Insertion into lipid bilayer of truncated pHLIP® peptide

The investigation of pH-dependent membrane-associated folding has both fundamental interest and practical applications for targeting of acidic tumors and specific delivery of therapeutic molecules across membrane of cancer cells. We and others investigated molecular mechanism and medical uses of class of water soluble membrane peptides, pH (Low) Insertion Peptides (pHLIP^® peptides). Here we employed optical spectroscopy methods to study interactions of the truncated pHLIP^® peptide (Short pHLIP^®) with lipid bilayer of membrane. Tryptophan fluorescence, CD and OCD data indicate on pH-triggered formation of transmembrane helical structure. Dual quenching and FRET assays demonstrated that Short pHLIP^® peptide spans lipid bilayer of membrane similar to Long pHLIP^® peptides. Truncated pHLIP^® peptides with multiple charged and protonatable residues in their sequences potentially can make these peptides to be less hydrophobic compared to Long pHLIP^® peptides, and might have utility in tumor imaging, and potentially, in pH-regulated cytoplasmic delivery of moderately hydrophobic drugs.     

How did the neurotransmitter cross the bilayer? A closer view

Plasma membrane neurotransmitter transporters for monoamines, GABA, glycine and excitatory amino acids are homologous to two sizable families of bacterial amino acid transporters. Recently, a high resolution structure was determined for a thermophilic glutamate transporter. Also, a bacterial tryptophan transporter related to the family of biogenic amine neurotransmitter transporters was functionally expressed. Structural insights from these and other bacterial transporters will help to rationalize the mechanisms for the increasingly complex functions that have been described for mammalian transporters, in addition to their modes of regulation. We touch on recent insights into the functions of neurotransmitter transporters in their physiological contexts.     

Molecular dynamics simulation of six β-blocker drugs passing across POPC bilayer

Six selected β-blocker drugs (alprenolol, atenolol, metoprolol, nadolol, pindolol and propranolol) passing across 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer were studied using all-atom molecular dynamics simulation. The free energy profiles can be divided into two groups, according to their shapes: the free energy curve of group one (atenolol, nadolol and pindolol) has an obvious minimum while that of the other group (propranolol, metoprolol and alprenolol) is flat inside membrane. Energy analysis shows that electrostatic interaction plays an important role for the first group drugs. The hydrogen bond analysis results also certify that the first group drugs form more hydrogen bonds than the other β-blockers. The calculated permeability sequence agrees with the experimental ones. Our calculation suggests that the permeability model using potential of mean force (PMF) method can be also applied to chemically similar compounds besides chemically diverse compounds.     

Binding of Aβ peptide creates lipid density depression in DMPC bilayer

Using isobaric–isothermal replica exchange molecular dynamics and all-atom explicit water model we study the impact of Aβ monomer binding on the equilibrium properties of DMPC bilayer. We found that partial insertion of Aβ peptide into the bilayer reduces the density of lipids in the binding “footprint” and indents the bilayer thus creating a lipid density depression. Our simulations also reveal thinning of the bilayer and a decrease in the area per lipid in the proximity of Aβ. Although structural analysis of lipid hydrophobic core detects disordering in the orientations of lipid tails, it also shows surprisingly minor structural perturbations in the tail conformations. Finally, partial insertion of Aβ monomer does not enhance water permeation through the DMPC bilayer and even causes considerable dehydration of the lipid–water interface. Therefore, we conclude that Aβ monomer bound to the DMPC bilayer fails to perturb the bilayer structure in both leaflets. Limited scope of structural perturbations in the DMPC bilayer caused by Aβ monomer may constitute the molecular basis of its low cytotoxicity.     

Cholesterol promotes the interaction of Alzheimer β-amyloid monomer with lipid bilayer

Cholesterol in the plasma membrane plays an important role in the pathogenesis of Alzheimer's disease, but the exact function of cholesterol in the regulation of amyloid-β (Aβ) generation, aggregation, and toxicity remains elusive. To gain insight into the bioactivity of cholesterol, we investigate the effect of cholesterol levels on the interaction of Aβ(1-42) monomer with the zwitterionic 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer containing different mole fractions of cholesterol from χ=0, 0.2, to 0.4 using all-atom molecular dynamics simulations. Simulation results show that an increased cholesterol level alters the structure, dynamics, and surface chemistry of the lipid bilayer, leading to increased bilayer thickness, hydrophobic chain order, surface hydrophobicity, and decreased lipid mobility. All these effects significantly promote the binding of Aβ to the lipid bilayer. Mechanistically, the adsorption of Aβ on the bilayer is a cooperative process. First, charged residues act as anchors to establish the initial binding of Aβ to phosphate headgroups of the bilayer driven by electrostatic interactions, which further facilitates hydrophobic residues to reside on the bilayer. Once hydrophobic residues especially from C-terminus are locked on the bilayer, the interactions among charged residues, lipid bilayer, and calcium ions are optimized to provide additional attractive forces to stabilize Aβ adsorbed on or inserted into the lipid bilayer. Inclusion of cholesterol makes this binding process more energetically favorable. Upon adsorption on the bilayer, Aβ appears to preferentially adopt α-helical or unstructured conformation. This work supports that cholesterol acts as a promoter for Aβ--membrane interactions, which would facilitate Aβ aggregation and membrane insertion.     

Modeling analysis of the lipid bilayer–cytoskeleton coupling in erythrocyte membrane

Studies of thermal fluctuations in discocytes, echinocytes, and spherocytes suggest that the coupling between lipid bilayer and cytoskeleton can affect viscoelastic behavior of single erythrocyte membranes. To test this hypothesis, we developed a 3D constitutive model describing viscoelastic behavior of erythrocyte membranes, at long relaxation times \(t \in [0.20\,\mathrm {s}, 1.05\,\mathrm {s}]\) and short relaxation times \(t \in [0.03\,\mathrm {s}, 0.20\,\mathrm {s}]\) . The model was constructed using combination of spring and spring pot rheological elements arranged in parallel. The rearrangement of cytoskeleton induced by changing the bending state of lipid bilayer was described by a modified Eyring model. The model predictions point to an anomalous nature of energy dissipation and an ordered harmonic nature of the coupling mechanism described by a series of fractional derivatives of the order n \(\alpha \) (where \( n \in [- 1, 2]\) ). As a result, the stress generated within the lipid bilayer is related to the rate of change of the irreversible stress within the cytoskeleton.     

Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO₂ and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P₂ (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P₂ and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.     

The effect of hydrophobicity of β-lactam antibiotics on their phospholipid bilayer permeability

Phospholipid bilayer permeability of β-lactam antibiotics was determined using liposomes enclosing β-lactamase. There was good correlation between the permeability and hydrophobicity within the analogous β-lactams. However, the effect of hydrophobic character on the permeability parameter was very different between the groups. Moderately hydrophilic penicillins such as benzylpenicillin and ampicillin showed very high permeability compared with cephalosporins. Penicillins having hindered side chains such as oxacillin and methicillin showed moderate permeability taking into account their hydrophobicity. These observations are suggestive of outer membrane permeation of these β-lactams via routes other than the porin pore, especially in porin-deficient mutants of gram-negative bacteria.     

Is lipid bilayer binding a common property of inhibitor cysteine knot ion-channel blockers?

Recent studies of several ICK ion-channel blockers suggest that lipid bilayer interactions play a prominent role in their actions. Structural similarities led to the hypothesis that bilayer interactions are important for the entire ICK family. We have tested this hypothesis by performing direct measurements of the free energy of bilayer partitioning (DeltaG) of several peptide blockers using our novel quenching-enhanced fluorescence titration protocol. We show that various ICK peptides demonstrate markedly different modes of interaction with large unilamellar lipid vesicles. The mechanosensitive channel blocker, GsMTx4, and its active diastereomeric analog, D-GsMTx4, bind strongly to both anionic and zwitterionic membranes. One potassium channel gating modifier, rHpTx2gs, interacts negligibly with both types of vesicles at physiological pH, whereas another, SGTx1, interacts only with anionic lipids. The slope of DeltaG dependence on surface potential is very shallow for both GsMTx4 and D-GsMTx4, indicating complex interplay of their hydrophobic and electrostatic interactions with lipid. In contrast, a cell-volume regulator, GsMTx1, and SGTx1 exhibit a very steep DeltaG dependence on surface potential, resulting in a strong binding only for membranes rich in anionic lipids. The high variability of 5 kcal/mole in observed DeltaG shows that bilayer partitioning is not a universal property of the ICK peptides interacting with ion channels.     

Nystatin-induced increase in photocurrent in the system ‘bacteriorhodopsin proteoliposome/bilayer planar membrane’

Proteoliposomes were reconstituted from bacteriorhodopsin sheets, asolectin and cholesterol with or without nystatin. Bacteriorhodopsin-mediated electrogenesis was monitored using (1) a proteoliposome suspension and phenyldicarbaundecaborane (PCB^?) probe or (2) proteoliposomes associated with planar bilayer membrane and orthodox electrometer techniques. In the light, PCB^? was shown to be taken up by proteoliposomes. The PCB^? uptake was inhibited by addition of nystatin to an incubation mixture with proteoliposomes if they were reconstituted in the presence of nystatin. Extraproteoliposomal nystatin was without influence if nystatin was omitted from the reconstitution mixture. The nystatin-containing proteoliposomes were associated with a planar bilayer asolectin membrane in the presence of Ca²⁺. It was found that in such a system, bacteriorhodopsin generated a photocurrent charging the proteoliposome-containing (cis-side) compartment negatively and the trans-side compartment positively. The photoresponse was shown to be increased several-fold by addition of nystatin to the trans-side solution. Nystatin addition was ineffective if proteoliposomes were reconstituted without nystatin. Taking into account that nystatin forms ion-permeable pores in a membrane only if present on both sides of the membrane and that this membrane is bilayer, one can explain the above data assuming that (1) the intraproteoliposomal solution does not mix with the extraproteoliposomal one when proteoliposomes are attached to a planar black membrane and (2) the attached proteoliposomes are separated from the trans-side bathing solution by a bimolecular membrane. If this is the case, nystatin in the trans-side bathing solution and inside the attached proteoliposome can form pores across that part of the planar membrane which separates the proteoliposome interior from the trans-side solution. Through these pores, H⁺ (pumped by bacteriorhodopsin from the cis-side solution into the proteoliposome interior) or some other intraproteoliposomal ions can be equilibrated with those in the trans-side solution. As a result, the bacteriorhodopsin-generated photocurrent increases.     

Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?

In the eye lens, the oxygen partial pressure is very low and the cholesterol (Chol) content in cell membranes is very high. Disturbance of these quantities results in cataract development. In human lens membranes, both bulk phospholipid-Chol domains and the pure Chol bilayer domains (CBDs) were experimentally detected. It is hypothesized that the CBD constitutes a significant barrier to oxygen transport into the lens. Transmembrane profiles of the oxygen diffusion-concentration product, obtained with electron paramagnetic resonance spin-labeling methods, allow evaluation of the oxygen permeability (P_M) of phospholipid membranes but not the CBD. Molecular dynamics simulation can independently provide components of the product across any bilayer domain, thus allowing evaluation of the P_M across the CBD. Therefore, to test the hypothesis, MD simulation was used. Three bilayers containing palmitoyl-oleoyl-phosphorylcholine (POPC) and Chol were built. The pure Chol bilayer modeled the CBD, the 1:1 POPC-Chol bilayer modeled the bulk membrane in which the CBD is embedded, and the POPC bilayer was a reference. To each model, 200 oxygen molecules were added. After equilibration, the oxygen concentration and diffusion profiles were calculated for each model and multiplied by each other. From the respective product profiles, the P_M of each bilayer was calculated. Favorable comparison with experimental data available only for the POPC and POPC-Chol bilayers validated these bilayer models and allowed the conclusion that oxygen permeation across the CBD is ~ 10 smaller than across the bulk membrane, supporting the hypothesis that the CBD is a barrier to oxygen transport into the eye lens.     

A model for the lateral diffusion of “stiff” chains in a lipid bilayer

We present random walk models for the diffusive motion of lipid probe molecules in a lipid bilayer and calculate the diffusion constants for probes spanning the entire bilayer and for probes extending through one lipid layer only. The stiffness of such molecules can explain the observed value of 2/3 for the ratio of these diffusion constants.     

Interactions of amyloid-β peptides on lipid bilayer studied by single molecule imaging and tracking

The amyloid-β peptides (Aβ40 and Aβ42) feature prominently in the synaptic dysfunction and neuronal loss associated with Alzheimer's disease (AD). This has been proposed to be due either to interactions between Aβ and cell surface receptors affecting cell signaling, or to the formation of calcium-permeable channels in the membrane that disrupt calcium homeostasis. In both mechanisms the cell membrane is the primary cellular structure with which Aβ interacts. Aβ concentrations in human bodily fluids are very low (pM-nM) rendering studies of the size, composition, cellular binding sites and mechanism of action of the oligomers formed in vivo very challenging. Most studies, therefore, have utilized Aβ oligomers prepared at micromolar peptide concentrations, where Aβ forms oligomeric species which possess easily observable cell toxicity. Such toxicity has not been observed when nM concentrations of peptide are used in the experiment highlighting the importance of employing physiologically relevant peptide concentrations for the results to be of biological significance. In this paper single-molecule microscopy was used to monitor Aβ oligomer formation and diffusion on a supported lipid bilayer at nanomolar peptide concentrations. Aβ monomers, the dominant species in solution, tightly associate with the membrane and are highly mobile whereas trimers and higher-order oligomers are largely immobile. Aβ dimers exist in a mixture of mobile and immobile states. Oligomer growth on the membrane is more rapid for Aβ40 than for the more amyloidogenic Aβ42 but is largely inhibited for a 1:1 Aβ40:Aβ42 mixture. The mechanism underlying these Aβ40-Aβ42 interactions may feature in Alzheimer's pathology.     

Depth of α-synuclein in a bilayer determined by fluorescence, neutron reflectometry, and computation

α-Synuclein (α-syn) membrane interactions are implicated in the pathogenesis of Parkinson's disease. Fluorescence and neutron reflectometry (NR) measurements reveal that α-syn penetrates ～9–14 Å into the outer leaflet of the bilayer, with a substantial portion of the membrane-bound polypeptide extending into the aqueous solvent. For the first time, to our knowledge, we used NR to obtain direct quantitative evidence of α-syn-induced membrane thinning. To examine the effect of specific residues on membrane penetration depths, we used a series of W4-containing N-terminal peptides. We identified that the first 15 residues (P15) nearly recapitulate the features of the full-length protein (i.e., partition constants, molecular mobility, and insertion of the W4 side chain into the bilayer), and found that as few as the first four N-terminal residues are sufficient for vesicle binding. Although at least one imperfect amphipathic repeat sequence (KAKEGV) is required for α-helical formation, secondary structural formation has little effect on membrane affinity. To develop an N-terminal α-syn model for bilayer interactions, we performed molecular-dynamics simulations of the P15 peptide submerged in a bilayer. The simulation results are highly consistent with experimental data indicating a broad low-energy region (8.5–14.5 Å) for W4 insertion.     

Transmembrane helix–helix interactions are modulated by the sequence context and by lipid bilayer properties

Folding of polytopic transmembrane proteins involves interactions of individual transmembrane helices, and multiple TM helix–helix interactions need to be controlled and aligned to result in the final TM protein structure. While defined interaction motifs, such as the GxxxG motif, might be critically involved in transmembrane helix–helix interactions, the sequence context as well as lipid bilayer properties significantly modulate the strength of a sequence specific transmembrane helix–helix interaction. Structures of 11 transmembrane helix dimers have been described today, and the influence of the sequence context as well as of the detergent and lipid environment on a sequence specific dimerization is discussed in light of the available structural information. This article is part of a Special Issue entitled: Protein Folding in Membranes.