Peptide Partitioning Research Articles

Thermodynamics of Membrane Partitioning and Folding of an Anionic Cell-Penetrating Peptide

Thermodynamics of Membrane Partitioning and Folding of an Anionic Cell-Penetrating Peptide

Partition Coefficient of Arginine between Translocon Interior and Lipid Phase

Depending on its hydrophobicity, the protein translocation complex sorts polypeptide segments into the membrane or into the aqueous solution. Its short residence time in the channel limits polypeptide sampling between the aqueous lumen and the membrane interior, giving rise to an out-of-equilibrium condition. Moreover, reduced mobility and orientational restrictions of confined intraluminal water molecules are bound to further bias peptide partition into the membrane, rendering good agreement between the physico-chemical and biological hydrophobicity scales all the more surprising. Here we report the equilibrium partition coefficient of arginine derived on the single molecule level by monitoring -repetitive lateral movement of a stalled polypeptide segment between the SecYEG lumen and the lipid interior. Polypeptide movements in the direction normal to the membrane were inhibited by sandwiching the transmembrane sequence between a SecA binding sequence and the sequence of the calmodulin binding protein. With calmodulin and SecA bound to opposite sides of the membrane, polypeptide residence within the SecYEG pore was indicated by channel opening, whereas episodes of residence in the hydrophobic membrane interior corresponded to channel closings. Translocon purification and reconstitution into planar lipid bilayers were performed as described previously.1-2 Comparison to amino acid partitioning in a biphasic system (physico-chemical hydrophobicity scale) allowed us to address the effects of water confinement by the SecYEG pore, while comparison to the biological hydrophobicity scale reveals out-of-equilibrium effects. 1. Knyazev, D. G.; ⋯; Pohl, P., The bacterial translocon SecYEG opens upon ribosome binding. J. Biol. Chem. 2013, 288 (25), 17941-6. 2. Knyazev, D. G.;⋯ ; Pohl, P., Ion conductivity of the bacterial translocation channel SecYEG engaged in translocation. J. Biol. Chem. 2014, 289 (35), 24611-6.

Antimicrobial Peptide K0-W6-Hya1 Induces Stable Structurally Modified Lipid Domains in Anionic Membranes.

Considering the known different mode of action of antimicrobial peptides in zwitterionic and anionic cell membranes, the present work compares the action of the antimicrobial peptide K0-W6-Hya1 (KIFGAIWPLALGALKNLIK-NH2) with zwitterionic and negatively charged model membranes, namely, liposomes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, and a mixture of the two. Differential scanning calorimetry (DSC), steady state fluorescence of the Trp residue, dynamic light scattering (DLS), and measurement of the leakage of an entrapped fluorescent dye (carboxyfluorescein, CF) were performed with large unilamellar vesicles (LUVs). All techniques evidenced the different action of the peptide in zwitterionic and anionic vesicles. Trp fluorescence spectroscopy shows that the differences are related not only to the partition of the cationic peptide in zwitterionic and anionic membranes, but also to the different penetration depth of the peptide into the lipid bilayers: Trp goes deeper into negatively charged membranes, both in the gel and fluid phases, than into zwitterionic ones. DSC shows that the peptide is strongly attached to anionic bilayers, giving rise to the coexistence of two different lipid regions, one depleted of peptide and another one peptide-disturbed, possibly a stable or transient polar pore, considering the leakage of CF. This contrasts with the homogeneous effect produced by the peptide in zwitterionic membranes, probably related to peptide-membrane diffusion. Moreover, in mixed bilayers (PC:PG), the peptide sequesters negatively charged lipids, creating peptide-rich anionic lipid regions, strongly disturbing the membrane. The distinct structural interaction displayed by the peptide in PC and PG membranes could be related to the different mechanisms of action of the peptide in anionic prokaryotic and zwitterionic eukaryotic cell membranes.

Partitioning-dependent conversion of polyelectrolyte assemblies in an aqueous two-phase system.

Partitioning refers to the distribution of solute molecules in the two immiscible phases of a mixture of two solutions, such as an aqueous two-phase system (ATPS). The partitioning of RNA and peptide has been adjusted in situ to facilitate their assembly into intracellular membraneless organelles. Despite the immense potential of this approach in artificial systems, a partitioning-dependent assembly of macromolecules has been limited, due to the sophisticated processing associated with their in situ modification. Here we demonstrate an approach to direct the assembly of polyelectrolytes in an ATPS through varying their partitioning via pH changes. Microcapsules can be converted to microgel particles as the polyelectrolytes selectively partition to different emulsion phases when changing pH. Such partitioning-dependence can also be equally applied for complexing hydrophilic nanoparticles with polyelectrolytes in an ATPS. By enabling access of hydrophilic materials across the aqueous interface freely, the ATPS allows modification of their intrinsic properties in situ; this advantage will inspire more versatile control over the partitioning of hydrophilic materials and will create new multi-functional biomaterials.

Separation of Antioxidant Peptides from Pepsin Hydrolysate of Whey Protein Isolate by ATPS of EOPO Co-polymer (UCON)/Phosphate

An aqueous two-phase system (ATPS) consisting of poly(ethylene glycol-ran-propylene glycol) monobutyl ether (UCON)/phosphate was developed for the separation of the antioxidant peptides from pepsin hydrolysate of Whey Protein Isolate (WPI). The efficiency of the separation was evaluated based on the DPPH radical scavenging activity, ABTS radical scavenging activity and ferric reducing antioxidant power (FRAP) of the separated peptides. The effects of some parameters on the partition of antioxidant peptides were investigated. An efficient separation of antioxidant peptides was achieved using ATPS with pH of 4.0, 4 mL of UCON solution (40%, w/w), 4 mL of KH2PO4 solution (15.5%, w/w), 2 mL of WPI hydrolysate and 0.40 g/10 mL of NaCl. Reversed-phase high-performance liquid chromatography (RP-HPLC), amino acid analyzer and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to characterize the purified peptides separated by the ATPS. The peptides in top phase were less polar than those in bottom phase. More antioxidative and hydrophobic amino acids were extracted to the top phase of ATPS, and the peptides with the amino acid sequences with antioxidant activities moved to the top phase as well. In conclusion, antioxidant peptides were successfully separated from the WPI hydrolysate by UCON/phosphate ATPS.

The influence of mild acidity on lysyl-phosphatidylglycerol biosynthesis and lipid membrane physico-chemical properties in methicillin-resistant Staphylococcus aureus

The increased biosynthesis of lysyl-phosphatidylglycerol in Staphylococcus aureus when cultured under conditions of mild acidity and the resultant increased proportion of this lipid in the plasma membrane of the bacterium, alters the physico-chemical properties of lipid bilayers in a manner which is itself dependent upon environmental pH. Clinically relevant strains of S. aureus, both methicillin susceptible and resistant, all exhibited increased lysyl-phosphatidylglycerol biosynthesis in response to mild environmental acidity, albeit to differing degrees, from ∼30% to ∼55% total phospholipid. Polar lipid extracts from these bacteria were analysed by 31P NMR and reconstituted into vesicles and monolayers, which were characterised by zeta potential measurements and Langmuir isotherms respectively. A combination of increased lysyl-phosphatidylglycerol content and mild environmental acidity were found to synergistically neutralise the charge of the membranes, in one instance altering the zeta potential from −56mV to +21mV, and induce closer packing between the lipids. Challenge of reconstituted S. aureus lipid model membranes by the antimicrobial peptide magainin 2 F5W was examined using monolayer subphase injection and neutron diffraction, and revealed that ionisation of the headgroup α-amine of lysyl-phosphatidylglycerol at pH 5.5, which reduced the magnitude of the peptide-lipid interaction by 80%, was more important for resisting peptide partitioning than increased lipid content alone. The significance of these results is discussed in relation to how colonising mildly acidic environments such as human mucosa may be facilitated by increased lysyl-phosphatidylglycerol biosynthesis and the implications of this for further biophysical analysis of the role of this lipid in bacterial membranes.

Phase Composition Control in Microsphere-Supported Biomembrane Systems.

The popularization of studies in membrane protein lipid phase coexistence has prompted the development of new techniques to construct and study biomimetic systems with cholesterol-rich lipid microdomains. Here, microsphere-supported biomembranes with integrated α-helical peptides, referred to as proteolipobeads (PLBs), were used to model peptide/protein partitioning within DOPC/DPPC/cholesterol phase-separated membranes. Due to the appearance of compositional heterogeneity and impurities in the formation of model PLB assemblies, fluorescence-activated cell sorting (FACS) was used to characterize and sort PLB populations on the basis of disordered phase (Ld) content. In addition, spectral imaging was used to assess the partitioning of FITC-labeled α-helical peptide between fluorescently labeled Ld phase and unlabeled ordered phase (Lo) phase lipid microdomains. The apparent peptide partition coefficient, Kp,app, was measured to be 0.89 ± 0.06, indicating a slight preference of the peptide for the Lo phase. A biomimetic motif of the Lo phase concentration enhancement of the biotinyl-peptide ligand display in proteolipobeads was also observed. Finally, peptide mobility was measured by FRAP separately in each lipid phase, yielding diffusivities of 0.036 ± 0.005 and 0.014 ± 0.003 μm2/s in the Ld and Lo phases, respectively.

Molecular Basis for the Role of Cationic Residues in Antimicrobial Peptide Interactions

Antimicrobial peptides (AMPs) play a vital role in the innate immune response and represent promising templates for developing broad-spectrum alternatives to conventional antibiotics. Most AMPs are charged amino acid sequences that interact more strongly with negatively charged prokaryotic membranes than net neutral eukaryotic ones. Both AMPs and synthetic analogues with arginine-like guanidinium groups show a greater toxic effect against bacteria than those with lysine-like amine groups, though the atomistic mechanism for this increase in potency remains unclear. To examine this, we have conducted comparative molecular dynamics simulations of a model prokaryotic membrane system (POPC/POPG) interacting with two different mutants of a prototypical AMP, KR-12: one with all lysine residues mutated to arginine (R-KR12), and one with all arginine residues mutated to lysine (K-KR12). Simulations show that both peptides partition analogously to the bilayer and display similar preferences for forming hydrogen bonds with the anionic POPGs. However, R-KR12 has a significantly higher propensity for hydrogen bonding with the bilayer than K-KR12, as well as stronger peptide-bilayer hydrogen bonds, resulting in considerably longer interaction times. Additional simulations with single and double methylated R-KR12 mutants show that the strength of these hydrogen bonds, rather than the number of bonds formed, is responsible for the extensive peptide-bilayer interaction seen in the R-KR12 system. Finally, free energy simulations reveal that both peptides are unstructured in solution and adopt a highly helical amphipathic structure when inserted into the bilayer. Overall, these results help elucidate the greater toxicity of arginine-rich AMPs and offer potential insights for designing more potent analogues in the future.

Structural Thermodynamics of myr-Src(2–19) Binding to Phospholipid Membranes

Many proteins are anchored to lipid bilayer membranes through a combination of hydrophobic and electrostatic interactions. In the case of the membrane-bound nonreceptor tyrosine kinase Src from Rous sarcoma virus, these interactions are mediated by an N-terminal myristoyl chain and an adjacent cluster of six basic amino-acid residues, respectively. In contrast with the acyl modifications of other lipid-anchored proteins, the myristoyl chain of Src does not match the host lipid bilayer in terms of chain conformation and dynamics, which is attributed to a tradeoff between hydrophobic burial of the myristoyl chain and repulsion of the peptidic moiety from the phospholipid headgroup region. Here, we combine thermodynamic information obtained from isothermal titration calorimetry with structural data derived from 2H, 13C, and 31P solid-state nuclear magnetic resonance spectroscopy to decipher the hydrophobic and electrostatic contributions governing the interactions of a myristoylated Src peptide with zwitterionic and anionic membranes made from lauroyl (C12:0) or myristoyl (C14:0) lipids. Although the latter are expected to enable better hydrophobic matching, the Src peptide partitions more avidly into the shorter-chain lipid analog because this does not require the myristoyl chain to stretch extensively to avoid unfavorable peptide/headgroup interactions. Moreover, we find that Coulombic and intrinsic contributions to membrane binding are not additive, because the presence of anionic lipids enhances membrane binding more strongly than would be expected on the basis of simple Coulombic attraction.

Peptide Folding in Translocon-Like Pores.

The cellular translocon, present in all three domains of life, is one of the most versatile and important biological nanopores. This complex molecular apparatus is directly responsible for the secretion of globular proteins across membranes as well as the insertion of integral membrane proteins into lipid bilayers. Recently determined structures of the archaean SecY translocon reveal an hour-glass-shaped pore, which accommodates the nascent peptide chain during translocation. While these structures provide important insights into ribosome binding to the translocon, threading of the nascent chain into the channel, and lateral gate opening for releasing the folded helical peptide into the membrane bilayer, the exact folding pathway of the peptide inside the protein-conducting channel during translocation and prior to the lateral release into the bilayer remains elusive. In the present study, we use molecular dynamics simulations to investigate atomic resolution peptide folding in hour-glass-shaped pore models that are based on the SecY translocon channel structure. The theoretical setup allows systematic variation of key determinants of folding, in particular the degree of confinement of the peptide and the hydration level of the pore. A 27-residue hydrophobic peptide was studied that is preferentially inserted into membranes by the translocon. Our results show that both pore diameter as well as channel hydration are important determinants for folding efficiency and helical stability of the peptide, therefore providing important insights into translocon gating and lateral peptide partitioning.

Interfacial peptide partitioning and undiminished antioxidative and emulsifying activity of oxidatively stressed soy protein hydrolysate in an O/W emulsion

Peptides as surfactants are able to neutralize radicals, but their ability to maintain physical and chemical stability of food emulsions when oxidatively stressed is not clear. In this study, the distribution of peptides present in hydroxyl radical-stressed soy protein hydrolysate (SPH) at the interface and the inhibition of lipid oxidation in an oil-in-water (O/W) emulsion were investigated. O/W emulsions (10 g/100 g soybean oil, 20 mg/mL protein, 1 g Tween 20/100 g emulsion) were prepared under 138 MPa and stored for 14 days. Morphology of emulsion droplets was observed using phase contrast, confocal laser scanning, and transmission electron microscopy. The stability of all emulsions gradually decreased during storage due to flocculation but not coalescence. Oxidized nonhydrolyzed soy protein (SP) and SPH, despite 3–4-fold increases in carbonyls, retained up to 74% of their power to inhibit lipid oxidation in emulsions when compared with nonoxidized counterparts. Nonpolar peptides in SPH had a preferred adsorption at the interface while oxidation decreased the selectivity. In conclusion, oxidation that produces 4-fold carbonyl increases modifies the distribution of peptides without significantly affecting their function as emulsifiers and does not eliminate their antioxidant capacity.

The role of tryptophans on the cellular uptake and membrane interaction of arginine-rich cell penetrating peptides

Cell-penetrating peptides (CPP) are able to efficiently transport cargos across cell membranes without being cytotoxic to cells, thus present a great potential in drug delivery and diagnosis. While the role of cationic residues in CPPs has been well studied, that of Trp is still not clear. Herein 7 peptide analogs of RW9 (RRWWRRWRR, an efficient CPP) were synthesized in which Trp were systematically replaced by Phe residues. Quantification of cellular uptake reveals that substitution of Trp by Phe strongly reduces the internalization of all peptides despite the fact that they strongly accumulate in the cell membrane. Cellular internalization and biophysical studies show that not only the number of Trp residues but also their positioning in the helix and the size of the hydrophobic face they form are important for their internalization efficacy, the highest uptake occurring for the analog with 3 Trp residues. Using CD and ATR-FTIR spectroscopy we observe that all peptides became structured in contact with lipids, mainly in α-helix. Intrinsic tryptophan fluorescence studies indicate that all peptides partition in the membrane in about the same manner (Kp~105) and that they are located just below the lipid headgroups (~10Å) with slightly different insertion depths for the different analogs. Plasmon Waveguide Resonance studies reveal a direct correlation between the number of Trp residues and the reversibility of the interaction following membrane washing. Thus a more interfacial location of the CPP renders the interaction with the membrane more adjustable and transitory enhancing its internalization ability.

Interfacial adsorption of peptides in oil-in-water emulsions costabilized by Tween 20 and antioxidative potato peptides.

Previous studies have shown that soybean oil-in-water (O/W) emulsions prepared with potato protein hydrolysate (PPH) are remarkably stable against oxidative changes. It was hypothesized that partitioning of peptides at the emulsion interface plays an important role in this phenomenon. The present study was conducted to examine the structural characteristics of the interfacial membrane. As revealed by atomic force microscopy, oil droplets costabilized with PPH and Tween 20 were more uniform than those stabilized with Tween 20 only (control). Confocal laser scanning microscopy images indicated the existence of peptides directly anchored into the interfacial membrane. The adsorbed peptides were mostly short oligopeptides composed of two to seven amino acids, of which Ser-Phe-Asp-Leu(Ile)-Lys matched the sequence of patatin. The adsorption of these peptides appeared to both improve the integrity of the interface and contribute to the oxidative stability of the emulsions. Furthermore, cryogenic transmission electron microscopy illustrated the morphology of the interfacial membrane as a noncontinuous short fibril structure. Partitioning of antioxidative peptides in the interfacial membrane provided steric hindrances and electrostatic effects to inhibit oxidation.

The p10 FAST protein fusion peptide functions as a cystine noose to induce cholesterol-dependent liposome fusion without liposome tubulation

The reovirus p10 fusion-associated small transmembrane (FAST) proteins are the smallest known membrane fusion proteins, and evolved specifically to mediate cell–cell, rather than virus–cell, membrane fusion. The 36–40-residue ectodomains of avian reovirus (ARV) and Nelson Bay reovirus (NBV) p10 contain an essential intramolecular disulfide bond required for both cell–cell fusion and lipid mixing between liposomes. To more clearly define the functional, biochemical and biophysical features of this novel fusion peptide, synthetic peptides representing the p10 ectodomains of ARV and NBV were analyzed by solution-state NMR spectroscopy, circular dichroism spectroscopy, fluorescence spectroscopy-based hydrophobicity analysis, and liposome binding and fusion assays. Results indicate that disulfide bond formation promotes exposure of hydrophobic residues, as indicated by bis-ANS binding and time-dependent peptide aggregation under aqueous conditions, implying the disulfide bond creates a small, geometrically constrained, cystine noose. Noose formation is required for peptide partitioning into liposome membranes and liposome lipid mixing, and electron microscopy revealed that liposome–liposome fusion occurs in the absence of liposome tubulation. In addition, p10 fusion peptide activity, but not membrane partitioning, is dependent on membrane cholesterol.

Residue specific partitioning of KL4 into phospholipid bilayers

KL4, which has demonstrated success in the treatment of respiratory distress, is a synthetic helical, amphipathic peptide mimetic of lung surfactant protein B. The unusual periodicity of charged residues within KL4 and its relatively high hydrophobicity distinguish it from canonical amphipathic helical peptides. Here we utilized site specific spin labeling of both lipids and the peptide coupled with EPR spectroscopy to discern the effects of KL4 on lipid dynamics, the residue specific dynamics of hydrophobic regions within KL4, and the partitioning depths of specific KL4 residues into the DPPC/POPG and POPC/POPG lipid bilayers under physiologically relevant conditions. KL4 induces alterations in acyl chain dynamics in a lipid-dependent manner, with the peptide partitioning more deeply into DPPC-rich bilayers. Combined with an earlier NMR study of changes in lipid dynamics on addition of KL4 (V.C. Antharam et al., 2009), we are able to distinguish how KL4 affects both collective bilayer motions and intramolecular acyl chain dynamics in a lipid-dependent manner. EPR power saturation results for spin labeled lipids demonstrate that KL4 also alters the accessibility profiles of paramagnetic colliders in a lipid-dependent manner. Measurements of dynamics and depth parameters for individual spin-labeled residues within KL4 are consistent with a model where the peptide partitions deeply into the lipid bilayers but lies parallel to the bilayer interface in both lipid environments; the depth of partitioning is dependent on the degree of lipid acyl chain saturation within the bilayer.

Quantitative understanding of pH- and salt-mediated conformational folding of histidine-containing, β-hairpin-like peptides, through single-molecule probing with protein nanopores.

Inter-amino acid residues electrostatic interactions contribute to the conformational stability of peptides and proteins, influence their folding pathways, and are critically important to a multitude of problems in biology including the onset of misfolding diseases. By varying the pH and ionic strength, the inter-amino acid residues electrostatic interactions of histidine-containing, β-hairpin-like peptides alter their folding behavior, and we studied this through quantifying, at the unimolecular level, the frequency, dwell-times of translocation events, and amplitude of blockades associated with interactions between such peptides and the α-hemolysin (α-HL) protein. Acidic buffers were shown to dramatically decrease the rate of peptide capture by the α-HL protein, through the interplay of enthalpic and entropic contributions brought about on the free energy barrier, which controls the peptides-α-HL association rate. We found that in acidic buffers, the amplitude of the blockage induced by an α-HL, β-barrel-residing peptide is smaller than the value seen at neutral pH, and this supports our interpretation of the pH-induced change in the conformation of the peptide, which behaves as a less-stable hairpin at acidic pH values that obstructs, to a lesser extent, the protein pore. This is also confirmed by the fact that the dissociation rate of such model peptide from the α-HL's β-barrel is higher at acidic, as compared to neutral, pH values. Experiments performed in low-salt buffers revealed the dramatic decrease of the peptide capture rate by the α-HL protein, most likely caused by the increase in the radius of counterions cloud around the peptide that hinders peptide partition into the α-barrel, and histidines protonation at low pH bolsters this effect. Reduced electrostatic screening in low-salt buffers, at neutral pH, leads to a decrease in peptides effective cross-sectional areas and an increase of their mobility inside the α-HL pore, due most likely to the chain stretching augmentation, via increased inter-residues electrostatic interactions.

Improvement of HIV fusion inhibitor C34 efficacy by membrane anchoring and enhanced exposure

The aim of the present work was to evaluate the interaction of two new HIV fusion inhibitors {HIVP3 [C34-polyethylene glycol (PEG)₄-cholesterol] and HIVP4 [(C34-PEG₄)₂-cholesterol]} with membrane model systems and human blood cells in order to clarify where and how the fusion inhibitors locate, allowing us to understand their mechanism of action at the molecular level, and which strategies may be followed to increase efficacy. Lipid vesicles with defined compositions were used for peptide partition and localization studies, based on the intrinsic fluorescence of HIVP3 and HIVP4. Lipid monolayers were employed in surface pressure studies. Finally, human erythrocytes and peripheral blood mononuclear cells (PBMCs) isolated from blood samples were used in dipole potential assays. Membrane partition, dipole potential and surface pressure assays indicate that the new fusion inhibitors interact preferentially with cholesterol-rich liquid-ordered membranes, mimicking biological membrane microdomains known as lipid rafts. HIVP3 and HIVP4 are able to interact with human erythrocytes and PBMCs to a similar degree as a previously described simpler drug with monomeric C34 and lacking the PEG spacer, C34-cholesterol. However, the pocket-binding domain (PBD) of both HIVP3 and HIVP4 is more exposed to the aqueous environment than in C34-cholesterol. The present data allow us to conclude that more efficient blocking of HIV entry results from the synergism between the membranotropic behaviour and the enhanced exposure of the PBD.

A Benchmark Dataset Comprising Partition and Distribution Coefficients of Linear Peptides

Peptides have a dominant role in biology; yet the study of their physical properties is at best sporadic. Peptide quantitative structure-activity relationship (QSAR) lags far behind the QSAR analysis of drug-like organic small molecules. Traditionally, QSAR has focussed on experimentally determined partition coefficients as the main descriptor of hydrophobicity. A partition coefficient () is the ratio between the concentrations of an uncharged chemical substance in two immiscible phases: most typically water and an organic solvent, usually 1-octanol. A distribution coefficient () is the equivalent ratio for charged molecules. We report here a compilation of partition and distribution coefficients for linear peptides compiled from literature reports, suitable for the development and benchmarking of peptide and prediction algorithms.

Thermodynamics of Partitioning of Substance P in Isotropic Acidic Bicelles

The temperature dependence of the partition coefficients of a neuropeptide, substance P (SP), in isotropic acidic bicelles was investigated by using a pulsed field gradient nuclear magnetic resonance diffusion technique. The addition of negatively charged dimyristoylphosphatidylserine to the neutral bicelle changed the SP partitioning a little, which implies that the hydrophobic interaction between the hydrophobic residues of SP and the acyl chains of lipid molecules is the major interaction while the electrostatic interaction is minor in SP binding in a lipid membrane. From the temperature dependence of the partition coefficients, thermodynamic functions were calculated. The partitioning of SP into the acidic bicelles is enthalpy-driven, as it is for small unilamellar vesicles and dodecylphosphocholine micelles, while peptide partitioning into a large unilamellar vesicle is entropy-driven. This may mean that the size of lipid membranes is a more important factor for peptide binding than the surface curvature and surface charge density.

Amphipathic Lipid Packing Sensor Motifs: Probing Bilayer Defects with Hydrophobic Residues

Sensing membrane curvature allows fine-tuning of complex reactions that occur at the surface of membrane-bound organelles. One of the most sensitive membrane curvature sensors, the Amphipathic Lipid Packing Sensor (ALPS) motif, does not seem to recognize the curved surface geometry of membranes per se; rather, it recognizes defects in lipid packing that arise from membrane bending. In a companion paper, we show that these defects can be mimicked by introducing conical lipids in a flat lipid bilayer, in agreement with experimental observations. Here, we use molecular-dynamics (MD) simulations to characterize ALPS binding to such lipid bilayers. The ALPS motif recognizes lipid-packing defects by a conserved mechanism: peptide partitioning is driven by the insertion of hydrophobic residues into large packing defects that are preformed in the bilayer. This insertion induces only minor modifications in the statistical distribution of the free packing defects. ALPS insertion is severely hampered when monounsaturated lipids are replaced by saturated lipids, leading to a decrease in packing defects. We propose that the hypersensitivity of ALPS motifs to lipid packing defects results from the repetitive use of hydrophobic insertions along the monotonous ALPS sequence.