Amphiphilic Helical Peptides Research Articles

Synthesis of Alternating Polyisocyanate Copolymers by Anionic Polymerization for Mimicking Amphiphilic Helical Peptides.

The amphiphilic conformation of α-helical peptides has important biological functions, such as ion transport, antifreeze, and innate immunity, which can be mimicked by alternating polyisocyanate copolymers. We synthesized poly(allyl isocyanate-alt-(S)-(-)-α-methylbenzyl isocyanate (P(AIC-alt-SMBIC)) and ammonium-containing P(AIC-alt-SMBIC) (N-P(AIC-alt-SMBIC)), ensuring the amphiphilic helical conformation. The benzyl group of SMBIC plays an important role in alternating copolymerization with its steric and electron-withdrawing effects, while AIC provides an alkene group capable of introducing a customized functional group. The P(AIC-alt-SMBIC) with predominantly alternating sequence was acquired at fSMBIC /fAIC =8 with a controlled molecular weight and narrow dispersity. N-P(AIC-alt-SMBIC)s were synthesized from thiol-ene radical addition with P(AIC-alt-SMBIC).

Synthesis of Alternating Polyisocyanate Copolymers by Anionic Polymerization for Mimicking Amphiphilic Helical Peptides

AbstractThe amphiphilic conformation of α‐helical peptides has important biological functions, such as ion transport, antifreeze, and innate immunity, which can be mimicked by alternating polyisocyanate copolymers. We synthesized poly(allyl isocyanate‐alt‐(S)‐(−)‐α‐methylbenzyl isocyanate (P(AIC‐alt‐SMBIC)) and ammonium‐containing P(AIC‐alt‐SMBIC) (N‐P(AIC‐alt‐SMBIC)), ensuring the amphiphilic helical conformation. The benzyl group of SMBIC plays an important role in alternating copolymerization with its steric and electron‐withdrawing effects, while AIC provides an alkene group capable of introducing a customized functional group. The P(AIC‐alt‐SMBIC) with predominantly alternating sequence was acquired at fSMBIC/fAIC=8 with a controlled molecular weight and narrow dispersity. N‐P(AIC‐alt‐SMBIC)s were synthesized from thiol‐ene radical addition with P(AIC‐alt‐SMBIC).

Loosening of Lipid Packing by Cell-Surface Recruitment of Amphiphilic Peptides by Coiled-Coil Tethering.

Lipid packing has a strong influence on the formation and structural dynamics of cell membranes. Techniques to modulate lipid packing may thus enable modification of cellular functions and events. An 18-residue amphiphilic helical peptide derived from the N-terminal segment of epsin-1 (EpN18) is reported to induce positive membrane curvature and to loosen lipid packing in the cell membrane. In this study, it is shown that EpN18, crosslinked to a leucine-zipper peptide K4, is recruited to the cell surface by interacting with a cell-surface-expressed E3 leucine-zipper segment. Cell-surface tethering markedly enhanced loosening of lipid packing, which led to the promotion of membrane translocation of octaarginine. The loosening of lipid packing by EpN18 was also confirmed by analyzing the generalized polarization value with a membrane-environment-sensitive dye, 2-hydroxy-3-{2-[(2-hydroxyethyl)dimethylamino]ethyl}-4-{2-[6-(dibutylamino)-2-naphthyl]ethenyl}pyridiniumdibromide (di-4-ANEPPDHQ). This approach thus shows promise for the control of lipid packing and related cellular events.

Best of Two Worlds? How MD Simulations of Amphiphilic Helical Peptides in Membranes Can Complement Data from Oriented Solid-State NMR.

The membrane alignment of helical amphiphilic peptides in oriented phospholipid bilayers can be obtained as ensemble and time averages from solid state 2H NMR by fitting the quadrupolar splittings to ideal α-helices. At the same time, molecular dynamics (MD) simulations can provide atomistic insight into peptide-membrane systems. Here, we evaluate the potential of MD simulations to complement the experimental NMR data that is available on three exemplary systems: the natural antimicrobial peptide PGLa and the two designer-made peptides MSI-103 and KIA14, whose sequences were derived from PGLa. Each peptide was simulated for 1 μs in a DMPC lipid bilayer. We calculated from the MD simulations the local angles which define the side chain geometry with respect to the peptide helix. The peptide orientation was then calculated (i) directly from the simulation, (ii) from back-calculated MD-derived NMR splittings, and (iii) from experimental 2H NMR splittings. Our findings are that (1) the membrane orientation and secondary structure of the peptides found in the NMR analysis are generally well reproduced by the simulations; (2) the geometry of the side chains with respect to the helix backbone can deviate significantly from the ideal structure depending on the specific residue, but on average all side chains have the same orientation; and (3) for all of our peptides, the azimuthal rotation angle found from the MD-derived splittings is about 15° smaller than the experimental value.

Peptide Membrane Displaying Membrane Fusion and Fission with Lipid–Raft–Like Organization

Organelles in cells take a wide range of morphologies and convert one morphology to the other for specific biological events. We are trying to construct such complex morphologies allowing transformation from one to the other by using molecular assemblies comprising amphiphilic helical peptides. One key player is poly(sarcosine)–b–(L–Leu- Aib)n having a hydrophilic block of poly(sarcosine) and a helical hydrophobic block of (L–Leu–Aib)n. The driving forces for making these molecular assemblies can be modulated widely by helical block length, knobs-into-holes packing of side chains between helix surfaces, and stereocomplex formation between right- and left–handed helices. Under properly designed amphiphilic polypeptides and their combinations, we can construct morphologies of sheet, tube, and vesicles and even the combination of tube and vesicle, a round–bottomed flask shape. There were several combinations of amphiphilic polypeptides to take the round–bottomed flask morphology. The chimeric morphology is a direct consequence of phase separation of the component polypeptides in one consecutive membrane. The distribution of the components into the round–bottomed or neck moiety was dependent on the combination meaning that lateral diffusion of the components in the membrane led to the phase separation. The vesicle composed of poly(sarcosine)– b–(L–Leu–Aib)6 and poly(sarcosine)–b–(D–Leu–Aib)6 allowed membrane fusion and fission above the phase transition temperature with itself and other vesicle. The monolayer elasticity of vesicles was variable in accordance with the component amphiphilic polypeptides, and the elasticity similar to liposome can be prepared.

Converting One-Face α-Helix Mimetics into Amphiphilic α-Helix Mimetics as Potent Inhibitors of Protein-Protein Interactions.

Many biologically active α-helical peptides adopt amphiphilic helical structures that contain hydrophobic residues on one side and hydrophilic residues on the other side. Therefore, α-helix mimetics capable of mimicking such amphiphilic helical peptides should possess higher binding affinity and specificity to target proteins. Here we describe an efficient method for generating amphiphilic α-helix mimetics. One-face α-helix mimetics having hydrophobic side chains on one side was readily converted into amphiphilic α-helix mimetics by introducing appropriate charged residues on the opposite side. We also demonstrate that such two-face amphiphilic α-helix mimetics indeed show remarkably improved binding affinity to a target protein, compared to one-face hydrophobic α-helix mimetics. We believe that generating a large combinatorial library of these amphiphilic α-helix mimetics can be valuable for rapid discovery of highly potent and specific modulators of protein-protein interactions.

Selective disruption of each part of Janus molecular assemblies by lateral diffusion of stimuli-responsive amphiphilic peptides.

Stimuli-responsive Janus-type assemblies with a round-bottom flask shape are prepared from amphiphilic helical peptides using the patchwork self-assembly technique. It can disassemble selectively at the neck or the round-bottom part of the Janus assemblies at low pH upon heat treatment accompanied by the lateral diffusion of the peptides.

Surfactant-like properties of an amphiphilic α-helical peptide leading to lipid nanodisc formation.

Nanodiscs are self-assembled discoidal nanoparticles composed of amphiphilic α-helical scaffold proteins or peptides that wrap themselves around the circumference of a lipid bilayer in a beltlike manner. In this study, an amphiphilic helical peptide that mimics helix 10 of human apoA-I was newly synthesized by solid phase peptide synthesis using Fmoc chemistry, and its physicochemical properties, including surface tension, self-association, and solubilization abilities, were evaluated and related directly to nanodisc formation. The synthesized peptide having hydrophobic and hydrophilic faces behaves like a general surfactant, affording a critical association concentration (CAC) of 2.7 × 10(-5) M and a γCAC of 51.2 mN m(-1) in aqueous solution. Interestingly, only a peptide solution above its CAC was able to microsolubilize L-α-dimyristoylphosphatidylcholine (DMPC) vesicles, and lipid nanodiscs with an average diameter of 9.5 ± 2.7 nm were observed by dynamic light scattering and negative stain transmission electron microscopy. Moreover, the ζ potentials of the lipid nanodiscs were measured for the first time as a function of pH, and the values changed from positive (20 mV) to negative (-30 mV). In particular, nanodisc solutions at acidic pH 4 (20 mV) or basic pH 9 (-20 mV) were found to be stable for more than 6 months as a result of the electrostatic repulsion between the particles.

The Discreet Charm of the Curve

The Discreet Charm of the Curve

Versatile peptide rafts for conjugate morphologies by self-assembling amphiphilic helical peptides

Amphiphilic helical peptides with opposite helicities and different hydrophobic helical block lengths were combined to prepare self-assemblies in water for constructing novel morphologies. (Sar)25-b-(L-Leu-Aib)6 and (Sar)25-b-(D-Leu-Aib)6 (sarcosine (Sar) chain, a hydrophobic helix of leucine (Leu) and α-aminoisobutyric acid (Aib)) have been reported to self-assemble into well-defined nanotubes, but a 1:1 mixture of these components self-assembles into vesicles. Their mixtures at molar ratios 8:2 and 2:8 have yielded round-bottom flask-shaped molecular assemblies, which are an AB-type conjugate morphology of a vesicle (A) and a nanotube (B). Here, we expand the conjugate morphologies to new AB-type, ABA-type, ABA′-type and A3B-type. The new AB-type conjugate morphology had a nano-test-tube shape. Two types of the nano-test-tube-shaped self-assemblies were prepared using different combinations of nanotubes and vesicles. One thin nanotube was sealed with vesicles at both open ends to generate the ABA-type conjugate morphology with a dumbbell shape. When the round-bottom flask-shaped self-assembly was combined with a small vesicle, the ABA′-type conjugate morphology was obtained. The combination of a three-neck nanotube with a vesicle produced the A3B-type morphology. These self-assemblies were thermodynamically stable owing to the phase separation in their fused membranes. Therefore, these self-assemblies are termed ‘peptide rafts’ as an analogy to ‘lipid rafts’. Amphiphilic peptides herein also induce phase separation in peptide membranes, ‘peptide rafts’. Amphiphilic helical peptides self-assembled into nanotubes by themselves, but into vesicles with 1:1 mixture of their stereocomplex. Their mixtures also yielded a round-bottom flask morphology, an AB-type conjugate morphology of a nanotube and a vesicle, due to phase separation in peptide membrane. The open mouth of the round-bottom flask was further combined with other peptide vesicles to generate asymmetric dumbbell morphology, ABC-type conjugate morphology of a nanotube and two vesicles.

Lipid shape is a key factor for membrane interactions of amphipathic helical peptides

The membrane alignment of the amphiphilic α-helical model peptide MSI-103 (sequence [KIAGKIA]3-NH2) was examined by solid state 2H-NMR in different lipid systems by systematically varying the acyl chain length and degree of saturation, the lipid head group type, and the peptide-to-lipid molar ratio. In liquid crystalline phosphatidylcholine (PC) lipids with saturated chains, the amphiphilic helix changes its orientation from a surface-bound “S-state” to a tilted “T-state” with increasing peptide concentration. In PC lipids with unsaturated chains, on the other hand, the S-state is found throughout all concentrations. Using phosphatidylethanolamine lipids with a small head group or by addition of lyso-lipids with only one acyl chain, the spontaneous curvature of the bilayer was purposefully changed. In the first case with a negative curvature only the S-state was found, whereas in systems with a positive curvature the peptide preferred the obliquely immersed T-state at high concentration. The orientation of MSI-103 thus correlates very well with the shape of the lipid molecules constituting the membrane. Lipid charge, on the other hand, was found to affect only the initial electrostatic attraction to the membrane surface but not the alignment preferences. In bilayers that are “sealed” with 20% cholesterol, MSI-103 cannot bind in a well-oriented manner and forms immobilized aggregates instead. We conclude that the curvature properties of a membrane are a key factor in the interactions of amphiphilic helical peptides in general, whose re-alignment and immersion preferences may thus be inferred in a straightforward manner from the lipid-shape concept.

Temperature-Triggered Fusion of Vesicles Composed of Right-Handed and Left-Handed Amphiphilic Helical Peptides

Vesicles prepared from a mixture of (Sar)(25)-b-(L-Leu-Aib)(6) (SLL) and (Sar)(25)-b-(D-Leu-Aib)(6) (SDL) fused with themselves upon heating to 90 °C. The vesicles also fused with (Sar)(28)-b-(L-Leu-Aib)(8) vesicles upon heating to 90 °C. The temperature-triggered fusion was due to the phase transition of the mixed membrane of SLL and SDL at 90 °C and should be driven by the bending energy stored in the stereocomplex membrane upon taking a vesicular structure.

Rational design of peptide nanotubes for varying diameters and lengths

Amphiphilic helical peptides (Sar)(m) -b-(L-Leu-Aib)(n) (m = 22-25; n = 7, 8, 10) with a hydrophobic block as a right-handed helix were synthesized and their mixtures with (Sar)(25) -b-(D-Leu-Aib)(6) containing the hydrophobic block as a left-handed helix were examined in their molecular assembly formation. The single component (Sar)(25) -b-(D-Leu-Aib)(6) forms peptide nanotubes of 70 nm diameter and 200 nm length. The two-component mixtures of (Sar)(25) -b-(D-Leu-Aib)(6) with (Sar)(24) -b-(L-Leu-Aib)(7) , (Sar)(22) -b-(L-Leu-Aib)(8) , and (Sar)(25) -b-(L-Leu-Aib)(10) yield peptide nanotubes of varying dimensions with 200 nm diameter and 400 nm length, 70 nm diameter and several micrometer length (maximum 30 µm), and 70 nm diameter and 100-600 nm length, respectively. The right-handed and the left-handed helix were thus found to be molecularly mixed due to the stereo-complex formation and to generate nanotubes of different sizes. When the mismatch of the hydrophobic helical length between the two components was of four residues, the longest nanotube was generated. Correspondingly, the hydrophobic helical segments have to interdigitate with an anti-parallel orientation at the hydrophobic core region of the nanotube.

Different mechanisms of action of antimicrobial peptides: insights from fluorescence spectroscopy experiments and molecular dynamics simulations

Most antimicrobial peptides exert their activity by interacting with bacterial membranes, thus perturbing their permeability. They are investigated as a possible solution to the insurgence of bacteria resistant to the presently available antibiotic drugs. However, several different models have been proposed for their mechanism of membrane perturbation, and the molecular details of this process are still debated. Here, we compare fluorescence spectroscopy experiments and molecular dynamics (MD) simulations regarding the association with lipid bilayers and lipid perturbation for two different amphiphilic helical antimicrobial peptides, PMAP-23 and trichogin GA IV. PMAP-23, a cationic peptide member of the cathelicidin family, is considered to induce membrane permeability according to the Shai-Matsuzaki-Huang "carpet" model, while trichogin GA IV is a neutral peptide, member of the peptaibol family. Although several lines of evidence suggest a "barrel-stave" mechanism of pore formation for the latter peptide, its length is only half the normal thickness of a lipid bilayer. Both fluorescence spectroscopy experiments and MD simulations indicated that PMAP-23 associates with membranes close to their surface and parallel to it, and in this arrangement it causes a severe perturbation to the bilayer, both regarding its surface tension and lipid order. By contrast, trichogin GA IV can undergo a transition from a surface-bound state to a transmembrane orientation. In the first arrangement, it does not cause any strong membrane perturbation, while in the second orientation it might be able to span the bilayer from one side to the other, despite its relatively short length, by causing a significant thinning of the membrane.

Molecular Dynamics Study of a Nanotube-Binding Amphiphilic Helical Peptide at Different Water/Hydrophobic Interfaces

Many potential applications of single-walled carbon nanotubes (SWNTs) require that they be isolated from one another. This may be accomplished through covalent or noncovalent SWNT functionalization. The noncovalent approach preserves the intrinsic electrical, optical, and mechanical properties of SWNTs and can be achieved by dispersing SWNTs in aqueous solution using surfactants, polymers, or biomacromolecules like DNA or polypeptides. The designed amphiphilic helical peptide nano-1, which contains hydrophobic valine and aromatic phenylalanine residues for interaction with SWNTs and glutamic acid and lysine residues for water solubility, has been shown to debundle and disperse SWNTs, although the details of the peptide-SWNT interactions await elucidation. Here we use fully atomistic molecular dynamics simulations to investigate the nano-1 peptide at three different water/hydrophobic interfaces: water/oil, water/graphite, and water/SWNT. The amphiphilic nature of the peptide is characterized by its secondary structure, peptide-water hydrogen bonding, and peptide-hydrophobic surface van der Waals energy. We show that nano-1 has reduced amphiphilic character at the water/oil interface because the peptide helix penetrates into the hydrophobic phase. The peptide alpha-helix cannot match its hydrophobic face to the rigid planar graphite surface without partially unfolding. In contrast, nano-1 can curve on the SWNT surface in an alpha-helical conformation to simultaneously maximize its hydrophobic contacts with the SWNT and its hydrogen bonds with water. The molecular insight into the peptide conformation at the various hydrophobic surfaces provides guidelines for future peptide design.

Conformation and Membrane Orientation of Amphiphilic Helical Peptides by Oriented Circular Dichroism

Oriented circular dichroism (OCD) was used to characterize and compare in a quantitative manner the secondary structure and concentration dependent realignment of the antimicrobial peptides PGLa and MSI-103, and of the structurally related cell-penetrating peptide MAP in aligned phospholipid bilayers. All these peptides adopt an amphiphilic α-helical conformation, and from solid-state NMR analysis they are known to bind to membranes in two distinct orientations depending on their concentration. At low peptide/lipid (P/L) ratio the helices are aligned parallel to membrane surface (S-state), but with increasing concentration they realign to a tilted orientation (T-state), getting immersed into the membrane with an oblique angle supposedly as a result of dimer-formation. In macroscopically aligned liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers the two limiting states are represented by distinct OCD spectra, and all spectra at intermediate peptide concentrations can be described by a linear combination of these two line shapes. The corresponding fraction of molecules occupying the T-state was determined by fitting the intermediate spectra with a superposition of the two extreme line shapes. By plotting this fraction versus 1/(P/L), the threshold P/L* ratio for realignment was extracted for each of the three related peptides. Despite their structural similarity distinctly different thresholds were obtained, namely for MSI-103 realignment starts already at a low P/L of ∼1:236, for a MAP derivative (using a nonaggregating analog containing a D-amino acid) the transition begins at P/L ∼1:156, whereas PGLa needs the highest concentration to flip into T-state at P/L ∼1:85. Analysis of the original MAP sequence (containing only L-amino acids) gave OCD spectra compatible with β-pleated conformation, suggesting that this peptide starts to aggregate with increasing concentration, unlike the other helical peptides. All these changes in peptide conformation and membrane alignment observed here by OCD seem to be functionally relevant, as they can be correlated with the membrane perturbing activities of the three antimicrobial and cell-penetrating sequences.

Ion-Channel Formation Assisted by Electrostatic Interhelical Interactions in Covalently Dimerized Amphiphilic Helical Peptides

An ultimate goal of synthetic ion-channel peptide design is to construct stable and functional ion-conducting pores. It is expected that specific interhelical interactions would facilitate the association of helices in phospholipid membranes and the successive helix-bundle formation. In the present study, we rationally designed helix-bundle ion channels using the synthetic hybrid peptide K20E20, a disulfide dimer of cationic- and anionic-amphiphilic helices Ac-CGG-(BKBA) 5-NH 2 and Ac-CGG-(BEBA) 5-NH 2. Circular dichroism (CD) measurements in aqueous media implied helix stabilization in the peptide caused by the interhelical electrostatic interactions. In addition, CD spectra recorded in the presence of DPPC liposomes and dye-leakage measurements suggested a high degree of association of peptide monomers in phospholipid membranes as well as high affinities between peptide and lipid bilayers. These features allowed ion-channel formation at extremely low peptide concentrations (as low as 1 nM). According to electrophysiological analyses, stable helix bundles were constructed of six peptide helices by association of three K20E20 molecules. Helix-helix association in lipid membranes, peptide-membrane interactions, and ion-channel formation of K20E20 peptides were all facilitated by intramolecular electrostatic interactions between the helices of the hybrid peptide and were pH-dependent. Conductance through K20E20 ion channels decreased under acidic conditions because of the interruption of the salt bridges.

Effect of Electron-Donating and Electron-Withdrawing Groups on Peptide/Single-Walled Carbon Nanotube Interactions

Nano-1, a designed peptide, has been demonstrated to efficiently disperse individual single-walled carbon nanotubes (SWNTs) by folding into an amphiphilic alpha-helix wherein the phenylalanine (Phe) residues on the hydrophobic face of the helix interact via pi-stacking with the aromatic surface of the SWNT. In this study, the ability of electron-donating (hydroxyl) and electron-withdrawing (nitro) groups on the phenyl ring of Phe to affect the interactions between the peptide and SWNTs is examined by substituting the Phe residues in the nano-1 sequence with tyrosine and p-nitro-phenylalanine, respectively. Atomic force microscopy measurements and optical absorption spectroscopy revealed that the ability to disperse individual SWNTs increases with increasing electron density of the aromatic residue on the hydrophobic face of the amphiphilic helical peptides. Scanning tunneling spectroscopy (STS) and Raman analyses were used to examine the effect of noncovalent protein functionalization on the electronic properties of SWNTs. Small shifts in the Raman G band peak for the peptide/SWNT composites, as well as weak features that appear near the Fermi energy (Ef) in the STS dI/dV spectra of the peptide-coated SWNTs, are suggestive of a weak charge-transfer interaction between the peptides and the SWNTs.

Amphiphilic Helical Peptide Enhances the Uptake of Single-Walled Carbon Nanotubes by Living Cells

The success of many projected applications of carbon nano-tubes (CNTs) to living cells, such as intracellular sensors and nanovectors, will depend on how many CNTs are taken up by cells. Here we report the enhanced uptake by HeLa cells of single-walled CNTs coated with a designed peptide termed nano-1. Atomic force microscopy showed that the dispersions were composed of individual and small bundles of nano-1 CNTs with 0.7- to 32-nm diameters and 100- to 400-nm lengths. Spectroscopic characterizations revealed that nano-1 disperses CNTs in a non-covalent fashion that preserves CNT optical properties. Elemental analyses indicated that our sample preparation protocol involving sonication and centrifugation effectively eliminated metal impurities associated with CNT manufacturing processes. We further showed that the purified CNT dispersions are taken up by HeLa cells in a time- and temperature-dependent fashion, and that they do not affect the HeLa cell growth rate, evidence that the CNTs inside cells are not toxic under these conditions. Finally, we discovered that approximately 6-fold more CNTs are taken up by cells in the presence of nano-1 compared with medium containing serum but no peptide. The fact that coating CNTs with a peptide enhances uptake offers a strategy for improving the performance of applications that require CNTs to be inside cells.

Phosphorylation and Stabilization of ATP Binding Cassette Transporter A1 by Synthetic Amphiphilic Helical Peptides

To investigate structural requirement of helical apolipoprotein to phosphorylate and stabilize ATP-binding cassette transporter A1 (ABCA1), synthetic peptides (Remaley, A. T., Thomas, F., Stonik, J. A., Demosky, S. J., Bark, S. E., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Patterson, A. P., Eggerman, T. L., Santamarina-Fojo, S., and Brewer, H. B. (2003) J. Lipid Res. 44, 828-836) were examined for these activities. L37pA, an L amino acid peptide that contains two class-A amphiphilic helices, and D37pA, the same peptide with all D amino acids, both removed cholesterol and phospholipid from differentiated THP-1 cells more than apolipoproteins (apos) A-I, A-II, and E. Both peptides also mediated lipid release from human fibroblasts WI-38 similar to apoA-I. L2D37pA, an L-peptide whose valine and tyrosine were replaced with D amino acids also promoted lipid release from WI-38 but less so with THP-1, whereas L3D37pA, in which alanine, lysine, and asparatic acid were replaced with D amino acids was ineffective in lipid release for both cell lines. ABCA1 protein in THP-1 and WT-38 was stabilized against proteolytic degradation by apoA-I, apoA-II, and apoE and by all the peptides tested except for L3D37pA, and ABCA1 phosphorylation closely correlated with its stabilization. The analysis of the relationship among these parameters indicated that removal of phospholipid triggers signals for phosphorylation and stabilization of ABCA1. We thus concluded that an amphiphilic helical motif is the minimum structural requirement for a protein to stabilize ABCA1 against proteolytic degradation.