©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Effect of the Arrangement of Tandem Repeating Units of Class A Amphipathic -Helixes on Lipid Interaction (*)

(Received for publication, August 1, 1994; and in revised form, September 26, 1994)

Vinod K. Mishra (1) Mayakonda N. Palgunachari (1) Sissel Lund-Katz (2) Michael C. Phillips (2) Jere P. Segrest (1) G. M. Anantharamaiah (1)(§)

From the  (1)Departments of Medicine and Biochemistry and the Atherosclerosis Research Unit, University of Alabama at Birmingham Medical Center, Birmingham, Alabama 35294 and (2)Biochemistry Department, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha-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(18) reversed-phase high performance liquid chromatography column decreased in the order 36A > 37aA > 37pA, consistent with the lengths of the nonpolar faces of the alpha-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.


INTRODUCTION

Exchangeable apolipoproteins possess the periodic pattern of an alpha-helix with well demarcated polar and nonpolar faces. This periodicity of amino acid arrangement to produce multiple amphipathic alpha-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 alpha-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 alpha-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 alpha-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 alpha-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).




EXPERIMENTAL PROCEDURES

Materials

DMPC (^1)and EYPC, purity >99%, were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Potassium iodide (KI) and acrylamide (>99.9%) were purchased from Fisher and Bio-Rad, respectively. N-Acetyltryptophanamide was obtained from Novabiochem (La Jolla, CA). (1S)-(+)-10-Camphorsulfonic acid (99%), 2,2,2-trifluoroethanol (TFE; 99.5+%, NMR grade), and guanidine hydrochloride (GdnHCl; 99%) were obtained from Aldrich. All other chemicals used were of highest purity commercially available.

Peptide Synthesis

The peptides were synthesized by Fmoc (9-fluorenyl)methoxycarbonyl) solid-phase synthesis(17) . Crude peptides were purified on a preparative Vydac C(4) column (22-mm inside diamter times 25 cm; particle size, 10 µm) by reversed-phase (RP)-HPLC using water (0.1% trifluoroacetic acid, solvent A) and acetonitrile (0.1% trifluoroacetic acid, solvent B). The purities of the peptides, as well as their retention times, were determined by analytical RP-HPLC using a Vydac C(18) column (4.6-mm inside diameter times 25 cm, particle size 5 µm). The gradient employed was 25-70% solvent B in 46 min; the flow rate was 1.2 ml/min. The purity of the peptides as judged by the analytical RP-HPLC was >95%. The composition and masses of the peptides were ascertained by amino acid analysis and electrospray mass spectrometry, respectively.

Preparation of Peptide Solutions

Peptide solutions were prepared by dissolving purified peptide in 6 M GdnHCl solution and dialyzing this solution against phosphate-buffered saline (PBS, pH 7.4; 6.45 mM Na(2)HPO(4)bullet7H(2)O, 1.47 mM KH(2)PO(4), 136.89 mM NaCl, 2.68 mM) KCl in a 1000 M(r) cut-off dialysis tube (Spectrum, Houston, TX). The concentrations of the peptide solutions were determined in 6 M GdnHCl solution by measuring absorbance at 280 nm ( = 14600 M cm). Peptide solutions were stored at 4 °C.

Preparation of Phospholipid Multilamellar Vesicles and Peptide/Lipid Complexes

A chloroform solution of the phospholipid was transferred into a test tube. The solvent was removed under a stream of nitrogen. The residual solvent was removed by storing the tube overnight under high vacuum in a vacuum oven at room temperature. The lipid film thus deposited onto the walls of the glass tube was hydrated by adding appropriate amount of PBS and vortexing the tube at room temperature to form multilamellar vesicles. Peptide/lipid complexes were prepared by adding a measured volume of peptide solution in PBS to a measured volume of the lipid multilamellar vesicles. The mixture was diluted appropriately by adding PBS to obtain the desired lipid to peptide molar ratio. This mixture was incubated overnight at room temperature. In the case of DMPC at a lipid/peptide molar ratio of 20:1, the above treatment resulted in a clear solution of the peptide/lipid complexes. This ratio was selected so as to have no detectable free peptide in the mixture(18) .

Circular Dichroism Studies

CD spectra were recorded using an AVIV 62DS spectropolarimeter (Lakewood, NJ). Details of the measurements have been described previously(19) . A thermoelectric controller was used to vary temperature. The temperature of the solution was monitored by inserting the probe inside the cell. Peptide solutions were incubated for 5 min at a particular temperature before recording their CD spectra. The helical contents of the peptides were estimated from their mean residue ellipticities at 222 nm(20) .

Interaction of Peptides with Phospholipid Monolayers

The relative affinities of the peptides for the lipid-water interface were investigated using a surface balance technique. Employing former procedures(21, 22) , an insoluble monolayer of EYPC was spread at the air-water interface (85 cm^2) in a circular Teflon dish containing 80 ml of phosphate-buffered saline (pH 7.0) at room temperature. The surface pressure () was monitored by the Wilhelmy plate technique using a mica plate connected to a Cahn RTL recording electrobalance. Sufficient EYPC was spread from 9:1 (v/v) hexane:ethanol solution to give an initial surface pressure ((i)) in the range 5-35 dynes/cm. Peptides dissolved in the above buffer containing 1.5 M GdnHCl were injected into the subphase to give an initial concentration of 5 times 10g/dl; a small Teflon tube which projected through the monolayer into aqueous subphase was used for this injection so that the phosphatidylcholine monolayer was not disrupted. The presence of GdnHCl ensured that the peptide molecules were initially present as random coil monomers. The peptide molecules renatured in the subphase as the GdnHCl was diluted to a final concentration of leq 1 mM. The solution was stirred continuously with a magnetic stirrer and the increase in surface pressure (Delta) with time was recorded until a steady-state value of Delta was obtained. The steady-state Delta values were plotted as a function of (i) A linear extrapolation to Delta = 0 dyne/cm gave the exclusion pressure, or the value of (i) at which the peptides were no longer able to penetrate into the EYPC monolayer.

Differential Scanning Calorimetry (DSC)

DSC studies were carried out using high sensitivity Microcal MC-2 scanning calorimeter (Micro Cal, Inc., Amherst, MA) as described previously(19) .

Fluorescence Measurements

Fluorescence emission spectra were recorded using a SLM 8100 Spectrofluorometer (SLM Instruments, Inc., Urbana, IL) as described previously(19) . Details of the fluorescence quenching experiments and the treatment of the data have been described earlier(19) . All the experiments were carried out at 25 °C.


RESULTS

Circular Dichroism Studies

Secondary structures of the peptides in different environments were determined by CD spectroscopy. Fig. 2shows the CD spectra of the three peptides in different environments. All the three peptides show CD spectra characteristic of predominantly alpha-helical conformation(23) . In buffer (Fig. 2A), the helical contents of 36A, 37aA, and 37pA were 88, 83, and 73%, respectively. In 50% TFE (Fig. 2B), the helical contents of 36A, 37aA, and 37pA were estimated to be 74, 72, and 70%, respectively; the lowering of the alpha-helical content in 50% TFE compared to that in buffer is presumably due to the fact that TFE can disrupt hydrophobic interactions and thus can act as denaturant of tertiary and quaternary structures(24, 25) . The CD spectra of the peptides in the presence of DMPC are shown in Fig. 2C. In the presence of DMPC, the alpha-helical contents of 36A, 37aA, and 37pA were estimated to be 89, 95, and 73%, respectively.




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 (circle), 36AbulletDMPC complex (bullet), 37aA in PBS (box), 37aAbulletDMPC complex (), 37pA in PBS (up triangle), 37pAbulletDMPC complex ().





RP-HPLC

RP-HPLC has been used successfully as a probe to study the secondary structure adopted by putative amphipathic helical peptides(27, 28) . The retention times of the peptides on an analytical C(18) column were determined by RP-HPLC. The peptides 36A, 37aA, and 37pA eluted from the column at 36.4, 30.0, and 27.6 min, respectively (Fig. 4). The retention time of the parent peptide 18A was 19.5 min (Fig. 4).


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).



Surface Pressure Measurements

The relative abilities of the three peptides to penetrate an EYPC monolayer were determined by surface pressure measurements. The exclusion pressures for 36A, 37aA, and 37pA were measured to be 35 ± 1, 41 ± 1, and 42 ± 1 dynes/cm, respectively (Fig. 5A). The exclusion pressure for the parent 18A peptide was 30 ± 1 dynes/cm. The various methods of linking 18A helixes had different effects on the rates at which the surface pressure increases associated with peptide penetration occurred (Fig. 5B). Thus, relative to the 18A molecule, 37pA penetrated at about the same rate with the time required to achieve half the maximal increase in surface pressure being close to 10 min for both peptides. The rates of 37aA and 36A penetration were approximately two and four times slower, respectively; the times to reach half the maximum surface pressure increases were about 17 and 35 min for 37aA and 36A, respectively. Of course, the absolute rates of interaction of the peptides with the EYPC monolayer were also dependent on the rate of mixing (stirring) in the subphase.



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 (bullet), 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 (bullet), 18A (), 37aA (), 37pA(). The EYPC monolayer was spread at an initial surface pressure of 10 dynes/cm.



DSC Studies

The effects of the peptides on the thermotropic phase transition properties of DMPC multilamellar vesicles were investigated by DSC at two different lipid/peptide molar ratios (100:1 and 50:1). At these lipid/peptide molar ratios none of the peptides clarified the turbidity due to DMPC vesicles. The heating endotherms of the DMPC vesicles alone and the lipid/peptide mixtures (lipid/peptide molar ratio 100:1) are shown in Fig. 6. DMPC vesicles alone showed endothermic transitions occurring at 13.7 and 23.3 °C which correspond to the pretransition and the gel to liquid-crystalline phase transition, respectively(29) . Addition of the peptides to DMPC vesicles broadened the gel to liquid-crystalline phase transition as well as reduced its enthalpy (Fig. 6). These effects are similar to that observed with other lipid-associating amphipathic helical peptides(30, 31) . However, the peptides showed different capacities to reduce the enthalpy of the gel to liquid-crystalline phase transition of the DMPC vesicles (Fig. 6, Table 2). It is evident from Fig. 6and Table 2that while 36A is least effective, 37pA is most effective in reducing the transition enthalpy of DMPC vesicles. Interestingly, 36A is less effective than the parent peptide 18A in reducing the transition enthalpy of DMPC vesicles (Fig. 6, Table 2).


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).





Fluorescence Studies

The fluorescence emission spectra of the peptides in PBS and in the presence of DMPC are shown in Fig. 7. In PBS, the emission maxima of the three peptides are 341 nm for 36A, 338 nm for 37aA, and 339 nm for 37pA. The peptide 18A has its emission maximum at 349 nm (Fig. 7). The emission maximum of tryptophan is sensitive to its microenvironment and a blue shift is observed when the tryptophan is transferred from a medium of high polarity to a medium of low polarity(32) . The emission maximum of all the peptides shows a blue shift in the presence of DMPC indicating partitioning of the tryptophan residues of the peptides into the membrane. However, the extent of the blue shift observed is not identical for all the peptides. While 37aA and 37pA show similar blue shifts in their tryptophan emission maxima (331 nm and 332 nm, respectively), 36A shows a smaller blue shift (337 nm). Interestingly, 18A shows a larger blue shift (335 nm) compared to 36A (Fig. 7). This indicates that the tryptophan residues in 36A are located in a more polar environment compared to the tryptophan residues in the other peptides. It is interesting to note that the Trp fluorescence emission intensity of 36A in the presence of DMPC is less than that in buffer, whereas for all the other peptides Trp fluorescence emission intensity is more in the presence of the lipid compared with that in buffer.


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 (circle), 36AbulletDMPC complex (bullet), 18A in PBS (down triangle), 18AbulletDMPC complex (), 37aA in PBS (box), 37aAbulletDMPC complex (), 37pA in PBS (up triangle), 37pabulletDMPC 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(0) 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 (circle), 36AbulletDMPC complex (bullet), 18A in PBS (down triangle), 18AbulletDMPC complex, () 37aA in PBS (box), 37aAbulletDMPC complex (), 37pA in PBS (up triangle), 37pAbulletDMPC complex().






DISCUSSION

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 alpha-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(18) 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 alpha-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 alpha-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.


FOOTNOTES

*
This research was supported in part by National Institutes of Health Program Project grants HL34343 and HL22633. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed. Tel.: 205-934-4420; Fax: 205-975-8079.

(^1)
The abbreviations used are: DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; EYPC, egg yolk L-alpha-phosphatidylcholine; PBS, phosphate-buffered saline; RP, reversed-phase; HPLC, high performance liquid chromatography; DSC, differential scanning calorimetry; GdnHCl, guanidine hydrochloride.


ACKNOWLEDGEMENTS

We are grateful to Dr. Donald D. Muccio, Chemistry Department, University of Alabama at Birmingham Medical Center, for the use of the spectropolarimeter.


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