Lipid-mediated Interaction Research Articles

Structural Basis for the Lipid-Mediated Interaction of Tubulin with VDAC Revealed by Neutron Reflectometry

A preponderance of evidence suggests that the mitochondrial outer membrane (MOM) binds dimeric tubulin, which blocks the flux of metabolites through VDAC, the major MOM channel, by inserting its negatively charged, disordered C-terminal tail into the VDAC pore. The discovery of this novel regulatory mechanism of mitochondrial respiration has raised several fundamental questions, including how tubulin binds to the MOM surface, whether its membrane-bound conformation is consistent with the accessibility of its C-termini to the membrane surface, and what role mitochondrial lipids assume in mediating the tubulin-VDAC interaction. Here we report neutron reflectivity (NR) studies on tubulin-coated and VDAC-containing biomimetic membranes. In combination with molecular dynamics simulations, these reveal, first, that dimeric tubulin is anchored to the membrane surface by an amphipathic a-helix of the a-tubulin subunit in an orientation that presents the C-termini of both tubulin subunits to the membrane surface. The structural model is supported by electrophysiological evidence using recombinant tubulin constructs in which C-terminal tails bound to either subunit are individually observed in a VDAC nanopore. Second, we demonstrate using NR that surface-immobilized VDAC can be reconstituted into a complete lipid membrane, thus creating a “protein-tethered bilayer lipid membrane” (ptBLM) over a large sample area. The ptBLM architecture allows dynamic changes of the solution environment, while NR reveals the corresponding structural response of the protein and its lipid environment. This allows us to experimentally observe the effects of pH-induced changes in VDAC's structure on the local lipid environment. Molecular dynamics simulations are in good agreement with NR experiments. We speculate that local curvature or lipid packing stress induced by membrane-embedded VDAC and its structural dynamics serves to localize cytosolic regulators such as tubulin to the vicinity of the channel.

Immobilization of Neurotrophin Peptides on Gold Nanoparticles by Direct and Lipid-Mediated Interaction: A New Multipotential Therapeutic Nanoplatform for CNS Disorders.

Neurotrophins are essential proteins for the development and maintenance of neural functions as well as promising drugs in neurodegenerative disorders. Current limits in their effective clinical applications can be overwhelmed by the combined use of peptidomimetic and nanomedicine approaches. Indeed, neurotrophin-mimicking peptides may allow minimizing the adverse side effects of the whole protein drug. Moreover, the immobilization of such peptides on nanomaterials may offer additional advantages, including protection against degradation, enhanced permeability of barrier membranes, and intrinsic therapeutic properties of the nanoparticles (e.g., antiangiogenic and plasmonic features of gold nanoparticles (AuNPs)). In the present article, we scrutinize the functionalization of spherical AuNPs of diameter 12 nm by peptides because of the N-terminal domains of the nerve growth factor (NGF) and the brain-derived neurotrophic factor (BDNF), NGF1-14 and BDNF1-12, respectively. The hybrid gold–peptide nanobiointerface was investigated, both in the direct physisorption and in the lipid-bilayer-mediated adsorption processes, by a multitechnique study that included UV–vis and X-ray photoelectron spectroscopies, dynamic light scattering, zeta-potential analyses, and atomic force microscopy. Both peptide- and lipid-dependent features were identified, to have a modulation in the peptide coverage of nanoparticles as well as in the cellular uptake of NGF and BDNF peptides, as investigated by confocal microscopy. The promising potentials of the neurotrophins to cross the blood–brain barrier were demonstrated.

Lipid-Mediated Interaction of Double-Knot Toxin with TRPV1 Channels

The Transient Receptor Potential V1 (TRPV1) channel is a cation channel that opens in response to diverse chemical and physical stimuli. These stimuli include capsaicin, temperature, pH and bioactive lipids. Recently, a novel peptide named Double cystine Knot Toxin (DkTx) was purified from tarantula venom and shown to activate the channel from the extracellar site of the membrane and proposed to bind the pore region (Bohlen CJ., et al, 2010). In order to characterize DkTx-TRPV1 interactions, we expressed and purified recombinant His-tagged DkTx in E. coli and 1D4-tagged TRPV1 in Saccharomyces cerevisiae. Expressed TRPV1 binds the specific TRPV1 agonist, 3H- resiniferatoxin (RTx), with high affinity (Kd∼500 pM) and in the presence of DkTx the affinity of RTx increases by ∼4-fold, indicating that both RTx and DkTx interact with the TRPV1 channel in membranes, but at distinct sites that interact allosterically. Pull-down experiments with full-length and truncated TRPV1 (containing the pore-forming S5-S6 segments and the C-terminus suggest that DkTx interacts with both detergent purified channel constructs, supporting an interaction of the toxin with the external pore region. DkTx and TRPV1 constructs do not, however, co-elute as a complex in size-exclusion or affinity chromatography, suggesting that in detergent solution the interactions are weak. To investigate the strength of TRPV1 interactions with DkTx in detergent micelles we utilized surface plasmon resonance with TRPV1 deposited on a gold chip using 1D4 tag. DkTx interacts with TRPV1 in detergent micelles, but with much lower apparent affinity than observed in membranes. The isolated K2 lobe of DkTx exhibited higher affinity than K1, in agreement with the previous electrophysiology studies. These results support the notion that DkTx interacts with the external pore of TRPV1 and suggest that the membrane lipids play a critical role in toxin-channel interaction.

Functional Consequences of Lipid-Mediated Interaction Between Rhodopsin Molecules

In the retina, rhodopsin is densely packed in a rod outer segment disk membrane rich in phospholipids with PC and PE headgroups. Increasing rhodopsin packing density in model PC membranes has been shown to alter metarhodopsin-II (MII) formation. This observation is deemed to be the consequence of rhodopsin association promoted by non-specific properties of the membrane. Here, we studied the effect of rhodopsin packing density on MII formation in membranes characterized by different intrinsic curvature and different interfacial hydrogen bonding propensity. Rhodopsin was reconstituted into a series of POPC bilayers doped with DOPC, di-and mono-methylated DOPE or DOPE at rhodopsin/lipid ratios ranging from 1:250 to 1:70. The level of rhodopsin activation and rate of MII formation were determined by steady-state and time-resolved UV/vis spectroscopy. In PC membranes, lower rhodopsin concentrations shift the MI/MII equilibrium towards MII and result in a faster rate of MII formation, in agreement with previous findings. On the contrary, in membranes rich in lipids with PE headgroups, the MII concentration is independent of rhodopsin packing density and rates of MII formation, while reduced, show only a slight dependence on rhodopsin crowding. In addition, at low or high protein density, the amount of MII formed depends to a larger extent on the ability of the annular PE headgroups to establish hydrogen bonds with the MII state than on changes in membrane curvature elasticity. These results show clearly that MII formation and interaction between rhodopsin molecules depend strongly on interactions between annular lipids and rhodopsin, highlighting the fundamental role of the first layer of lipids surrounding the protein.

Membrane-mediated repulsion between gramicidin pores

We investigated the X-ray scattering signal of highly aligned multilayers of the zwitterionic lipid 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine containing pores formed by the antimicrobial peptide gramicidin as a function of the peptide/lipid ratio. We are able to obtain information on the structure factor of the pore fluid, which then yields the interaction potential between pores in the plane of the bilayers. Aside from a hard core with a radius close to the geometric radius of the pore, we find a repulsive exponential lipid-mediated interaction with a decay length of 2.5 Å and an amplitude that decreases with the pore concentration, in agreement with the hydrophobic matching hypothesis. In dilute systems, the contact value of this interaction is about 30 k BT . Similar results are obtained for gramicidin pores inserted within bilayers formed by the nonionic surfactant pentaethylene glycol monododecyl ether.

Interaction of Alamethicin Pores in DMPC Bilayers

We have investigated the x-ray scattering signal of highly aligned multilayers of the zwitterionic lipid 1,2-dimyristoyl- sn-glycero-3-phosphatidylcholine containing pores formed by the antimicrobial peptide alamethicin as a function of the peptide/lipid ratio. We are able to obtain information on the structure factor of the pore fluid, which then yields the interaction potential between pores in the plane of the bilayers. Aside from a hard core with a radius corresponding to the geometric radius of the pore, we find a repulsive lipid-mediated interaction with a range of ∼30 Å and a contact value of 2.4 k B T. This result is in qualitative agreement with recent theoretical models.

Lipid-Mediated Interactions between Intrinsic Membrane Proteins: Dependence on Protein Size and Lipid Composition

The present study is an application of an approach recently developed by the authors for describing the structure of the hydrocarbon chains of lipid-bilayer membranes (LBMs) around embedded protein inclusions (Lagüe et al., 2000 Biophys. J. 79:2867–2879). The approach is based on statistical mechanical integral equation theories developed for the study of dense liquids. First, the configurations extracted from molecular dynamics simulations of pure LBMs are used to extract the lateral density-density response function. Different pure LBMs composed of different lipid molecules were considered: dioleoyl phosphatidylcholine (DOPC), palmitoyl-oleoyl phosphatidylcholine (POPC), dipalmitoyl phosphatidylcholine (DPPC), and dimyristoyl phosphatidylcholine (DMPC). The results for the lateral density-density response function was then used as input in the integral equation theory. Numerical calculations were performed for protein inclusions of three different sizes. For the sake of simplicity, protein inclusions are represented as hard smooth cylinders excluding the lipid hydrocarbon core from a small cylinder of 2.5 Å radius, corresponding roughly to one aliphatic chain, a medium cylinder of 5 Å radius, corresponding to one α-helix, and a larger cylinder of 9 Å radius, representing a small protein such as the gramicidin channel. The lipid-mediated interaction between protein inclusions was calculated using a closed-form expression for the configuration-dependent free energy. This interaction was found to be repulsive at intermediate range and attractive at short range for two small cylinders in POPC, DPPC, and DMPC bilayers, whereas it oscillates between attractive and repulsive values in DOPC bilayers. For medium size cylinders, it is again repulsive at intermediate range and attractive at short range, but for every model LBM considered here. In the case of a large cylinder, the lipid-mediated interaction was shown to be repulsive for both short and long ranges for the DOPC, POPC, and DPPC bilayers, whereas it is again repulsive and attractive for DMPC bilayers. The results indicate that the packing of the hydrocarbon chains around protein inclusions in LBMs gives rise to a generic (i.e., nonspecific) lipid-mediated interaction which favors the association of two α-helices and depends on the lipid composition of the membrane.

A molecular model for lipid-mediated interaction between proteins in membranes

The loss of conformational freedom experienced by lipid chains in the vicinity of one, or two, impenetrable walls, representing the surfaces of hydrophobic transmembrane proteins, is calculated using a mean-field molecular-level chain packing theory. The hydrophobic thickness of the protein is set equal to that of the unperturbed lipid membrane (i.e., no “ hydrophobic mismatch”). The probability distributions of chain conformations, at all distances from the walls, are calculated by generating all conformations according to the rotational-isomeric-state model, and subjecting the system free energy to the requirement that the hydrophobic core of the membrane is liquid-like, and hence uniformly packed by chain segments. As long as the two protein surfaces are far apart, their interaction zones do not overlap, each extending over several molecular diameters. When the interaction zones begin to overlap, inter-protein repulsion sets in. At some intermediate distance the interaction turns strongly attractive, resulting from the depletion of (highly constrained) lipid tails from the volume separating the two surfaces. The chains confined between the hydrophobic surfaces are tilted away from the walls. Their tilt angle decreases monotonically with the distance from the walls, and with the distance between the walls. A nonmonotonic variation of the lipid-mediated interaction free energy between hydrophobic surfaces in membranes is also obtained using a simple, analytical, model in which chain conformations are grouped according to their director (end-to-end vector) orientations.

Lipid-mediated interactions between intrinsic molecules in bilayer membranes

The effect of intrinsic molecules (impurities) dissolved in phospholipid bilayers on their lipid enviroment and the related lipid-mediated interactions between such molecules are examined in terms of a ten-state model for lipid chain configurations and interchain interactions. The numerical parameters used in the calculations were previously obtained by fitting to experimental data. Numerical results are presented for the case when the impurity is a cholesterol molecule, a protein, or a vacancy and when the lipid bilayer is either in the gel or fluid (liquids crystal) phase. The calculations indicate that proteins fluidize their lipid environment when the bilayer is in the gel phase. The resulting lipid-mediated interaction is shown to be short range and to have a different behavior below and above the main gel–fluid phase transition. The relation to previous theories is discussed in the final section.

Lipid-mediated protein interaction in membranes

This study describes the effects ensuing from a non-specific interaction between membrane integral proteins and the surrounding lipids. The results are obtained using an appropriate molecular field theory to describe the ordering of membrane lipids. The modification of the lipid structure near a protein molecule, while most pronounced within the annulus of the first neighbour molecules, extends two or three layers beyond the annulus. The ordering of lipids within the annulus has a modified temperature dependence, and becomes a continuous function of temperature for low lipid/protein ratios. The change in order of lipid molecules surrounding a protein leads to an indirect, lipid-mediated interaction between membrane integral proteins. This interaction depends sensitively on the bulk lipid order. Under favourable circumstances, it gives rise to protein aggregation.