies have also been done to understand the minimal length of the amphipathic helix needed to interact with phospholipid bilayers(31) . While these studies have given insights into the structural features of exchangeable apolipoproteins responsible for their lipid association, not much attention has been paid to the involvement of amphipathic helical segments longer than 22 residues which, due to helix length considerations alone, may have different lipid-associating properties.
In the present study, we have compared
the lipid-associating properties of three synthetic class A amphipathic
helical peptides to determine the optimal arrangement of the
amphipathic helical segments for lipid association. Results of the CD
studies indicate that the peptides 36A, 37aA, and 37pA adopt a
predominantly -helical conformation in different environments (Fig. 2). It is interesting to note that although in solution
36A possesses the most stable helical conformation while 37pA forms the
least stable helix, in the presence of DMPC this order is reversed (Fig. 3, Table 1). Accommodation of a Pro residue in the
middle of a helix results in two less hydrogen bonds and a bend near
the Pro(37) . In addition, the presence of a Pro residue in
37pA might facilitate the formation of a turn between the two
amphipathic helical segments. Thus, 37pA is expected to form a less
stable helix in solution in the absence of any other helix-stabilizing
interactions. Interaction with DMPC results in stabilization of the
37pA helix compared to 37aA and 36A helixes (Fig. 3, Table 1).
Separation of peptides by RP-HPLC is primarily due
to the different hydrophobic interactions of the peptides with the
alkyl groups of the stationary
phase(27, 38, 39) . It has been shown that
the retention time in RP-HPLC increases progressively with the length
of the hydrophobic face of the helix(27) . Fig. 1shows
that the faces of the two 18-residue long putative amphipathic helical
segments in 36A are aligned to form a longer continuous hydrophobic
face whereas, in the 37-residue peptides, incorporation of either a Pro
or an Ala residue displaces the faces of the helical segments by 100
°. The difference in retention times of 37pA and 37aA might arise
because, as has been mentioned above, Pro in 37pA may facilitate the
formation of a turn between the two amphipathic helical segments
whereas this is unlikely in 37aA. Since the spacer residue in either
37aA or 37pA gives rise to a discontinuous hydrophobic face, as
compared to the continuous longer hydrophobic face in 36A, the longer
retention time of 36A on a C RP-HPLC column compared to
37aA and 37pA is expected (Fig. 4). As expected, the parent
peptide 18A, in which the length of the hydrophobic face is the
smallest, has the shortest retention time (Fig. 4).
The
results of the surface pressure measurements indicate that 37pA and
37aA are both equally able to penetrate an EYPC monolayer (Fig. 5A). The 36A molecule that contains two 18A
amphipathic helixes linked directly is significantly less able to
penetrate the monolayer because it is excluded at a pressure of 35
dynes/cm (Fig. 5A). However, the longer helix in 36A
confers higher surface activity than that of the parent 18A molecule
which is excluded at surface pressures greater than 30 dynes/cm. The
monolayer data suggest that relative to either 5-turn (18A) or 10-turn
-helixes (36A), more complete insertion of the helixes among the
EYPC molecules occurs if there are two amphipathic helical segments
present with their nonpolar faces separated by Pro (37pA) or twisted
out of register (37aA) (Fig. 1). The disruption of the helixes
at five turns also influences the rate of penetration into the
phospholipid monolayer (Fig. 5B). Thus, the presence of
Pro between the two 18A helixes permits as rapid penetration as occurs
with the shorter 18A molecule; this rapid adsorption of polypeptides
containing multiple amphipathic helixes separated by Pro residues may
have physiological significance in apolipoprotein exchange between
lipoprotein particles. Lengthening the
-helix presumably increases
the molecular rigidity and slows penetration. It is interesting that
the presence of two, out-of-register, 5-turn amphipathic helixes in
37aA halves the rate of penetration whereas the presence of a 10-turn
helix with the nonpolar face in register along its length reduces the
rate of penetration of 36A approximately 4-fold.
Lipid-associating amphipathic helixes have been shown to modify the thermotropic phase transition properties of the phospholipid vesicles (30, 31) . Results of the DSC studies indicated that while 37pA is the most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of DMPC multilamellar vesicles, 36A is the least effective (Fig. 6, Table 2). Reduction in the enthalpy of the gel to liquid-crystalline phase transition of the DMPC vesicles in the presence of the peptides reflects lowering in the amount of energy required to melt the acyl chains of the phospholipid molecules because of the perturbation of the bilayer structure. It follows that two five-turn helixes joined by a Pro residue disrupt the acyl chain packing of the DMPC bilayer more than the 10-turn helix with a long nonpolar face (36A). Interestingly, 36A the dimer of 18A, is less effective than 18A in reducing the transition enthalpy of DMPC vesicles (Fig. 6, Table 2).
The blue shift in the tryptophan emission maximum and reduced accessibility of the tryptophan to aqueous phase quenchers like iodide and acrylamide in the presence of the lipid have been used as criteria for the lipid affinity of the peptide(40) . The smallest blue shift in the tryptophan emission maximum in the presence of DMPC was observed in the case of 36A compared to the other peptides (Fig. 7, Table 3). The results of the quenching experiments show that in the presence of DMPC, while tryptophan residues in 37pA are least exposed to aqueous phase quenchers, in 36A they are most exposed (Fig. 8, A and B, Table 3). In agreement with the DSC data, these results also indicate that the shielding of Trp residues in 36A in the DMPC bilayer is even less than that of 18A (Fig. 8, A and B, Table 3). It is interesting to note that in 36A the Trp fluorescence emission intensity in the presence of DMPC is less than that in buffer. The higher emission intensity of 36A in buffer presumably results because of stronger peptide-peptide hydrophobic interaction which leads to greater self-association and shielding of Trp residues from the aqueous phase. In the presence of DMPC, it is possible that while Trp20 is in a more hydrophobic environment compared to that in buffer, Trp2, which is close to the N-terminal end, may be exposed to aqueous phase. This is likely because the length of the helix in 36A is presumably longer than that required to fit onto the lipid bilayer. Thus, while in all the other peptides there is an increase in the fluorescence emission intensity in the presence of the lipid compared with that in buffer, in the case of 36A there is a decrease in the emission intensity.
Taken together, these results indicate that among the three peptides 37pA has optimal lipid-associating properties. A possible explanation for this observation is as follows. As has already been mentioned, the presence of a Pro residue in 37pA may facilitate the formation of a turn between the two amphipathic helical segments both of which can associate with the lipid. In 37aA and 36A, however, such a bending is unlikely because there is no helix-disrupting residue between the two 18A segments. This seems to result in a reduction in the hydrophobic surface area of the amphipathic helical segments which is in contact with the lipid perhaps due to a mismatch with the thickness of the lipid bilayer. The resultant increase in the hydrophobic surface area of the amphipathic helical segments which is not in contact with the lipid, and therefore might be exposed to aqueous phase, creates an energetically unfavorable situation. Differences in the lipid-associating properties of 37aA and 36A can probably be explained by the presence of a face-aligned long amphipathic helix in 36A enhancing self-association via strong peptide-peptide hydrophobic interaction. In 37aA, because the faces of the amphipathic helical segments are not aligned, such interaction is presumably less favorable for self-association. The results of the present study clearly show that longer (10 turns) amphipathic helixes (36A and 37aA) associate less well with lipids such as DMPC and EYPC than the five-turn helixes joined by Pro (37pA).
In this connection it is interesting to note that a long class A amphipathic helix present in the carboxyl-terminal end of apolipoprotein E (amino acid residues 202-243) has been shown not to associate with the lipid(41, 42) . The authors propose that this is most probably because of the low hydrophobicity of this segment(41) . Our results also indicate that the lipid-associating properties of longer class A amphipathic helixes are impaired. However, this effect is not attributable to the lower hydrophobicity of the amphipathic helixes in the case of peptides studied here. Results of this study show that the arrangement of amphipathic helixes plays an important role in determining the lipid-associating properties of the peptides.