THE JOURNAL OF BIOLOGICAL CHEMISTRY
`O 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 269, No. 10, Issue of March 11, pp. 7185-7191, 1994
`Printed in U.SA.
`
`Interactions of Synthetic Peptide Analogs of the Class A
`Amphipathic Helix with Lipids
`EVIDENCE FOR THE SNORKEL HYP0THESIS*
`
`(Received for publication, October 12, 1993, and in revised form, December 6, 1993)
`
`Vinod K. Mishra, Mayakonda N. Palgunachari, Jere P. Segrest, and G. M. Anantharamaiaht
`From the Departments of Medicine and Biochemistry and the Atherosclerosis Research Unit, University of
`Alabama-Birmingham Medical Center, Birmingham, Alabama 35294
`
`Class A amphipathic helixes present in exchangeable
`plasma apolipoproteins are characterized by the loca-
`tion of positively charged amino acid residues at the
`non-polar-polar interface and negatively charged amino
`acid residues at the center of the polar face. The objec-
`tives of the present study were: (i) to investigate the role
`of hydrocarbon side chain length of the interfacial posi-
`tively charged amino acid residues in the lipid affinity
`of class A amphipathic helixes, and (ii) to investigate the
`importance of the nature of interfacial charge in the
`lipid affinity of class A amphipathic helixes. Toward this
`end, lipid interactions of the following two analogs of
`the class A amphipathic helix, Ac-18A-NH2 (acetyl-Asp-
`Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-
`Lys-Glu-Ala-Phe-NH2), and Ac-18A(Lys>Haa)-NH2
`(acetyl-Asp-Trp-Leu-Haa-Ala-Phe-Tyr-Asp-Haa-Val-Ala-
`Glu-Haa-Leu-Haa-Glu-Ala-Phe-NH2) (Haa = homoamino-
`alanine), were studied. The side chain of Haa has two
`CH2 groups less than that of lysine. The lipid affinities of
`these two peptide analogs were compared with that of
`Ac-18R-NH2, an analog of Ac-18A-NH2 with positions of
`the charged amino acid residues reversed. The tech-
`niques used in these studies were circular dichroism,
`fluorescence spectroscopy, right-angle light scattering
`measurements, and differential scanning calorimetry.
`The results of these studies indicated the following rank
`order of lipid affinity: Ac-18A-NH2 > Ac-18A(Lys>Haa)-
`NH2 > Ac-18R-NH2. These results are in agreement with
`the "snorkel" model proposed earlier to explain the
`higher lipid affinity of class A amphipathic helixes (Seg-
`rest, J. P., Loof, H. D., Dohlman, J. G., Brouillette, C. G.,
`and Anantharamaiah, G. M. (1990) Proteins Struct.
`Funct. Genetics 8, 103-117). In addition, it was observed
`from the differential scanning calorimetry studies that
`Ac-18A-NH2 and Ac-18A(Lys>Haa)-NH2 interact more
`strongly than Ac-18R-NH2 with negatively charged di-
`myristoyl phosphatidylglycerol. The weaker interaction
`of Ac-18R-NH2 with dimyristoyl phosphatidylglycerol is
`suggested to be due to electrostatic repulsion between
`the negatively charged lipid and the interfacial negative
`charges of the peptide.
`
`Exchangeable plasma apolipoproteins are protein detergents
`and are responsible for carrying lipids in circulation in the form
`of lipoproteins. There is now considerable evidence that the
`structural motif that is responsible for their detergent property
`
`* This research was supported in part by National Institutes of
`Health Program Project HL34343. The costs of publication of this ar-
`ticle were defrayed in part by the payment of page charges. This article
`must therefore be hereby marked "advertisement" in accordance with 18
`U.S.C. Section 1734 solely to indicate this fact.
`To whom correspondence should be addressed.
`
`is the amphipathic a-helix (1). While many other proteins and
`biologically active peptides have been found to possess this
`secondary structural motif, the exchangeable apolipoproteins
`are unique in that they possess multiple copies of amphipathic
`a-helixes with positively charged amino acid residues at the
`non-polar-polar interface and negatively charged amino acid
`residues at the center of the polar face (2). The amphipathic
`a-helixes with this kind of distribution of charged amino acid
`residues in the polar face have been classified as class A (3-5).
`The lipid-associating properties of exchangeable apolipopro-
`teins are believed to reside predominantly in the amphipathic
`helical domains possessing the class A motif (3).
`On the basis of the key structural features predicted for the
`lipid-associating properties of the amphipathic helix (1, 2),
`many laboratories, including ours, have studied structural and
`functional properties of de novo designed peptide analogs of the
`amphipathic helix (6-16). We have addressed the question
`whether or not the location of charged amino acid residues on
`the polar face of the class A amphipathic helical peptide play a
`role in determining its lipid affinity (17-19). A model peptide
`with 18 amino acid residues was designed to mimic the general
`features of the amphipathic helix described in the original
`model (2, 17). The model peptide 18A, with primary sequence
`Asp-Trp- Leu-Lys-Ala-Phe-Tyr-Asp- Lys-Val -Al a-Glu-Ly s- Leu-
`Lys-Glu-Ala-Phe, in the helical wheel representation has posi-
`tively charged Lys residues at the non-polar-polar interface and
`negatively charged Asp and Glu at the center of the polar face
`(17). To address the question of contribution of interfacial Lys
`residues to the lipid affinity of 18A, a peptide with the same
`amino acid composition, but the positions of charged amino acid
`residues reversed, was synthesized. The peptide reverse-18A
`(18R) had the following amino acid sequence: Lys-Trp-Leu-Asp-
`Ala-Phe-Tyr-Lys-Asp-Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala-Phe. A
`helical wheel representation of this sequence shows negatively
`charged amino acid residues Asp and Glu at the non-polar-
`polar interface and positively charged Lys residues at the cen-
`ter of the polar face (17). Studies on these two peptides and
`their analogs showed that class A amphipathic peptides with
`positively charged residues at the non-polar-polar interface and
`negatively charged residues at the center of the polar face have
`higher lipid affinity compared with the corresponding charge-
`reversed peptide analogs (17-20).
`To explain the higher lipid affinity of class A amphipathic
`helixes, the "snorkel" model was proposed (3). The bulk of the
`van der Waals surface area of the positively charged lysine and
`arginine residues is hydrophobic. In the snorkel model, it was
`proposed that the interfacial positively charged amino acid
`residues of the peptide, when associated with lipid, extend
`toward the polar face of the helix to insert their charged moi-
`eties into the aqueous environment for solvation. Thus, in the
`snorkel orientation, the entire uncharged van der Weals sur-
`face of the class A amphipathic helix is buried within the hy-
`
`7185
`
`This is an Open Access article under the CC BY license.
`
`MYLAN EXHIBIT - 1030
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`7186
`
`Lipid Interactions of Amphipathic Helical Peptides
`
`drophobic interior of a lipid bilayer.
`Apolipoprotein A-I (apoA-I),' the major protein constituent of
`plasma high density lipoproteins, and class A amphipathic hel-
`ical peptide analogs have been shown to stabilize the bilayer
`structure of phospholipids, and these properties were corre-
`lated to their ability to protect against lytic peptide-induced
`erythrocyte lysis (21). These molecules also exhibit anti-viral
`and anti-inflammatory properties and protect fatty acid con-
`taining dye-entrapped phospholipid vesicles from albumin-in-
`duced leakage (22-24). These properties were attributed to the
`snorkeling effect of interfacial Lys residues in these molecules.
`This snorkeling effect was thought to create a "wedge" cross-
`sectional shape which is responsible for the stabilization of
`phospholipid vesicles (21, 24). It was, therefore, important to
`determine if indeed the longer hydrocarbon side chain of inter-
`facial Lys residues increases the lipid-associating ability, which
`is a key determinant of the properties of apoA-1 and the corre-
`sponding peptide analogs.
`Earlier, we reported that N- and C-terminal protection of 18A
`drastically increased its helicity as well as lipid affinity and the
`resulting peptide, Ac-18A-NH2 (acetyl-Asp-Trp-Leu-Lys-Ala-
`Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe-NH2),
`closely mimicked the properties of apoA-I (25). In the present
`report, we compare the lipid affinity of Ac-18A(Lys>Haa)-NH2
`(acetyl-Asp-Trp-Leu-Haa-Ala-Phe-Tyr-Asp-Haa-Val-Ala-Glu-
`Haa-Leu-Haa-Glu-Ala-Phe-NH2) with that of Ac-18A-NH2 and
`Ac-18R-NH2
`(acetyl-Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-
`Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala-Phe-NH2). The side chain of
`Haa residue has two methylene groups less than that of Lys.
`The peptide Ac-18A(Lys>Haa)-N112 was designed to investigate
`the role of longer hydrocarbon side chain of the interfacial
`lysine residues in the lipid affinity of Ac-18A-NH2. The three
`peptides were studied for their ability to interact with dimy-
`ristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidyl-
`glycerol (DMPG), and egg yolk phosphatidylcholine (EYPC)
`multilamellar vesicles. The following criteria were used for the
`lipid affinity of these peptides (26-28): (i) increase in the a -hel-
`ical structure in the presence of the lipid as detected by far-UV
`CD spectra; (ii) blue-shift in the tryptophan emission maxi-
`mum and shielding from the aqueous-phase quenchers, iodide
`and acrylamide, in the presence of the lipid as monitored by
`steady-state fluorescence spectroscopy; (iii) clarification of the
`turbidity due to EYPC multilamellar vesicles as measured by
`right-angle light scattering; and (iv) reduction in the transition
`enthalpy and broadening in the gel to liquid-crystalline phase
`transition of the DMPC and DMPG multilamellar vesicles as
`measured by differential scanning calorimetry (DSC).
`
`EXPERIMENTAL PROCEDURES
`Materials—Synthesis and purification of the peptide Ac-18A-NH2
`has been described earlier (25). Peptide Ac-18R-NH2 was synthesized
`and purified following similar methodology (20). For the synthesis of the
`peptide Ac-18A(Lys>Haa)-NH2, Boc-Haa(Z), instead of Boc-Lys(2-C1Z),
`was used. The amino acid derivative was prepared using the procedure
`described elsewhere (29). The other steps in the synthesis and purifi-
`cation were similar to those described earlier for Ac-18A-NH2 (25).
`DMPC, DMPG, and EYPC, purity > 99%, were purchased from
`Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further pu-
`rification. Potassium iodide (KO was obtained from Fisher Scientific
`Company, and acrylamide (>99.9%) was purchased from Bio-Rad. (1S )-
`(TFE,
`(99%), 2,2,2-trifluoroethanol
`(+)-10-Camphorsulfonic acid
`
`The abbreviations used are: apoA-I, apolipoprotein A-I; Boc, terti-
`ary-butyloxycarbonyl; Z, benzyloxycarbonyl; (2C1)Z, 2-chlorobenzyloxy-
`carbo-nyl; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG,
`1,2-dimyristoyl-sn-glycero-3-lphospho-rac-(1-glyceronlisodium salt);
`EYPC, egg yolk t-a-phosphatidylcholine; NATA, N-acetyltryptophan-
`amide; PBS, phosphate-buffered saline; TFE, 2,2,2-trifluoroethanol;
`DSC, differential scanning calorimetry; Haa, homoaminoalanine; Ac,
`acetyl; NH2, carboxamide.
`
`99.5+%, NMR grade), and guanidine hydrochloride (99%) were obtained
`from Aldrich. All other chemicals were of highest purity commercially
`available.
`Preparation of Peptide Solutions—Peptide solutions were prepared
`by dissolving the peptide in 4 M guanidine hydrochloride and dialyzing
`against phosphate-buffered saline (PBS, pH 7.4; KH2PO4 1.47 ms,
`Na2HPO4.7H2O 6.45 ms, NaCl 136.89 rnm, KCl 2.68 mm) extensively
`(overnight, with at least three buffer changes). All the studies described
`below were done in the same buffer. Peptide concentrations were deter-
`mined in 4 M guanidine hydrochloride by measuring absorbance at 280
`nm (€ 20. = 7300 m-1 cm-').
`Circular Dichroism—The CD spectra were recorded with an AVIV
`62DS spectropolarimeter interfaced to a personal computer. The instru-
`ment was calibrated with (1S )-(+)-10-camphorsulfonic acid (30, 31). The
`CD spectra were measured from 260 to 190 nm every nm with 1 s
`averaging per point, and a 2-nm bandwidth. An 0.01-cm path length cell
`was used for obtaining the spectra. All CD spectra were signal averaged
`by adding four scans, base-line corrected, and smoothed. All the CD
`spectra were recorded at 25 °C. Temperature was regulated with a
`Lauda RS2 circulating water bath. Final peptide concentrations of 100
`pi or less were used for obtaining the CD spectra. DMPC multilamellar
`vesicles suspension was prepared by dissolving a known amount of lipid
`(-10 mg) in ethanol in a test tube and evaporating the solvent slowly
`under a stream of dry nitrogen. The residual solvent was removed by
`storing the tube under high vacuum overnight in a vacuum oven at
`25 °C. To the dried lipid film appropriate volume of buffer was added to
`give a final lipid concentration of 14.7 mm. The lipid film was hydrated
`by vortexing the mixture for 30 min at room temperature. Peptide-
`DMPC complexes for CD studies were prepared as follows. Appropriate
`volume of peptide solution in buffer was added to the DMPC multila-
`mellar vesicles suspension to give lipid to peptide molar ratio of 20:1.
`The mixture was incubated overnight at room temperature. This
`yielded a clear solution of the peptide-DMPC complex.
`The mean residue ellipticity, lO1mRE(deg-cm2.dmol-1), was calculated
`using the following equation:
`
`[O1,
`
` = MRW.0/10.c.1
`
`where, MRW is mean residue weight of the peptide, O is the ob(sEergVeld)
`ellipticity in degrees, c is the concentration of the peptide in g/rril, and
`1 is the path length of the cell in centimeters. The percent helicity of the
`peptide was estimated from the following equation:
`
`(Eq. 2)
`
`% a helix = (l0 i222 + 3,000)/(36,000 + 3,000)
`where, [01222 is the mean residue ellipticity at 222 nm (32).
`fluorescence emission
`Fluorescence Measurements-Steady-state
`spectra were recorded in the ratiometric mode on an SLM 8000C photon
`counting spectrofluorometer (SLM Instruments, Inc., Urbana, IL) at
`25 °C. Excitation and emission slit widths were both 4 nm. An excitation
`wavelength of 280 nm was used for recording emission spectra. Wave-
`lengths at which maximum emission occurred (AOOO,) were determined
`by positioning the cursor manually at the peak maximum and reading
`the corresponding wavelength. The final peptide concentration used
`was 14 gm (A280 = 0.1).
`Fluorescence quenching experiments were performed using 295-nm
`excitation wavelength in order to minimize absorptive screening by the
`quenchers used. Aliquots (10 ul) of freshly prepared potassium iodide (4
`M) or acrylamide (4 M) stock solutions were added to a constantly stirred
`and thermostated (25 °C) 2-ml peptide solution. After every addition of
`the quencher, the emission spectra were recorded from 310 to 450 nm,
`and the emission intensity at Am. were determined as described above.
`Stock solution of potassium iodide contained 1 mm sodium thiosulfate
`(Na2S2O3) in order to prevent I3 formation (33). For acrylamide quench-
`ing, corrections for inner filter effects (€ 295 = 0.25 m--1 cm-1, for acryl-
`amide) were made (34). The fluorescence quenching data were analyzed
`according to the Stern-Volmer equation for the collisional quenching
`(35):
`
`FVF = 1 + Ksv[Q] = 70/r= 1 + iz,t0[Q]
`
`(Eq. 3)
`
`where, F. and F are fluorescence intensities in the absence and pres-
`ence of quencher, respectively, K. is Stern-Volmer constant for the
`collisional quenching process, fQJ is quencher concentration, +0 and r
`are fluorescence lifetimes of the flurophore in the absence and presence
`of the quencher, respectively, and kq is the rate constant for the bimo-
`lecular quenching process. The above equation predicts a linear plot of
`Fo/F (or +0/+) versus [Q] for a homogeneously emitting solution. The
`slope of this plot yields the value of K.„.
`
`

`

`Lipid Interactions of Amphipathic Helical Peptides
`
`7187
`
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`Wavelength (nm)
`Wavelength (nm)
`Flo 1. Far-UV CD spectra of peptides (---100 um) in buffer (PBS, pH 7.4) (A), 50% (v/v) TFE/PBS (B), and DMPC complex (lipid/
`peptide molar ratio 20:1) (C). Ac-18A-NH2 (O), Ac-18A(Lys>Haa)-NH2 (A), Ac-18R-NI-12 (0).
`
`Wavelength (nm)
`
`Right-angle Light Scattering Measurements—The association of the
`peptides with EYPC multilamellar vesicles was followed by monitoring
`the rate of clarification of the turbidity due to the vesicles (36). The rate
`of turbidity clarification was measured by right-angle light scattering
`using SLM 8000C photon counting spectrofluorometer with both exci-
`tation and emission monochromators set at 400 nm. Data were acquired
`using slow time based acquisition. EYPC multilamellar vesicles sus-
`pension was prepared as described above for the DMPC. The sample
`containing 105 pm EYPC and an equimolar amount of the peptide was
`maintained at 25 °C and was continuously stirred. Complete dissolution
`of the EYPC vesicles was achieved by the addition of Triton X-100 to the
`vesicles suspension at a final concentration of 1 mm.
`Differential Scanning Calorimetry—The high sensitivity DSC stud-
`ies were performed in Microcal MC-2 scanning calorimeter (MicroCal,
`Inc., Amherst, MA) at the scan rate of 20`h- ' at an instrumental sen-
`sitivity of 1 and with a filtering constant of 10 s. The lipid multilamellar
`vesicles and the peptide-lipid mixtures for DSC were prepared as fol-
`lows. DMPC or DMPG (-2 mg) was dissolved in chloroform in a test
`tube and dried by slow evaporation under a stream of dry nitrogen. The
`residual solvent was removed under high vacuum in the vacuum oven
`as described above. To the dried lipid film either buffer alone or buffer
`containing the peptide was added to obtain lipid/peptide molar ratio
`100:1. The lipid was hydrated by vortexing at room temperature for 30
`min. The suspension was degassed for 30 min, and an aliquot of L2 ml
`of suspension was used and run against buffer in the reference cell.
`Four consecutive scans with 60 min equilibration time between each
`scan were run for the same sample. No significant changes in the
`thermograms were observed between the first scan and the fourth scan.
`The observed transitions were analyzed using software provided by
`MicroCal, Inc., Amherst, MA.
`
`RESULTS
`CD Studies—Secondary structures of the peptides were de-
`termined by recording their far-UV CD spectra in buffer (PBS,
`pH 7.4), in 50% (v/v) TFE/PBS, and in the presence of DMPC
`(lipid/peptide molar ratio 20:1). The CD spectra are shown in
`Fig. 1. The CD spectrum of an a-helix is characterized by two
`negative bands, one at 222 nm and another at 208 nm, and a
`positive band at 192 nm (37). The CD spectrum of Ac-18A-NH2
`in buffer (Fig. 1A) indicates that the peptide is largely helical.
`As estimated from mean residue ellipticity at 222 nm [O1222,
`the peptide is 55% a-helical. The CD spectrum of Ac-18R-NH2
`indicates that it is less helical (43%) than Ac-18A-NH2. The CD
`spectrum of Ac-18A(Lys>Haa)-NH2 in buffer is distinct from
`the CD spectra of the other two peptides because of its greatly
`reduced mean residue ellipticity values as well as a large shift
`in the zero cross-over point (wavelength at which mean residue
`ellipticity is zero). This suggests a largely non-helical structure
`of the peptide in buffer. The peptide was estimated to have 22%
`a-helical structure.
`TFE is a most commonly used structure-inducing, hydropho-
`bic, cosolvent (38). CD spectra of the three peptides in 50% (v/v)
`TFE/PBS were recorded to estimate their a-helical contents in
`a relatively hydrophobic environment. As is evident from Fig.
`
`O
`
`0
`
`325
`
`350
`375
`100
`Wavelength loin)
`
`• 5
`
`450
`
`FIG. 2. Fluorescence emission spectra of peptides in buffer
`(PBS, pH 7.4) and peptide-DMPC complexes (lipid/peptide mo-
`lar ratio 20:1). Peptide concentration = 14 pm, excitation wavelength =
`280 nm. Ac-18A-NH2 in buffer (O), Ac-18A-NH2-DMPC complex (6),
`Ac-18A(Lys>Haa)-NH2 in buffer (z), Ac-18A(Lys>Haa)-NH2-DMPC
`complex (A), Ac-18R-NH2 in buffer (0), Ac-18R-NIL-DMPC complex
`( • ).
`
`1B, among the three peptides, Ac-18A-NH2 has the maximum
`a-helical structure (67%). The a-helical contents ofAc-18R-NH2
`and Ac-18A(Lys>Haa)-NH2 are less than that of Ac-18A-NH2
`(61 and 59%, respectively). However, a closer inspection of the
`CD spectra of the peptides in TFE reveals that while Ac-
`18A(Lys>Haa)-NH2 has a zero cross-over point (202.7 nm)
`nearly identical with that of Ac-18A-NH2 (202.6 nm), the zero
`cross-over point of Ac-18R-NH2 is shifted toward a shorter
`wavelength (202 nm). This indicates that Ac-18A(Lys>Haa)-
`NH2 has a higher a-helical content than Ac-18R-NH2 in this
`solvent system (39).
`The CD spectra of peptide-DMPC complexes (lipid/peptide
`molar ratio 20:1) are shown in Fig. 1C. When complexed with
`the lipid, all the three peptides are largely a-helical. The three
`peptides were estimated to have the following a-helical con-
`tents: Ac-18A-NH2
`72%, Ac-18R-NH2 62%, and Ac-
`18A(Lys>Haa)-NH2 56%. We would like to point out that the
`a-helical content of Ac-18A-NH2 in DMPC complex was earlier
`reported to be 92% (25). This value was recently found to be an
`overestimation because of the improper calibration of the spec-
`tropolarimeter.
`Fluorescence Measurements—The fluorescence emission
`maximum of tryptophan is highly sensitive to its microenviron-
`ment (40). The microenvironments of tryptophan residues in
`the peptides were probed by recording their emission spectra in
`buffer and in the presence of DMPC (lipid/peptide molar ratio
`20:1). The emission spectra are shown in Fig. 2. All three pep-
`tides disrupt DMPC vesicles completely at lipid/peptide molar
`ratio 20:1 resulting in an optically clear solution. In buffer
`
`

`

`7188
`
`Lipid Interactions of Amphipathic Helical Peptides
`
`TABLE I
`Tryptophan emission maxima and fluorescence quenching parameters of the peptides and the peptide-DMPC complexes
`Peptide concentration was 14 um; lipid/peptide molar ratio was 20:1. Fluorescence emission maxima were determined using excitation wave-
`length of 280 nm; quenching studies were done using excitation wavelength of 295 nm. Ksv is Stern-Volmer quenching constant determined from
`the slopes of the lines for the plots of Fo/F-1 versus [Q]; slopes were determined by linear regression analysis of the fluorescence quenching data
`using the least squares method.
`
`Peptide
`
`Ac-18A-NH2
`Ac-18A(Lys>Haa)-NH2
`Ac-18R-NH2
`NATA
`* Not determined.
`
`(nm)
`
`DMPC
`complex
`
`329
`331
`336
`ND°
`
`Buffer
`
`343
`344
`344
`350
`
`Ksv (hi')
`
`Buffer,
`iodide
`quenching
`
`5.7
`7.2
`8.0
`11.0
`
`DMPC
`complex,
`iodide
`quenching
`1.4
`3.7
`5.4
`ND
`
`Buffer,
`acrylamide
`quenching
`
`13.5
`15.5
`17.4
`27.7
`
`DMPC
`complex,
`acrylamide
`quenching
`2.6
`6.2
`10.3
`ND
`
`2.0
`
`0.
`
`O 40 GO 00 100 120 140 ISO 100 200 220
`
`[Iodide] mil
`
`0
`
`20 40 SO SO l00 120 I40 16U
`
`100 200 220
`
`tAcrylemldel mil
`Fin. 3. Stern-Volmer plots of fluorescence quenching of the
`peptides in buffer (PBS, pH 7.4), and peptide-DMPC complexes
`(lipid/peptide molar ratio 20:1) by iodide (A) and acrylamide (B).
`Peptide concentration = 14 pm, excitation wavelength = 295 nm. Ac-
`(O), Ac-18A-NH2-DMPC complex (0), Ac-
`in buffer
`18A-NH2
`18A(Lys>Haa)-NH2 in buffer (1k), Ac-18A(Lys>Haa)-NH2-DMPC com-
`plex (A), Ac-18R-NH2 in buffer ( 0), Ac-18R-NH2-DMPC complex( • ),
`NATA in buffer (E).
`
`alone, while Ac-18A-NH2 has its emission maximum (Amex) at
`343 nm, Ac-18R-NH2 and Ac-18A(Lys>Haa)-NH2 both have
`their emission maxima at 344 nm. When complexed with
`DMPC, all the three peptides show an increase in the emission
`intensity as well as a blue-shift in their emission maxima com-
`pared with those in buffer, indicating partitioning of trypto-
`phan into a more hydrophobic environment. However, the ex-
`tent of the blue-shift in the tryptophan emission maximum,
`compared with that in buffer, is different for the three peptides.
`In the presence of DMPC, while Ac-18A-NH2 emission maxi-
`mum shows a blue-shift of 14 nm, Ac-18A(Lys>Haa)-NH2 and
`Ac-18R-NH2 show blue-shifts of 13 and 8 nm, respectively
`(Table I). This indicates that the tryptophan residue in Ac-18R-
`NH2 is in a less hydrophobic environment than the tryptophan
`residues in Ac-18A-NH2 and Ac-18A(Lys>Haa)-NH2.
`
`O
`
`C
`
`SS
`
`O
`
`O
`
`0
`
`o
`
`400
`
`SOO
`
`1200
`
`'GOO
`
`2000
`
`,•••••.•
`
`Time (tlee)
`Fin. 4. Rate of decrease of scattered-light intensity measured
`at 90° from a 105 pm suspension of EYPC vesicles. Equimolar
`amounts of the peptides were added to the lipid suspension. Complete
`dissolution was achieved by adding Triton X-100 solution to the lipid
`suspension at a final concentration of 1 inm. Excitation and emission
`wavelengths both were 400 nm. EYPC alone (M), Ac-18A-NH2 (0), Ac-
`18A(Lys>Haa)-NH2 (A), Ac-18R-NH2 ( • ), Triton X-100 (El).
`
`To further probe the location of the tryptophan residues of
`the three peptides in the lipid bilayer, fluorescence quenching
`experiments were carried out. Iodide and acrylamide were used
`as aqueous-phase quenchers of the tryptophan fluorescence
`(35). While iodide is an anionic quencher, acrylamide is a neu-
`tral but polar quencher. Iodide is considered to have access only
`to surface tryptophans, whereas acrylamide has good access to
`all but the most highly buried tryptophan residues (41). The
`Stern-Volmer plots of the quenching of tryptophan fluorescence
`by iodide and acrylamide are shown in Fig. 3. For comparison,
`Stern-Volmer plots of fluorescence quenching of NATA (N-acet-
`yltryptophanamide), a model compound for free tryptophan, in
`buffer by iodide and acrylamide are also included. In buffer,
`compared with NATA, tryptophans in all the three peptides are
`less exposed to the quenchers. In the presence of DMPC, com-
`pared with buffer, tryptophans in all the three peptides become
`less accessible to the quenchers, suggesting shielding by the
`lipid bilayer. However, the extent of shielding from the quench-
`ers is different for the three peptides. In the presence of DMPC,
`compared with buffer, the following order was observed in the
`shielding of the tryptophan residue of the three peptides from
`the quenchers: Ac-18A-NH2 > Ac-18A(Lys>Haa)-NH2 > Ac-18R-
`NH2. Thus, among the three peptides, tryptophan in Ac-18R-
`NH2 experiences least shielding in going from buffer to the lipid
`bilayer. The Stern-Volmer quenching constants for a bimolecu-
`lar collisional quenching process were calculated from the ap-
`parent slopes of the plots of Fo/F-1 versus [Q] (slopes were
`calculated by linear regression analysis using the "least
`squares" method; 0.989 < r < 0.999) and are given in Table I.
`The Stern-Volmer plots of the fluorescence quenching by acryl-
`amide for the three peptides as well as that of NATA in solution
`
`

`

`Lipid Interactions of Amphipathic Helical Peptides
`
`7189
`
`absence and in the presence of the peptides by DSC. The heat-
`ing endotherms of the pure lipid vesicles and the peptide-lipid
`mixtures (lipid/peptide molar ratio 100:1) are shown in Fig. 5.
`At neutral pH, while DMPC is zwitterionic, DMPG is nega-
`tively charged. Interactions of the peptides with DMPG vesicles
`were studied to investigate possible electrostatic interactions
`between the positively charged amino acid residues of the pep-
`tides and the negative charges of the lipid. DMPC vesicles
`alone exhibited endothermic transitions at 13.7 and 23.1 °C,
`which correspond to the pretransition (lamellar to periodic gel)
`and the gel to liquid-crystalline (order-disorder) phase transi-
`tion of the lipid, respectively (43). Addition of peptides to the
`DMPC vesicles resulted in lowering of the transition enthalpy
`and broadening of the gel to liquid-crystalline phase transition
`of DMPC vesicles (Table II). Among the three peptides, Ac-18A-
`NH2 caused the maximal reduction in the enthalpy of the main
`phase transition, while Ac-18A(Lys>Haa)-NH2 is intermediate
`to Ac-18A-NH2 and Ac-18R-NH2 (Table II). None of the peptides
`changed the chain-melting temperature (T„) of DMPC vesicles
`by more than 0.2 °C. While the pretransition in the DMPC
`vesicles is observed with Ac-18A(Lys>Haa)-NH2 and Ac-18R-
`NH2, it is not detected in the presence of Ac-18A-NH2. Similar
`to DMPC vesicles, DMPG vesicles exhibited endothermic tran-
`sitions at 11.5 and 22.7 °C, corresponding to the pretransition
`and the main phase transition of the lipid, respectively (44).
`The effect of the three peptides on the phase transition prop-
`erties of the DMPG vesicles is similar to that observed with
`DMPC (Table II). The maximum change in T, of the lipid
`vesicles observed in the presence of the peptides is less than
`0.3 °C. The width at half of the peak height of the main phase
`transition in DMPG is more than that in DMPC (Table ID. This
`is presumably due to the poor packing of the lipid molecules in
`the bilayer because of electrostatic repulsion between the nega-
`tive charges in the head group of the phospholipid molecules
`(36).
`
`DISCUSSION
`A comparison of the CD spectra of the three peptides in the
`DMPC complex (Fig. 1C) shows the following rank order of
`helicity: Ac-18A-NH2 > Ac-18R-NH2 > Ac-18A(Lys>Haa)-NH2.
`While the same order is observed in the helicity of the three
`peptides in buffer (Fig. 1A), the helicity of Ac-18A(Lys>Haa)-
`NH2 in the presence of TFE is more than that of Ac-18R-NH2
`(Fig. 1B). In the presence of TFE, Ac-18A(Lys>Haa)-NH2 was
`estimated to have less helical structure than Ac-18R-NH2
`based on the mean residue ellipticity at 222 nm of the two
`peptides. However, comparison of the zero cross-over point of
`the two peptides revealed that Ac-18A(Lys>Haa)-NH2 has more
`helical structure than Ac-18R-NH2 (39). It has been pointed out
`that since the shape and zero cross-over of a CD spectrum
`directly reflect the proportional contribution of different sec-
`ondary structures, any genuine change in secondary structure
`would lead to a change in either the shape or the zero cross-
`over, or both, of that spectrum (39). Therefore, it is important to
`note that a comparison of helicity should not be based on ellip-
`ticity values alone. The results of the CD studies indicate that
`the lipid affinities of Ac-18R-NH2 and Ac-18A(Lys>Haa)-NH2
`are less than the lipid affinity of Ac-18A-NH2. The lipid affinity
`of Ac-18A(Lys>Haa)-NH2, based on the results of the CD stud-
`ies alone, is lower than that of Ac-18R-NI-12.
`A comparison of the fluorescence emission maxima of the
`peptides, however, reveals that in the DMPC complex, while
`tryptophan in Ac-18A(Lys>Haa)-NH2 is in a less hydrophobic
`environment than the tryptophan in Ac-18A-NH2, it is in a
`more hydrophobic environment than the tryptophan in Ac-18R-
`NH2 (Table I). These conclusions are further supported by the
`results from the fluorescence quenching studies (Fig. 3). The
`
`A
`
`(I)
`
`(a)
`
`Endothermic
`
`ID
`
`16
`
`20
`
`26
`
`50
`
`Temperature (cC)
`
`B
`
`(I)
`
`Ili)
`
`wn
`
`(JO
`
`Endothermic
`
`15
`
`20
`
`25
`
`30
`
`Temperature (CC)
`FIG. 5. DSC heating thermograms of lipids and lipid-peptide
`mixtures (lipid/peptide molar ratio 100:1). A, DMPC alone (1.5 ma)
`(i), DMPC + Ac-18R-NH2 (ii), DMPC + Ac-18A(Lys>Haa)-NH2 (iii),
`DMPC + Ac-18A-NH, (iv). B, DMPG alone (1.4 mM) (i), DMPG + Ac-
`18R-NH2 (ii), DMPG + Ac-18A(Lys>Haa)-NH2 (iii), DMPG -4- Ac-18A-
`N112 (it)).
`
`TABLE II
`Phase transition parameters for lipids and peptide-lipid mixtures as
`determined by DSC
`DMPC concentration 1.5 ma, DMPG concentration 1.4 ma, lipid/
`peptide molar ratio 100:1. T,,, is the temperature at which excess heat
`capacity is maximum; Alto is calorimetric enthalpy; AlivH is Van't Hoff
`enthalpy; AT1/2 is width of the transition at half of the peak heat capac-
`ity.
`
`Sample
`
`DMPC
`DMPC/Ac-18A-NH2
`DMPC/Ac-18A(Lys>Haa)-NHz
`DMPC/Ac-18R-NH2
`DMPG
`DMPG/Ac-18A-NI-12
`DMPG/Ac-18A(Lys>Haa)-NH2
`DMPG/Ac-18R-NH2
`
`T,,,
`oc
`23.1
`23.0
`23.3
`23.1
`22.3
`22.2
`22.0
`22.1
`
`AHvii
`
`ATv.
`
`kcal I mol
`5.4
`4284.4
`3.3
`1389.6
`4.1
`1566.8
`4.2
`2537.5
`5.5
`1171.1
`3.0
`480.4
`3.8
`568.4
`4.8
`674.3
`
`°C
`0.2
`0.6
`0.6
`0.3
`0.6
`0.8
`0.7
`0.4
`
`(Fig. 3B) curve upward, indicating the occurrence of static
`quenching (42).
`Right-angle Light Scattering Measurements—Interactions