Tue Journat or BioLocicaL CHEMISTRY
`© 1994 by The American Society for Biochemistry and MolecularBiology, Inc.
`
`Vol. 269, No. 10, Issue of March 11, pp. 7185-7191, 1994
`Printed in U.S.A.
`
`Interactions of Synthetic Peptide Analogs of the Class A
`Amphipathic Helix with Lipids
`EVIDENCE FOR THE SNORKEL HYPOTHESIS*
`
`(Received for publication, October 12, 1993, and in revised form, December6, 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 residuesat the center of the polar face. The objec-
`tives of the present study were:(i) to investigate the role
`of hydrocarbonside chain length of the interfacial posi-
`tively charged aminoacid residuesin 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-NH, (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)-NH,
`(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
`CH, groupsless than thatof lysine. The lipid affinities of
`these two peptide analogs were compared with that of
`Ac-18R-NHg, an analog of Ac-18A-NH, 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.
`Theresults of these studies indicated the following rank
`order of lipid affinity: Ac-18A-NHz > Ac-18A(Lys>Haa)-
`NH, > Ac-18R-NH2. These results are in agreement with
`the “snorkel” model proposed earlier to explain the
`higherlipid 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-NHz, and Ac-18A(Lys>Haa)-NH, interact more
`strongly than Ac-18R-NH, with negatively charged di-
`myristoyl phosphatidylglycerol. The weaker interaction
`of Ac-18R-NH, with dimyristoyl phosphatidylglycerol is
`suggested to be due to electrostatic repulsion between
`the negatively charged lipid and the interfacial negative
`chargesof the peptide.
`
`
`Exchangeable plasmaapolipoproteins 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 paymentof page charges. Thisarticle
`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 otherproteins and
`biologically active peptides have been found to possess this
`secondary structural motif, the exchangeable apolipoproteins
`are uniquein 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).
`Onthebasis of the key structural features predicted for the
`lipid-associating properties of the amphipathic helix (1, 2),
`manylaboratories, including ours, have studied structural and
`functional properties of de novo designed peptide analogsof the
`amphipathic helix (6-16). We have addressed the question
`whetheror not the location of charged amino acid residues on
`the polar face of the class A amphipathic helical peptide play a
`role in determiningits 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-Ala-Glu-Lys-Leu-
`Lys-Glu-Ala-Phe,in the helical wheel representation hasposi-
`tively charged Lys residuesat the non-polar-polar interface and
`negatively charged Asp and Glu at the centerof 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
`aminoacid composition,but the positions of charged aminoacid
`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 residuesat the non-polar-polar interface and
`negatively charged residuesat the centerof the polar face have
`higher lipid affinity compared with the corresponding charge-
`reversed peptide analogs (17-20).
`To explain the higherlipid 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 environmentfor solvation. Thus, in the
`snorkel orientation, the entire uncharged van der Waals sur-
`face of the class A amphipathic helix is buried within the hy-
`
`This is an Open Accessarticle under the CC BYlicense.
`
`7185
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`7186
`
`Lipid Interactions ofAmphipathic Helical Peptides
`
`99.5+%, NMRgrade), and guanidine hydrochloride (99%) were obtained
`drophobic interior of a lipid bilayer.
`from Aldrich. All other chemicals were of highest purity commercially
`Apolipoprotein A-I (apoA-I), the major protein constituent of
`available.
`plasmahigh density lipoproteins, and class A amphipathichel-
`Preparation of Peptide Solutions—Peptide solutions were prepared
`ical peptide analogs have been shownto stabilize the bilayer
`by dissolving the peptide in 4 m guanidine hydrochloride and dialyzing
`structure of phospholipids, and these properties were corre-
`against phosphate-buffered saline (PBS, pH 7.4; KH,PO, 1.47 mm,
`lated to their ability to protect against lytic peptide-induced
`Na,HPO,.7H20 6.45 ma, NaCl 136.89 mm, KC] 2.68 mm) extensively
`(overnight, with at least three buffer changes). All the studies described
`erythrocyte lysis (21). These molecules also exhibit anti-viral
`below were donein the samebuffer. Peptide concentrations were deter-
`and anti-inflammatory properties and protect fatty acid con-
`mined in 4 m guanidine hydrochloride by measuring absorbance at 280
`taining dye-entrapped phospholipid vesicles from albumin-in-
`nM (€gg9 = 7300 m7! cm-!).
`duced leakage (22-24). These properties were attributed to the
`Circular Dichroism—The CD spectra were recorded with an AVIV
`snorkeling effect of interfacial Lys residues in these molecules.
`62DSspectropolarimeter interfaced to a personal computer. Theinstru-
`This snorkeling effect was thought to create a “wedge” cross-
`mentwascalibrated with (1S)-(+)-10-camphorsulfonic acid (30, 31). The
`sectional shape which is responsible for the stabilization of
`CD spectra were measured from 260 to 190 nm every nm with 1 s
`averagingper point, and a 2-nm bandwidth. An 0.01-cm path lengthcell
`phospholipid vesicles (21, 24). It was, therefore, important to
`was used for obtaining the spectra. All CD spectra were signal averaged
`determine if indeed the longer hydrocarbon side chainof inter-
`by adding four scans, base-line corrected, and smoothed. All the CD
`facial Lys residuesincreases the lipid-associating ability, which
`spectra were recorded at 25°C. Temperature was regulated with a
`is a key determinantof the properties of apoA-I and thecorre-
`Lauda RS2circulating water bath. Final peptide concentrations of 100
`sponding peptide analogs.
`po or less were used for obtaining the CD spectra. DMPC multilamellar
`Earlier, we reported that N- and C-terminal protection of 18A
`vesicles suspension was prepared by dissolving a known amountoflipid
`drastically increasedits helicity as well as lipid affinity and the
`(~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
`resulting peptide, Ac-18A-NH, (acetyl-Asp-Trp-Leu-Lys-Ala-
`storing the tube under high vacuum overnight in a vacuum oven at
`Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe-NH,),
`25 °C. To the driedlipid film appropriate volumeof buffer was added to
`closely mimicked the properties of apoA-I (25). In the present
`giveafinal lipid concentration of 14.7 mm. Thelipid film was hydrated
`report, we comparethelipid affinity of Ac-18A(Lys>Haa)-NH,
`by vortexing the mixture for 30 min at room temperature. Peptide-
`(acetyl-Asp-Trp-Leu-Haa-Ala-Phe-Tyr-Asp-Haa-Val-Ala-Glu-
`DMPC complexes for CD studies were prepared as follows. Appropriate
`Haa-Leu-Haa-Glu-Ala-Phe-NHg,) with that of Ac-18A-NH. and
`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.
`Ac-18R-NHz__(acetyl-Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-
`The mixture was incubated overnight at room temperature. This
`Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala-Phe-NH,). The side chain of
`yielded a clear solution of the peptide-DMPC complex.
`Haa residue has two methylene groups less than that of Lys.
`The meanresidueellipticity, {O]mre(deg-cm?-dmol-}), was calculated
`The peptide Ac-18A(Lys>Haa)-NHg wasdesignedto investigate
`using the following equation:
`the role of longer hydrocarbon side chain of the interfacial
`lysine residues in the lipid affinity of Ac-18A-NHg. 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 presenceof the lipid as detected by far-UV
`CD spectra; (ii) blue-shift in the tryptophan emission maxi-
`mum andshielding 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 broadeningin the gel to liquid-crystalline phase
`transition of the DMPC and DMPG multilamellar vesicles as
`measuredbydifferential scanning calorimetry (DSC).
`
`[luz = MRW-0/10.c.1
`
`(Eq. 1)
`
`where, MRWis meanresidue weightof the peptide, © is the observed
`ellipticity in degrees, c is the concentration of the peptide in g/ml, and
`lis the path length of thecell in centimeters. The percenthelicity of the
`peptide was estimated from the following equation:
`
`% a helix = ({O@],o + 3,000)(36,000 + 3,000)
`
`(Eq. 2)
`
`EXPERIMENTAL PROCEDURES
`
`Materials—Synthesis and purification of the peptide Ac-18A-NH2
`has been described earlier (25). Peptide Ac-18R-NH,2 was synthesized
`and purified following similar methodology (20). For the synthesis ofthe
`peptide Ac-18A(Lys>Haa)-NHp, Boc-Haa(Z), instead of Boc-Lys(2-C1Z),
`was used. The aminoacid 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-NH, (25).
`DMPC, DMPG, and EYPC, purity > 99%, were purchased from
`Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further pu-
`rification. Potassium iodide (KI) was obtained from Fisher Scientific
`Company, and acrylamide (>99.9%) was purchased from Bio-Rad. (1S)-
`
`
`
`
`acid (99%), 2,2,2-trifluoroethanol(+)-10-Camphorsulfonic (TFE,
`
`1 The abbreviations used are: apoA-I, apolipoprotein A-I; Boc, terti-
`ary-butyloxycarbonyl, Z, benzyloxycarbonyl; (2Cl)Z, 2-chlorobenzyloxy-
`carbo-nyl; DMPC,1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG,
`1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)(sodium salt);
`EYPC, egg yolk L-a-phosphatidylcholine; NATA, N-acetyltryptophan-
`amide; PBS, phosphate-buffered saline; TFE, 2,2,2-trifluoroethanol;
`DSC, differential scanning calorimetry; Haa, homoaminoalanine; Ac,
`acetyl; NH», carboxamide.
`
`where,[Ole is the meanresidueellipticity at 222 nm (32).
`Fluorescence Measurements—Steady-state fluorescence emission
`spectra were recorded in the ratiometric mode on an SLM 8000C photon
`counting spectrofluorometer (SLM Instruments, Inc., Urbana, IL) at
`25 °C. Excitation and emissionslit widths were both 4 nm.An excitation
`wavelength of 280 nm wasusedfor recording emission spectra. Wave-
`lengths at which maximum emission occurred (Amax) were determined
`by positioning the cursor manually at the peak maximum andreading
`the corresponding wavelength. The final peptide concentration used
`was 14 pM (Aggy = 0.1).
`Fluorescence quenching experiments were performed using 295-nm
`excitation wavelength in order to minimize absorptive screening by the
`quenchersused.Aliquots (10 pl) of freshly prepared potassium iodide (4
`M) or acrylamide (4 M) stock solutions were addedto 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,
`andthe emission intensity at Ama, were determinedas described above.
`Stock solution of potassium iodide contained 1 mm sodium thiosulfate
`(Na,S,0O3)in order to preventI; formation (33). For acrylamide quench-
`ing, corrections for innerfilter effects (€295 = 0.25 m7? cm", for acryl-
`amide) were made(34). The fluorescence quenching data were analyzed
`according to the Stern-Volmer equation for the collisional quenching
`(35):
`
`(Eq. 3)
`F/F = 1 + Ksy[Q] = 19/7 = 1+ &71Q]
`where, Fy and F are fluorescence intensities in the absence and pres-
`ence of quencher, respectively, K,, is Stern-Volmer constant for the
`collisional quenching process, [QJ is quencher concentration, t) and T
`are fluorescence lifetimes of the flurophore in the absence and presence
`of the quencher, respectively, and k, is the rate constant for the bimo-
`lecular quenching process. The above equation predicts a linearplot of
`F)/F (or 70/1) versus [Q] for a homogeneously emitting solution. The
`slope of this plot yields the valueof K,,.
`
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`Lipid Interactions ofAmphipathic Helical Peptides
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`Fic. 1. Far-UV CD spectra of peptides (~100 pm) in buffer (PBS, pH 7.4) (A), 50% (v/v) TFE/PBS (B), and DMPC complex (lipid/
`peptide molarratio 20:1) (C). Ac-18A-NH, (O), Ac-18A(Lys>Haa)-NHe (A), Ac-18R-NH2 (©).
`
`Right-angle Light Scattering Measurements—Theassociation of the
`peptides with EYPC multilamellar vesicles was followed by monitoring
`the rateof 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 monochromatorsset 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 amountof the peptide was
`maintained at 25 °C and wascontinuously stirred. Complete dissolution
`of the EYPC vesicles was achieved by the addition of Triton X-100to the
`vesicles suspension at a final concentration of 1 mm.
`Differential Scanning Calorimetry—Thehighsensitivity DSC stud-
`ies were performed in Microcal MC-2 scanning calorimeter (MicroCal,
`Inc., Amherst, MA) at the scan rate of 20h"? at an instrumentalsen-
`sitivity of 1 and with a filtering constant of 10 s. The lipid multilamellar
`vesicles and the peptide-lipid mixtures for DSC were prepared asfol-
`lows. DMPC or DMPG (~2 mg) wasdissolved in chloroform in a test
`tube anddried by slow evaporation undera 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 temperaturefor 30
`min. The suspension was degassed for 30 min, and an aliquot of 1.2 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
`thermogramswereobserved betweenthefirst scan and the fourth scan.
`The observed transitions were analyzed using software provided by
`MicroCal, Inc., Amherst, MA.
`
`Emission
`
`‘300
`
`finens
`328.«S80:CS75COOSCHCCABT
`Wavelength (nm)
`
`Fic. 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 uM,excitation wavelength =
`280 nm. Ac-18A-NH, in buffer (©), Ac-18A-NH2-DMPC complex (@),
`Ac-18A(Lys>Haa)-NH,
`in buffer
`(A), Ac-18A(Lys>Haa)-NH2-DMPC
`complex (4), Ac-18R-NH, in buffer (©), Ac-18R-NH».-DMPC complex
`(@).
`
`RESULTS
`
`1B, amongthe three peptides, Ac-18A-NHz has the maximum
`a-helical structure (67%). The a-helical contents ofAc-18R-NH,
`and Ac-18A(Lys>Haa)-NHgare 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-NHp.(202.6 nm), the zero
`cross-over point of Ac-18R-NHy,is shifted toward a shorter
`CD Studies—Secondary structures of the peptides were de-
`terminedby recording their far-UV CD spectra in buffer (PBS,
`wavelength (202 nm). This indicates that Ac-18A(Lys>Haa)-
`pH 7.4), in 50% (v/v) TFE/PBS,andin the presence of DMPC
`NH,hasa higher a-helical content than Ac-18R-NHgin this
`(lipid/peptide molar ratio 20:1). The CD spectra are shown in
`solvent system (39).
`Fig. 1. The CD spectrum of an a-helix is characterized by two
`The CD spectra of peptide-DMPC complexes (lipid/peptide
`negative bands, one at 222 nm and another at 208 nm, and a
`molarratio 20:1) are shownin Fig. 1C. When complexed with
`positive band at 192 nm (37). The CD spectrum ofAc-18A-NH,
`the lipid, all the three peptides are largely a-helical. The three
`in buffer (Fig. 14) indicates that the peptide is largely helical.
`peptides were estimated to have the following a-helical con-
`As estimated from mean residueellipticity at 222 nm [Oloz0,
`
`tents: Ac-18A-NH, and_Ac-72%, Ac-18R-NH, 62%,
`
`
`the peptide is 55% a-helical. The CD spectrum of Ac-18R-NH2
`18A(Lys>Haa)-NH2 56%. We would like to point out that the
`indicates that it is less helical (43%) than Ac-18A-NH2. The CD
`a-helical content ofAc-18A-NH, in DMPC complex wasearlier
`spectrum of Ac-18A(Lys>Haa)-NHgin buffer is distinct from
`reported to be 92% (25). This value wasrecently found to be an
`the CD spectra of the other two peptides becauseofits greatly
`overestimation becauseof the impropercalibration of the spec-
`reduced meanresidueellipticity values as well as a large shift
`tropolarimeter.
`emission
`fluorescence
`Fluorescence Measurements—The
`in the zero cross-over point (wavelength at which mean residue
`ellipticity is zero). This suggests a largely non-helical structure
`maximumoftryptophanis highly sensitive to its microenviron-
`of the peptide in buffer. The peptide was estimated to have 22%
`ment (40). The microenvironments of tryptophan residues in
`a-helical structure.
`the peptides were probed by recording their emission spectra in
`TFEis a most commonly used structure-inducing, hydropho-
`buffer and in the presence of DMPC(lipid/peptide molarratio
`bic, cosolvent (38). CD spectra of the three peptides in 50% (v/v)
`20:1). The emission spectra are shownin Fig. 2. All three pep-
`TFE/PBSwererecorded to estimate their a-helical contents in
`tides disrupt DMPC vesicles completely at lipid/peptide molar
`a relatively hydrophobic environment. Asis evident from Fig.
`ratio 20:1 resulting in an optically clear solution. In buffer
`
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`7188
`
`Lipid Interactions of Amphipathic Helical Peptides
`Tasie I
`Tryptophan emission maxima andfluorescence quenching parameters of the peptides and the peptide-DMPC complexes
`Peptide concentration was 14 pM; 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. Kgy is Stern-Volmer quenching constant determined from
`the slopes of the lines for the plots of F,/F-1 versus [@Q]; slopes were determinedby linear regression analysis of the fluorescence quenching data
`using the least squares method.
`
`Amax (nm)
`Kgy (m7!)
`PC
`DMPC
`.
`Peptide
`DMPC
`Buffer,
`DM! 1
`Buffer,
`Buffer
`iodide
`complex,
`acrylamide
`complex,
`complex
`quenching
`ceaidez
`quenching
`aeryomice
`
`Ac-18A-NH2
`343
`329
`5.7
`14
`13.5
`2.6
`Ac-18A(Lys>Haa)-NH2
`344
`331
`7.2
`3.7
`15.5
`6.2
`Ac-18R-NH2
`344
`336
`8.0
`5.4
`17.4
`10.3
`NATA
`350
`ND-
`11.0
`ND
`27.7
`ND
`* Not determined.
`
`Intensity
`ScatteredLight
`
`Time (sec)
`
`Fic. 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 addedto the lipid suspension. Complete
`dissolution was achieved by adding Triton X-100 solution to the lipid
`suspension at a final concentration of 1 mm. Excitation and emission
`wavelengths both were 400 nm. EYPC alone (M), Ac-18A-NHp2 (@), Ac-
`18A(Lys>Haa)-NH,(A), Ac-18R-NH, (), Triton X-100 (C).
`
`To further probe the location of the tryptophan residues of
`the three peptides in the lipid bilayer, fluorescence quenching
`experiments werecarried out. Iodide and acrylamide were used
`as aqueous-phase quenchers of the tryptophan fluorescence
`(35). While iodide is an anionic quencher, acrylamideis 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-Volmerplots of the quenchingof tryptophan fluorescence
`by iodide and acrylamide are shownin Fig. 3. For comparison,
`Stern-Volmerplots of fluorescence quenching of NATA (N-acet-
`yltryptophanamide), a model compoundfor free tryptophan,in
`buffer by iodide and acrylamide are also included. In buffer,
`compared with NATA,tryptophansin all the three peptides are
`less exposed to the quenchers. In the presence of DMPC, com-
`pared with buffer, tryptophansin 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 tryptophanresidue of the three peptides from
`the quenchers: Ac-18A-NHz > Ac-18A(Lys>Haa)-NHg > Ac-18R-
`NHg. Thus, amongthethree peptides, tryptophan in Ac-18R-
`NHzexperiencesleast shielding in going from bufferto the lipid
`bilayer. The Stern-Volmer quenchingconstants for a bimolecu-
`lar collisional quenching process were calculated from the ap-
`parent slopes of the plots of F)/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-Volmerplots of the fluorescence quenchingbyacryl-
`amidefor the three peptidesas well as that of NATAin solution
`
`MSN Exhibit 1030 - Page 4 of 7
`MSNv.Bausch - IPR2023-00016
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`0.8
`
`2.0
`
`(F,/FJ-1
`
`20
`
`40
`
`60
`
`100 (20 140
`BO
`[Iodide] mM
`
`180 180 200 220
`
` a
`
`ot
`o
`20
`
`40
`
`—
`160 180 200 220
`100 120 140
`#60
`680
`{Acrylamide] mM
`
`Fic. 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-
`18A-NH,
`in buffer
`(©), Ac-18A-NH2-DMPC complex (@), Ac-
`18A(Lys>Haa)-NH, in buffer (A), Ac-18A(Lys>Haa)-NH2-DMPC com-
`plex (A), Ac-18R-NH, in buffer (©), Ac-18R-NH,-DMPC complex( @),
`
`NATAin buffer (().
`
`alone, while Ac-18A-NHghasits emission maximum (Aynjqx) at
`343 nm, Ac-18R-NHz and Ac-18A(Lys>Haa)-NH2 both have
`their emission maxima at 344 nm. When complexed with
`DMPC,all the three peptides show anincrease 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 thatin buffer, is different for the three peptides.
`In the presence of DMPC, while Ac-18A-NH2 emission maxi-
`mum showsa blue-shift of 14 nm, Ac-18A(Lys>Haa)-NH2 and
`Ac-18R-NH, show blue-shifts of 13 and 8 nm, respectively
`(Table I). This indicates that the tryptophanresidue in Ac-18R-
`NHis in a less hydrophobic environment than the tryptophan
`residues in Ac-18A-NHg and Ac-18A(Lys>Haa)-NHo2.
`
`

`

`A
`
`Temperature (°C)
`
`
`
`Temperature (°C)
`
`Fic. 5. DSC heating thermogramsoflipids and lipid-peptide
`mixtures(lipid/peptide molar ratio 100:1). A, DMPC alone (1.5 mm)
`(i), DMPC + Ac-18R-NH, (ii), DMPC + Ac-18A(Lys>Haa)-NHg (iii),
`DMPC+ Ac-18A-NH,(iv). B, DMPG alone (1.4 mm) (i), DMPG + Ac-
`18R-NH, (ii), DMPG + Ac-18A(Lys>Haa)-NHp (iii), DMPG + Ac-18A-
`NH,(iv).
`
`TasLe II
`Phase transition parameters for lipids and peptide-lipid mixtures as
`determined by DSC
`DMPC concentration 1.5 mm, DMPG concentration 1.4 mw, lipid/
`peptide molarratio 100:1. T,, is the temperature at which excess heat
`capacity is maximum; AH,is calorimetric enthalpy; AHyy is Van’t Hoff
`enthalpy; ATv. is width of the transition at half of the peak heat capac-
`ity.
`
`Sample
`
`DMPC
`DMPC/Ac-18A-NH2
`DMPC/Ac-18A(Lys>Haa)-NH,
`DMPC/Ac-18R-NH.
`DMPG
`DMPG/Ac-18A-NH,
`DMPG/Ac-18A(Lys>Haa)-NH2
`DMPG/Ac-18R-NH,
`
`Tm
`°c
`23.1
`23.0
`23.3
`23.1
`22.3
`22.2
`22.0
`22.1
`
`AA
`AH
`kcal/mol
`4284.4
`1389.6
`1566.8
`2537.5
`1171.1
`480.4
`568.4
`674.3
`
`5.4
`3.3
`4.1
`42
`5.5
`3.0
`3.8
`4.8
`
`AT
`°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 of
`the three peptides with EYPC were studied by monitoring the
`rate of clarification of the turbidity due to the lipid vesicles
`after addition of the peptide. The results of this study are
`shown in Fig. 4. As has been noted earlier (25), at equimolar
`lipid/peptide ratio, Ac-18A-NHg clarifies the lipid turbidity
`within 30 min. At an identical lipid/peptide molar ratio, while
`the rate of
`turbidity clarification was
`slower with Ac-
`18A(Lys>Haa)-NHg, Ac-18R-NHgfailed to clarify the lipid tur-
`bidity within the duration of the experiment (33 min). A slight
`increase in the turbidity of the lipid suspension was observed
`after the addition of Ac-18R-NHo. The reason for this is not
`clear at present.
`DSC Studies—The thermotropic phasetransition properties
`of DMPC and DMPGmultilamellar vesicles were studied in the
`
`Lipid Interactions ofAmphipathic Helical Peptides
`
`7189
`
`———>
`Endothermic
`Endothermic——>
`
`absence andin the presence of the peptides by DSC. The heat-
`ing endothermsofthe purelipid vesicles and the peptide-lipid
`mixtures(lipid/peptide molar ratio 100:1) are shownin Fig. 5.
`At neutral pH, while DMPC is zwitterionic, DMPG is nega-
`tively charged. Interactionsofthe peptides with DMPGvesicles
`were studied to investigate possible electrostatic interactions
`between thepositively charged aminoacid residuesof the pep-
`tides and the negative charges of the lipid. DMPC vesicles
`alone exhibited endothermic transitions at 13.7 and 23.1 °C,
`which correspondto the pretransition (lamellar to periodic gel)
`andthegel to liquid-crystalline (order-disorder) phase transi-
`tion of the lipid, respectively (43). Addition of peptides to the
`DMPCvesicles resulted in lowering of the transition enthalpy
`and broadeningofthe gel to liquid-crystalline phase transition
`of DMPCvesicles (Table II). Amongthe three peptides, Ac-18A-
`NHcaused the maximalreduction in the enthalpy of the main
`phasetransition, while Ac-18A(Lys>Haa)-NHgis intermediate
`to Ac-18A-NHp and Ac-18R-NH,(Table IT). Noneof the peptides
`changedthe chain-melting temperature(7,,,) of DMPC vesicles
`by more than 0.2 °C. While the pretransition in the DMPC
`vesicles is observed with Ac-18A(Lys>Haa)-NHg2 and Ac-18R-
`NHg,it is not detected in the presence of Ac-18A-NHg. Similar
`to DMPCvesicles, DMPGvesicles 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).
`Theeffect of the three peptides on the phase transition prop-
`erties of the DMPGvesicles 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 DMPGis more than that in DMPC (TableII). This
`is presumably dueto the poor packingof the lipid molecules in
`the bilayer because ofelectrostatic 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-NHgz > Ac-18R-NH, > Ac-18A(Lys>Haa)-NHp.
`While the sameorder is observed in the helicity of the three
`peptides in buffer (Fig. 1A), the helicity of Ac-18A(Lys>Haa)-
`NH,in the presence of TFE is more than that of Ac-18R-NH,
`(Fig. 1B). In the presence of TFE, Ac-18A(Lys>Haa)-NH, was
`estimated to have less helical structure than Ac-18R-NH2
`based on the meanresidueellipticity 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)-NH, has more
`helical structure than Ac-18R-NHg(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 importantto
`note that a comparison ofhelicity should not be based onellip-
`ticity values alone. The results of the CD studies indicate that
`the lipid affinities of Ac-18R-NHz and Ac-18A(Lys>Haa)-NH,
`areless than the lipid affinity ofAc-18A-NHg.Thelipid affinity
`of Ac-18A(Lys>Haa)-NHp, based on theresults of the CD stud-
`ies alone, is lower than that of Ac-18R-NHg.
`A comparison of the fluorescence emission maxima of the
`peptides, however, reveals that in the DMPC complex, while
`tryptophan in Ac-18A(Lys>Haa)-NH,gis in a less hydrophobic
`environment than the tryptophan in Ac-18A-NHg,it is in a
`more hydrophobic environmentthan the tryptophan in Ac-18R-
`NH,(Table I). These conclusions are further supported by the
`results from the fluorescence quenching stud