Amphipathic Α-helices Research Articles

Two rare variations, D478N and D478E, that occur at the same amino acid residue in nicotinic acetylcholine receptor (nAChR) α2 subunit influence nAChR function

There occur two rare variations, Asp(D)478Asn(N) and Asp(D)478Glu(E), in the putative cytoplasmic amphipathic α-helices of human nicotinic acetylcholine receptor (nAChR) α2 subunit as a result of mutation in the 1st (G → A: rs141072985) and 3rd (C → A: rs56344740) nucleotide of its 478th triplet codon (GAC). We assessed the effects of these two variations on the function of α2β2- and α2β4-nAChRs as they could alter the electronegativity and/or the structure of the cytoplasmic ‘portals’ (framed by subunit amphipathic α-helices) necessary for obligate ion permeation from extracellular space to cytoplasm. We injected decreasing ratio of subunit cRNAs (α:β; 10:1, 1:1 and 1:10) into Xenopus oocytes to express putative low-sensitivity (LS; 10:1), intermediate-sensitivity (IS; 1:1) and high sensitivity (HS; 1:10) isoforms of wild type and variant α2β2- and α2β4-nAChRs. Two-electrode voltage clamp analyses indicate that the agonist (ACh or nicotine) induced peak current responses (Imax) of α2β2-nAChR isoforms and those of α2β4-nAChR isoforms are increased (1.3–4.7-fold) as a result of D478E variation. The α2 subunit D478N variation only increases the Imax of IS (∼2-fold) or HS (1.4–2.1-fold) α2β2-nAChRs. Concentration–response curves constructed indicate no effect on agonist sensitivities of LS and HS isoforms of α2β2- or α2β4-nAChRs as a result of either variation in α2 subunit. Between the two variant nAChRs, α2(D478E)*-nAChR isoforms generally yield higher Imax than those of respective α2(D478N)*-nAChR isoforms. These effects could be attributed to alteration in cytoplasmic ‘portals’ and/or ion permeation through it owing to change in amino acid electronegativity (D → N) and side chain length (D → E) in nAChR α2 subunit.

Role of positions e and g in the fibrous assembly formation of an amphipathic α‐helix‐forming polypeptide

We previously characterized α3, a polypeptide that has a three times repeated sequence of seven amino acids (abcdefg: LETLAKA) and forms fibrous assemblies composed of amphipathic α-helices. Upon comparison of the amino acid sequences of α3 with other α-helix forming polypeptides, we proposed that the fibrous assemblies were formed due to the alanine (Ala) residues at positions e and g. Here, we characterized seven α3 analog polypeptides with serine (Ser), glycine (Gly), or charged residues substituted for Ala at positions e and g. The α-helix forming abilities of the substituted polypeptides were less than that of α3. The polypeptides with amino acid substitutions at position g and the polypeptide KEα3, in which Ala was substituted with charged amino acids, formed few fibrous assemblies. In contrast, polypeptides with Ala replaced by Ser at position e formed β-sheets under several conditions. These results show that Ala residues at position e and particularly at position g are involved in the formation of fibrous assemblies.

Apolipoprotein E mutations: a comparison between lipoprotein glomerulopathy and type III hyperlipoproteinemia

Apolipoprotein E (ApoE) serves as a ligand for the low-density lipoprotein (LDL) receptor and cell surface receptors of the LDL receptor gene family. More than 10 different causative apoE mutations associated with lipoprotein glomerulopathy (LPG) have been reported. ApoE polymorphisms including three common phenotypes (E2, E3, E4), and a variety of rare mutations can affect blood cholesterol and triglyceride levels. The N-terminal domain of apoE is folded into a four-helix bundle of amphipathic α-helices, and contains the receptor-binding domain in which most apoE mutations that cause LPG or dominant mode of type III hyperlipoproteinemia (HL) are located. No single apoE mutation has been reported that causes both LPG and the dominant mode of type III HL.

Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin and lipid binding.

Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), and it is required for proper NPC distribution and assembly. To characterize the Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a new Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between the Pom33 C-terminal domain (CTD) and Kap123. In silico analysis predicted the presence of two amphipathic α-helices within Pom33-CTD. Circular dichroism and liposome co-flotation assays showed that this domain is able to fold into α-helices in the presence of liposomes and preferentially binds to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, this domain behaves as a Kap123-dependent nuclear localization signal (NLS). Although deletion of Pom33 C-terminal domain (Pom33(ΔCTD)-GFP) impaired Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the α-helices did not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects targeting of Pom33 to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.

Fluorescence Correlation Spectroscopy Reveals Additional Information on Protein Insertions into Phospholipid Monolayers

The generation of curvature in phospholipid membranes is a crucial step in vesicle and tube formation. A variety of proteins in eukaryotic cells are able to induce such curved membrane structures and produce coated transport vesicles or tubular carriers. Membrane curvature can be generated in different ways.Sar1, which is a small GTPase of the RAS superfamily and a part of the coat protein complex II (COPII), and the N-BAR dimer of amphiphysin II, which is involved in clathrin-coated vesicle formation, both have amphipathic α-helices, which insert only into the proximal leaflet of a phospholipid membrane. Phospholipid monolayers are therefore convenient model systems for analyzing the protein-lipid interactions.While a typical Langmuir film balance setup allows to observe the interaction of proteins with phospholipid monolayers (by changing the surface pressure or the surface area),the determination of protein concentrations and diffusion properties is not possible.To overcome these limitations, we combine a Langmuir film balance with a confocal fluorescence correlation spectroscopy setup for optical detection. This setup, which was described for the first time by Gudmand et.al (Biophys. J., 2009, 96, 4598-4609), allows FCS measurements in a monolayer system.Here we show some basic interaction studies of Sar1 and N-BAR with phospholipid monolayers at different surface pressures. In all cases, FCS measurements were carried out to obtain additional information on the protein.

Membrane Interactions with ATRA Peptides

The “catastrophic threat” of antibiotic resistance has prompted research into biological methods of combating bacterial infection. One such pervasive strategy employs cationic antimicrobial peptides, CAMPs. These peptides use their structure to target and disrupt bacterial membranes. The cationic peptides are specifically attracted to bacteria because of the high anionic lipid content in the outer leaflet of bacterial membranes. Most have already been shown to have broad spectrum activity, adequate potency, and minimal resistance. Considering these peptides have been active against pathogens for millions of years and have not developed any broad resistance, they are of particular interest to study. One class of CAMPs, AHCAPs, forms amphipathic α-helices when in the presence of anionic membranes. The peptide NA-CATH, from the Naja atra snake, and its analog, ATRA-1A, show promising activity. We are currently examining the effect of lipid composition and peptide chirality (L and D isomers) on peptide activity and membrane interactions. Kinetic assays show increased activity with increasing peptide concentration and match the EC50 (50% of maximal dose) trends for zwitterionic/anionic lipid composition.

Structure Activity Relationship for a Synergistic Pair of Antimicrobial Peptides from the Magainin Family

The antimicrobial peptides Magainin 2 (Mag2) and PGLa are both found in the skin of the African frog Xenopus laevis. They show high antimicrobial activity against bacteria, fungi, and cancer cells.[1] Both peptides form amphipathic α-helices upon binding to a lipid bilayer. Previous studies revealed that mixtures of Mag2 and PGLa show synergistic effects,[2] and the formation of stable 1:1 heterodimeric peptide pores has been proposed as a possible mechanism.The membrane alignment of each of these two peptides per se has been determined with high accuracy using solid state 2H-, 15N-, and 19F-NMR spectroscopy. Using solid state 2H-NMR, we have previously characterized the orientation of PGLa on its own and in the presence of an equimolar amount of Mag2, and found an inserted transmembrane orientation of PGLa in DMPC/DMPG only in the presence of Mag2.[3] On the other hand, Mag2 always stays flat on the surface of the lipid bilayer, both on its own and in the presence of PGLa.The aim of the present study was to determine the detailed local interaction between the two peptides in the membrane-bound state, taking hydrophobic and charge interactions into account. Several mutations were introduced at potentially crucial positions of either peptide and investigated by 15N-NMR and biological assays. It was found that synergy is correlated with the insertion of PGLa, and important residues for synergy could thus be identified.

Insertion of apoLp-III into a lipid monolayer is more favorable for saturated, more ordered, acyl-chains

Neutral lipid transport in mammals is complicated involving many types of apolipoprotein. The exchangeable apolipoproteins mediate the transfer of hydrophobic lipids between tissues and particles, and bind to cell surface receptors. Amphipathic α-helices form a common structural motif that facilitates their lipid binding and exchangeability. ApoLp-III, the only exchangeable apolipoprotein found in insects, is a model amphipathic α-helix bundle protein and its three dimensional structure and function mimics that of the mammalian proteins apoE and apoAI. Even the intracellular exchangeable lipid droplet protein TIP47/perilipin 3 contains an α-helix bundle domain with high structural similarity to that of apoE and apoLp-III. Here, we investigated the interaction of apoLp-III from Locusta migratoria with lipid monolayers. Consistent with earlier work we find that insertion of apoLp-III into fluid lipid monolayers is highest for diacylglycerol. We observe a preference for saturated and more highly ordered lipids, suggesting a new mode of interaction for amphipathic α-helix bundles. X-ray reflectivity shows that apoLp-III unfolds at a hydrophobic interface and flexible loops connecting the amphipathic α-helices stay in solution. X-ray diffraction indicates that apoLp-III insertion into diacylglycerol monolayers induces additional ordering of saturated acyl-chains. These results thus shed important new insight into the protein–lipid interactions of a model exchangeable apolipoprotein with significant implications for its mammalian counterparts.

Coiled-Coil Response to Mechanical Force: Global Stability and Local Cracking

Coiled coils are important structural motifs formed by two or more amphipathic α-helices that twist into a supercoil. These motifs are found in a wide range of proteins, including motor proteins and structural proteins, that are known to transmit mechanical loads. We analyze atomically detailed simulations of coiled-coil cracking under load with Milestoning. Milestoning is an approach that captures the main features of the process in a network, quantifying kinetics and thermodynamics. A 112-residue segment of the β-myosin S2 domain was subjected to constant-magnitude (0–200 pN) and constant-direction tensile forces in molecular dynamics simulations. Twenty 20 ns straightforward simulations at several load levels revealed that initial single-residue cracking events (Ψ > 90°) at loads <100 pN were accompanied by rapid refolding without either intra- or interhelix unfolding propagation. Only initial unfolding events at the highest load (200 pN) regularly propagated along and between helices. Analysis of hydrophobic interactions and of interhelix hydrogen bonds did not show significant variation as a function of load. Unfolding events were overwhelmingly located in the vicinity of E929, a charged residue in a hydrophobic position of the heptad repeat. Milestoning network analysis of E929 cracking determined that the mean first-passage time ranges from 20 ns (200 pN) to 80 ns (50 pN), which is ∼20 times the mean first-passage time of an isolated helix with the same sequence.

Structural and Functional Characterization of the Recombinant Death Domain from Death-Associated Protein Kinase

Death-associated protein kinase (DAPk) is a calcium/calmodulin-regulated Ser/Thr-protein kinase that functions at an important point of integration for cell death signaling pathways. DAPk has a structurally unique multi-domain architecture, including a C-terminally positioned death domain (DD) that is a positive regulator of DAPk activity. In this study, recombinant DAPk-DD was observed to aggregate readily and could not be prepared in sufficient yield for structural analysis. However, DAPk-DD could be obtained as a soluble protein in the form of a translational fusion protein with the B1 domain of streptococcal protein G. In contrast to other DDs that adopt the canonical six amphipathic α-helices arranged in a compact fold, the DAPk-DD was found to possess surprisingly low regular secondary structure content and an absence of a stable globular fold, as determined by circular dichroism (CD), NMR spectroscopy and a temperature-dependent fluorescence assay. Furthermore, we measured the in vitro interaction between extracellular-regulated kinase-2 (ERK2) and various recombinant DAPk-DD constructs. Despite the low level of structural order, the recombinant DAPk-DD retained the ability to interact with ERK2 in a 1∶1 ratio with a K d in the low micromolar range. Only the full-length DAPk-DD could bind ERK2, indicating that the apparent ‘D-motif’ located in the putative sixth helix of DAPk-DD is not sufficient for ERK2 recognition. CD analysis revealed that binding of DAPk-DD to ERK2 is not accompanied by a significant change in secondary structure. Taken together our data argue that the DAPk-DD, when expressed in isolation, does not adopt a classical DD fold, yet in this state retains the capacity to interact with at least one of its binding partners. The lack of a stable globular structure for the DAPk-DD may reflect either that its folding would be supported by interactions absent in our experimental set-up, or a limitation in the structural bioinformatics assignment of the three-dimensional structure.

Changes in helical content or net charge of apolipoprotein C-I alter its affinity for lipid/water interfaces

Amphipathic α-helices mediate binding of exchangeable apolipoproteins to lipoproteins. To probe the role of α-helical structure in protein-lipid interactions, we used oil-drop tensiometry to characterize the interfacial behavior of apolipoprotein C-I (apoC-I) variants at triolein/water (TO/W) and 1-palmitoyl-2-oleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. ApoC-I, the smallest apolipoprotein, has two amphipathic α-helices. Mutants had single Pro or Ala substitutions that resulted in large differences in helical content in solution and on phospholipids. The ability of apoC-I to bind TO/W and POPC/TO/W interfaces correlated strongly with α-helical propensity. On binding these interfaces, peptides with higher helical propensity increased surface pressure to a greater extent. Likewise, peptide exclusion pressure at POPC/TO/W interfaces increased with greater helical propensity. ApoC-I retention on TO/W and POPC/TO/W interfaces correlated strongly with phospholipid-bound helical content. On compression of these interfaces, peptides with higher helical content were ejected at higher pressures. Substitution of Arg for Pro in the N-terminal α-helix altered net charge and reduced apoC-I affinity for POPC/TO/W interfaces. Our results suggest that peptide-lipid interactions drive α-helix binding to and retention on lipoproteins. Point mutations in small apolipoproteins could significantly change α-helical propensity or charge, thereby disrupting protein-lipid interactions and preventing the proteins from regulating lipoprotein catabolism at high surface pressures.

One common structural feature of “words” in protein sequences and human texts

Frequently discussed analogy between genetic and human texts is explored by comparison of alternation of polar and non-polar amino-acid residues in proteins and alternation of consonants and vowels in human texts. In human languages, the usage of possible combinations of consonants and vowels is influenced by pronounceability of the combinations. Similarly, oligopeptide composition of proteins is influenced by requirements of protein folding and stability. One special type of structure often present in proteins is amphipathic α-helices in which polar and non-polar amino acids alternate with the period 3.5 residues, not unlike alternation of consonants and vowels. In this study, we evaluated the contribution made by amphipathic alternations to the protein sequence texts (20–24%). Their proportion is lower than respective values for alternating words in human texts (57–89%). The proteomes (full sets of proteins for selected organisms) were transformed into ranked sequences of n-grams (words of length n), including periodical amphipathic structures. Similarly, human texts were transformed into sequences of alternating consonants and vowels. Analysis of the vocabularies shows that in both types of texts (human languages and proteins) the alternating words are dominant or highly preferred, thus, strengthening the analogy between these two types of texts. The contribution of amphipathic words in the upper parts of the ranked lists for 10 analyzed proteomes varies between 58 and 74%. In human texts respective values range between 90 and 100%.

Amphipathic α-Helix Mimetics Based on a 1,2-Diphenylacetylene Scaffold

In order to mimic amphipathic α-helices, a novel scaffold based on a 1,2-diphenylacetylene was designed. NMR and computational modeling confirmed that an intramolecular hydrogen bond favors conformations of the 1,2-diphenylacetylene that allow for accurate mimicry of the i, i + 7 and i + 2, i + 5 side chains found on opposing faces of an α-helix.

Surface pressure-dependent conformation change of apolipoprotein-derived amphipathic α-helices

Amphipathic α-helices (AαH) are the primary structural motif of exchangeable apolipoproteins. AαHs in exchangeable apolipoproteins adsorb, remodel, and desorb at the surface of plasma lipoproteins in response to changes in their size or composition. A triolein/water (TO/W) interface was used as a model surface to study adsorption and desorption of AαHs at a lipoprotein-like interface. We previously reported that AαH peptides spontaneously adsorb to a TO/W interface, but they only partially desorb from the surface when the excess peptide was removed from the system. This finding suggests that "exchangeable" apolipoproteins are in fact partially exchangeable and only desorb from a surface in response to compression or change in composition. Here, we develop a thermodynamic and kinetic model to describe this phenomenon based on the change in the interfacial pressure (Π) of the C-terminal 46 amino acids of apolipoprotein A-I (C46) at a TO/W interface. This model suggests that apolipoproteins have at least two interfacial conformations that are in a surface concentration and Π-dependent equilibrium. This two-state surface equilibrium model, which is based on experimental data and is consistent with dynamic changes in Π(t), provides insights into the selective metabolism and clearance of plasma lipoproteins and the process of lipoprotein remodeling.

Effects of Chain Length of an Amphipathic Polypeptide Carrying the Repeated Amino Acid Sequence (LETLAKA)n on α-Helix and Fibrous Assembly Formation

Polypeptide α3 (21 residues), with three repeats of a seven-amino-acid sequence (LETLAKA)(3), forms an amphipathic α-helix and a long fibrous assembly. Here, we investigated the ability of α3-series polypeptides (with 14-42 residues) of various chain lengths to form α-helices and fibrous assemblies. Polypeptide α2 (14 residues), with two same-sequence repeats, did not form an α-helix, but polypeptide α2L (15 residues; α2 with one additional leucine residue on its carboxyl terminal) did form an α-helix and fibrous assembly. Fibrous assembly formation was associated with polypeptides at least as long as polypeptide α2L and with five leucine residues, indicating that the C-terminal leucine has a critical element for stabilization of α-helix and fibril formation. In contrast, polypeptides α5 (35 residues) and α6 (42 residues) aggregated easily, although they formed α-helices. A 15-35-residue chain was required for fibrous assembly formation. Electron microscopy and X-ray fiber diffraction showed that the thinnest fibrous assemblies of polypeptides were about 20 Å and had periodicities coincident with the length of the α-helix in a longitudinal direction. These results indicated that the α-helix structures were orientated along the fibrous axis and assembled into a bundle. Furthermore, the width and length of fibrous assemblies changed with changes in the pH value, resulting in variations in the charged states of the residues. Our results suggest that the formation of fibrous assemblies of amphipathic α-helices is due to the assembly of bundles via the hydrophobic faces of the helices and extension with hydrophobic noncovalent bonds containing a leucine.

Synthetic Molecular Evolution of Pore-Forming Peptides by Iterative Combinatorial Library Screening

We previously reported the de novo design of a combinatorial peptide library that was subjected to high-throughput screening to identify membrane-permeabilizing antimicrobial peptides that have β-sheet-like secondary structure. Those peptides do not form discrete pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24-26-residue, 16,200-member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non-helical secondary structure. We used a new high-throughput assay to identify members that self-assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane-permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore-formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore-forming peptides known. Furthermore, they are not α-helical, which makes them unusual, as most of the highly potent pore-forming peptides are amphipathic α-helices. Here we demonstrate that this synthetic molecular evolution-based approach, taken together with the new high-throughput tools we have developed, enables the identification, refinement, and optimization of unique membrane active peptides.

Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10

The pre-channel state of helices 6, 7, and 10 (Val(447)-Gly(475) and Ile(508)-Ile(522)) of colicin E1 was investigated by a site-directed fluorescence labeling technique. A total of 44 cysteine variants were purified and covalently labeled with monobromobimane fluorescent probe. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. Using site-directed fluorescence labeling combined with our novel helical periodicity analysis method, the data revealed that helices 6, 7, and 10 are separate amphipathic α-helices with a calculated periodicity of T = 3.34 ± 0.08 for helix 6, T = 3.56 ± 0.03 for helix 7, and T = 2.99 ± 0.12 for helix 10 in the soluble state. In the membrane-bound state, the helical periodicity was determined to be T = 3.00 ± 0.15 for helix 6, T = 3.68 ± 0.03 for helix 7, and T = 3.47 ± 0.04 for helix 10. Dual fluorescence quencher analysis showed that both helices 6 and 7 adopt a tilted topology that correlates well with the analysis based on the fluorescence anisotropy profile. These data provide further support for the umbrella model of the colicin E1 channel domain.

Remodeling of Lipid Vesicles into Cylindrical Micelles by α-Synuclein in an Extended α-Helical Conformation

α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.

Apolipoprotein A-I helical structure and stability in discoidal high-density lipoprotein (HDL) particles by hydrogen exchange and mass spectrometry

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6nm) and small (7.8nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5kcal/mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.

Difference in lipid packing sensitivity of exchangeable apolipoproteins apoA-I and apoA-II: An important determinant for their distinctive role in lipid metabolism

Exchangeable apolipoproteins A-I and A-II play distinct roles in reverse cholesterol transport. ApoA-I interacts with phospholipids and cholesterol of the cell membrane to make high density lipoprotein particles whereas apolipoprotein A-II interacts with high density lipoprotein particles to release apolipoprotein A-I. The two proteins show a high activity at the aqueous solution/lipid interface and are characterized by a high content of amphipathic α-helices built upon repetition of the same structural motif. We set out to investigate to what extent the number of α-helix repeats of this structural motif modulates the affinity of the protein for lipids and the sensitivity to lipid packing. To this aim we have compared the insertion of apolipoproteins A-I and A-II in phospholipid monolayers formed on a Langmuir trough in conditions where lipid packing, surface pressure and charge were controlled. We also used atomic force microscopy to obtain high resolution topographic images of the surface at a resolution of several nanometers and performed statistical image analysis to calculate the spatial distribution and geometrical shape of apolipoproteins A-I and A-II clusters. Our data indicate that apolipoprotein A-I is sensitive to packing of zwitterionic lipids but insensitive to the packing of negatively charged lipids. Interestingly, apolipoprotein A-II proved to be insensitive to the packing of zwitterionic lipids. The different sensitivity to lipid packing provides clues as to why apolipoprotein A-II barely forms nascent high density lipoprotein particles while apolipoprotein A-I promotes their formation. We conclude that the different interfacial behaviors of apolipoprotein A-I and apolipoprotein A-II in lipidic monolayers are important determinants of their distinctive roles in lipid metabolism.